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500+ Quantitative Research Titles and Topics

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Quantitative research involves collecting and analyzing numerical data to identify patterns, trends, and relationships among variables. This method is widely used in social sciences, psychology , economics , and other fields where researchers aim to understand human behavior and phenomena through statistical analysis. If you are looking for a quantitative research topic, there are numerous areas to explore, from analyzing data on a specific population to studying the effects of a particular intervention or treatment. In this post, we will provide some ideas for quantitative research topics that may inspire you and help you narrow down your interests.

Quantitative Research Titles

Quantitative Research Titles are as follows:

Business and Economics

• “Statistical Analysis of Supply Chain Disruptions on Retail Sales”
• “Quantitative Examination of Consumer Loyalty Programs in the Fast Food Industry”
• “Predicting Stock Market Trends Using Machine Learning Algorithms”
• “Influence of Workplace Environment on Employee Productivity: A Quantitative Study”
• “Impact of Economic Policies on Small Businesses: A Regression Analysis”
• “Customer Satisfaction and Profit Margins: A Quantitative Correlation Study”
• “Analyzing the Role of Marketing in Brand Recognition: A Statistical Overview”
• “Quantitative Effects of Corporate Social Responsibility on Consumer Trust”
• “Price Elasticity of Demand for Luxury Goods: A Case Study”
• “The Relationship Between Fiscal Policy and Inflation Rates: A Time-Series Analysis”
• “Factors Influencing E-commerce Conversion Rates: A Quantitative Exploration”
• “Examining the Correlation Between Interest Rates and Consumer Spending”
• “Standardized Testing and Academic Performance: A Quantitative Evaluation”
• “Teaching Strategies and Student Learning Outcomes in Secondary Schools: A Quantitative Study”
• “The Relationship Between Extracurricular Activities and Academic Success”
• “Influence of Parental Involvement on Children’s Educational Achievements”
• “Digital Literacy in Primary Schools: A Quantitative Assessment”
• “Learning Outcomes in Blended vs. Traditional Classrooms: A Comparative Analysis”
• “Correlation Between Teacher Experience and Student Success Rates”
• “Analyzing the Impact of Classroom Technology on Reading Comprehension”
• “Gender Differences in STEM Fields: A Quantitative Analysis of Enrollment Data”
• “The Relationship Between Homework Load and Academic Burnout”
• “Assessment of Special Education Programs in Public Schools”
• “Role of Peer Tutoring in Improving Academic Performance: A Quantitative Study”

Medicine and Health Sciences

• “The Impact of Sleep Duration on Cardiovascular Health: A Cross-sectional Study”
• “Analyzing the Efficacy of Various Antidepressants: A Meta-Analysis”
• “Patient Satisfaction in Telehealth Services: A Quantitative Assessment”
• “Dietary Habits and Incidence of Heart Disease: A Quantitative Review”
• “Correlations Between Stress Levels and Immune System Functioning”
• “Smoking and Lung Function: A Quantitative Analysis”
• “Influence of Physical Activity on Mental Health in Older Adults”
• “Antibiotic Resistance Patterns in Community Hospitals: A Quantitative Study”
• “The Efficacy of Vaccination Programs in Controlling Disease Spread: A Time-Series Analysis”
• “Role of Social Determinants in Health Outcomes: A Quantitative Exploration”
• “Impact of Hospital Design on Patient Recovery Rates”
• “Quantitative Analysis of Dietary Choices and Obesity Rates in Children”

Social Sciences

• “Examining Social Inequality through Wage Distribution: A Quantitative Study”
• “Impact of Parental Divorce on Child Development: A Longitudinal Study”
• “Social Media and its Effect on Political Polarization: A Quantitative Analysis”
• “The Relationship Between Religion and Social Attitudes: A Statistical Overview”
• “Influence of Socioeconomic Status on Educational Achievement”
• “Quantifying the Effects of Community Programs on Crime Reduction”
• “Public Opinion and Immigration Policies: A Quantitative Exploration”
• “Analyzing the Gender Representation in Political Offices: A Quantitative Study”
• “Impact of Mass Media on Public Opinion: A Regression Analysis”
• “Influence of Urban Design on Social Interactions in Communities”
• “The Role of Social Support in Mental Health Outcomes: A Quantitative Analysis”
• “Examining the Relationship Between Substance Abuse and Employment Status”

Engineering and Technology

• “Performance Evaluation of Different Machine Learning Algorithms in Autonomous Vehicles”
• “Material Science: A Quantitative Analysis of Stress-Strain Properties in Various Alloys”
• “Impacts of Data Center Cooling Solutions on Energy Consumption”
• “Analyzing the Reliability of Renewable Energy Sources in Grid Management”
• “Optimization of 5G Network Performance: A Quantitative Assessment”
• “Quantifying the Effects of Aerodynamics on Fuel Efficiency in Commercial Airplanes”
• “The Relationship Between Software Complexity and Bug Frequency”
• “Machine Learning in Predictive Maintenance: A Quantitative Analysis”
• “Wearable Technologies and their Impact on Healthcare Monitoring”
• “Quantitative Assessment of Cybersecurity Measures in Financial Institutions”
• “Analysis of Noise Pollution from Urban Transportation Systems”
• “The Influence of Architectural Design on Energy Efficiency in Buildings”

Quantitative Research Topics

Quantitative Research Topics are as follows:

• The effects of social media on self-esteem among teenagers.
• A comparative study of academic achievement among students of single-sex and co-educational schools.
• The impact of gender on leadership styles in the workplace.
• The correlation between parental involvement and academic performance of students.
• The effect of mindfulness meditation on stress levels in college students.
• The relationship between employee motivation and job satisfaction.
• The effectiveness of online learning compared to traditional classroom learning.
• The correlation between sleep duration and academic performance among college students.
• The impact of exercise on mental health among adults.
• The relationship between social support and psychological well-being among cancer patients.
• The effect of caffeine consumption on sleep quality.
• A comparative study of the effectiveness of cognitive-behavioral therapy and pharmacotherapy in treating depression.
• The relationship between physical attractiveness and job opportunities.
• The correlation between smartphone addiction and academic performance among high school students.
• The impact of music on memory recall among adults.
• The effectiveness of parental control software in limiting children’s online activity.
• The relationship between social media use and body image dissatisfaction among young adults.
• The correlation between academic achievement and parental involvement among minority students.
• The impact of early childhood education on academic performance in later years.
• The effectiveness of employee training and development programs in improving organizational performance.
• The relationship between socioeconomic status and access to healthcare services.
• The correlation between social support and academic achievement among college students.
• The impact of technology on communication skills among children.
• The effectiveness of mindfulness-based stress reduction programs in reducing symptoms of anxiety and depression.
• The relationship between employee turnover and organizational culture.
• The correlation between job satisfaction and employee engagement.
• The impact of video game violence on aggressive behavior among children.
• The effectiveness of nutritional education in promoting healthy eating habits among adolescents.
• The relationship between bullying and academic performance among middle school students.
• The correlation between teacher expectations and student achievement.
• The impact of gender stereotypes on career choices among high school students.
• The effectiveness of anger management programs in reducing violent behavior.
• The relationship between social support and recovery from substance abuse.
• The correlation between parent-child communication and adolescent drug use.
• The impact of technology on family relationships.
• The effectiveness of smoking cessation programs in promoting long-term abstinence.
• The relationship between personality traits and academic achievement.
• The correlation between stress and job performance among healthcare professionals.
• The impact of online privacy concerns on social media use.
• The effectiveness of cognitive-behavioral therapy in treating anxiety disorders.
• The relationship between teacher feedback and student motivation.
• The correlation between physical activity and academic performance among elementary school students.
• The impact of parental divorce on academic achievement among children.
• The effectiveness of diversity training in improving workplace relationships.
• The relationship between childhood trauma and adult mental health.
• The correlation between parental involvement and substance abuse among adolescents.
• The impact of social media use on romantic relationships among young adults.
• The effectiveness of assertiveness training in improving communication skills.
• The relationship between parental expectations and academic achievement among high school students.
• The correlation between sleep quality and mood among adults.
• The impact of video game addiction on academic performance among college students.
• The effectiveness of group therapy in treating eating disorders.
• The relationship between job stress and job performance among teachers.
• The correlation between mindfulness and emotional regulation.
• The impact of social media use on self-esteem among college students.
• The effectiveness of parent-teacher communication in promoting academic achievement among elementary school students.
• The impact of renewable energy policies on carbon emissions
• The relationship between employee motivation and job performance
• The effectiveness of psychotherapy in treating eating disorders
• The correlation between physical activity and cognitive function in older adults
• The effect of childhood poverty on adult health outcomes
• The impact of urbanization on biodiversity conservation
• The relationship between work-life balance and employee job satisfaction
• The effectiveness of eye movement desensitization and reprocessing (EMDR) in treating trauma
• The correlation between parenting styles and child behavior
• The effect of social media on political polarization
• The impact of foreign aid on economic development
• The relationship between workplace diversity and organizational performance
• The effectiveness of dialectical behavior therapy in treating borderline personality disorder
• The correlation between childhood abuse and adult mental health outcomes
• The effect of sleep deprivation on cognitive function
• The impact of trade policies on international trade and economic growth
• The relationship between employee engagement and organizational commitment
• The effectiveness of cognitive therapy in treating postpartum depression
• The correlation between family meals and child obesity rates
• The effect of parental involvement in sports on child athletic performance
• The impact of social entrepreneurship on sustainable development
• The relationship between emotional labor and job burnout
• The effectiveness of art therapy in treating dementia
• The correlation between social media use and academic procrastination
• The effect of poverty on childhood educational attainment
• The impact of urban green spaces on mental health
• The relationship between job insecurity and employee well-being
• The effectiveness of virtual reality exposure therapy in treating anxiety disorders
• The correlation between childhood trauma and substance abuse
• The effect of screen time on children’s social skills
• The impact of trade unions on employee job satisfaction
• The relationship between cultural intelligence and cross-cultural communication
• The effectiveness of acceptance and commitment therapy in treating chronic pain
• The correlation between childhood obesity and adult health outcomes
• The effect of gender diversity on corporate performance
• The impact of environmental regulations on industry competitiveness.
• The impact of renewable energy policies on greenhouse gas emissions
• The relationship between workplace diversity and team performance
• The effectiveness of group therapy in treating substance abuse
• The correlation between parental involvement and social skills in early childhood
• The effect of technology use on sleep patterns
• The impact of government regulations on small business growth
• The relationship between job satisfaction and employee turnover
• The effectiveness of virtual reality therapy in treating anxiety disorders
• The correlation between parental involvement and academic motivation in adolescents
• The effect of social media on political engagement
• The impact of urbanization on mental health
• The relationship between corporate social responsibility and consumer trust
• The correlation between early childhood education and social-emotional development
• The effect of screen time on cognitive development in young children
• The impact of trade policies on global economic growth
• The relationship between workplace diversity and innovation
• The effectiveness of family therapy in treating eating disorders
• The correlation between parental involvement and college persistence
• The effect of social media on body image and self-esteem
• The impact of environmental regulations on business competitiveness
• The relationship between job autonomy and job satisfaction
• The effectiveness of virtual reality therapy in treating phobias
• The correlation between parental involvement and academic achievement in college
• The effect of social media on sleep quality
• The impact of immigration policies on social integration
• The relationship between workplace diversity and employee well-being
• The effectiveness of psychodynamic therapy in treating personality disorders
• The correlation between early childhood education and executive function skills
• The effect of parental involvement on STEM education outcomes
• The impact of trade policies on domestic employment rates
• The relationship between job insecurity and mental health
• The effectiveness of exposure therapy in treating PTSD
• The correlation between parental involvement and social mobility
• The effect of social media on intergroup relations
• The impact of urbanization on air pollution and respiratory health.
• The relationship between emotional intelligence and leadership effectiveness
• The effectiveness of cognitive-behavioral therapy in treating depression
• The correlation between early childhood education and language development
• The effect of parental involvement on academic achievement in STEM fields
• The impact of trade policies on income inequality
• The relationship between workplace diversity and customer satisfaction
• The effectiveness of mindfulness-based therapy in treating anxiety disorders
• The correlation between parental involvement and civic engagement in adolescents
• The effect of social media on mental health among teenagers
• The impact of public transportation policies on traffic congestion
• The relationship between job stress and job performance
• The effectiveness of group therapy in treating depression
• The correlation between early childhood education and cognitive development
• The effect of parental involvement on academic motivation in college
• The impact of environmental regulations on energy consumption
• The relationship between workplace diversity and employee engagement
• The effectiveness of art therapy in treating PTSD
• The correlation between parental involvement and academic success in vocational education
• The effect of social media on academic achievement in college
• The impact of tax policies on economic growth
• The relationship between job flexibility and work-life balance
• The effectiveness of acceptance and commitment therapy in treating anxiety disorders
• The correlation between early childhood education and social competence
• The effect of parental involvement on career readiness in high school
• The impact of immigration policies on crime rates
• The relationship between workplace diversity and employee retention
• The effectiveness of play therapy in treating trauma
• The correlation between parental involvement and academic success in online learning
• The effect of social media on body dissatisfaction among women
• The impact of urbanization on public health infrastructure
• The relationship between job satisfaction and job performance
• The effectiveness of eye movement desensitization and reprocessing therapy in treating PTSD
• The correlation between early childhood education and social skills in adolescence
• The effect of parental involvement on academic achievement in the arts
• The impact of trade policies on foreign investment
• The relationship between workplace diversity and decision-making
• The effectiveness of exposure and response prevention therapy in treating OCD
• The correlation between parental involvement and academic success in special education
• The impact of zoning laws on affordable housing
• The relationship between job design and employee motivation
• The effectiveness of cognitive rehabilitation therapy in treating traumatic brain injury
• The correlation between early childhood education and social-emotional learning
• The effect of parental involvement on academic achievement in foreign language learning
• The impact of trade policies on the environment
• The relationship between workplace diversity and creativity
• The effectiveness of emotion-focused therapy in treating relationship problems
• The correlation between parental involvement and academic success in music education
• The effect of social media on interpersonal communication skills
• The impact of public health campaigns on health behaviors
• The relationship between job resources and job stress
• The effectiveness of equine therapy in treating substance abuse
• The correlation between early childhood education and self-regulation
• The effect of parental involvement on academic achievement in physical education
• The impact of immigration policies on cultural assimilation
• The relationship between workplace diversity and conflict resolution
• The effectiveness of schema therapy in treating personality disorders
• The correlation between parental involvement and academic success in career and technical education
• The effect of social media on trust in government institutions
• The impact of urbanization on public transportation systems
• The relationship between job demands and job stress
• The correlation between early childhood education and executive functioning
• The effect of parental involvement on academic achievement in computer science
• The effectiveness of cognitive processing therapy in treating PTSD
• The correlation between parental involvement and academic success in homeschooling
• The effect of social media on cyberbullying behavior
• The impact of urbanization on air quality
• The effectiveness of dance therapy in treating anxiety disorders
• The correlation between early childhood education and math achievement
• The effect of parental involvement on academic achievement in health education
• The impact of global warming on agriculture
• The effectiveness of narrative therapy in treating depression
• The correlation between parental involvement and academic success in character education
• The effect of social media on political participation
• The impact of technology on job displacement
• The relationship between job resources and job satisfaction
• The effectiveness of art therapy in treating addiction
• The correlation between early childhood education and reading comprehension
• The effect of parental involvement on academic achievement in environmental education
• The impact of income inequality on social mobility
• The relationship between workplace diversity and organizational culture
• The effectiveness of solution-focused brief therapy in treating anxiety disorders
• The correlation between parental involvement and academic success in physical therapy education
• The effect of social media on misinformation
• The impact of green energy policies on economic growth
• The relationship between job demands and employee well-being
• The correlation between early childhood education and science achievement
• The effect of parental involvement on academic achievement in religious education
• The impact of gender diversity on corporate governance
• The relationship between workplace diversity and ethical decision-making
• The correlation between parental involvement and academic success in dental hygiene education
• The effect of social media on self-esteem among adolescents
• The impact of renewable energy policies on energy security
• The effect of parental involvement on academic achievement in social studies
• The impact of trade policies on job growth
• The relationship between workplace diversity and leadership styles
• The correlation between parental involvement and academic success in online vocational training
• The effect of social media on self-esteem among men
• The impact of urbanization on air pollution levels
• The effectiveness of music therapy in treating depression
• The correlation between early childhood education and math skills
• The effect of parental involvement on academic achievement in language arts
• The impact of immigration policies on labor market outcomes
• The effectiveness of hypnotherapy in treating phobias
• The effect of social media on political engagement among young adults
• The impact of urbanization on access to green spaces
• The relationship between job crafting and job satisfaction
• The effectiveness of exposure therapy in treating specific phobias
• The correlation between early childhood education and spatial reasoning
• The effect of parental involvement on academic achievement in business education
• The impact of trade policies on economic inequality
• The effectiveness of narrative therapy in treating PTSD
• The correlation between parental involvement and academic success in nursing education
• The effect of social media on sleep quality among adolescents
• The impact of urbanization on crime rates
• The relationship between job insecurity and turnover intentions
• The effectiveness of pet therapy in treating anxiety disorders
• The correlation between early childhood education and STEM skills
• The effect of parental involvement on academic achievement in culinary education
• The impact of immigration policies on housing affordability
• The relationship between workplace diversity and employee satisfaction
• The effectiveness of mindfulness-based stress reduction in treating chronic pain
• The correlation between parental involvement and academic success in art education
• The effect of social media on academic procrastination among college students
• The impact of urbanization on public safety services.

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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Quantitative Research

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In conducting quantitative research, you need to make sure you have the right numbers and the correct values for specific variables. This is because quantitative research focuses more on numeric and logical results. Quantitative studies report and understand numerical data to make further analysis of a given phenomenon. This research organizes and computes statistics from current and prospect clients to make business forecasts for your company. Quantitative analysis  examples also uses methods like polls, surveys, and sampling to gather information that can help complete your investigation.

31+ Quantitative Research Examples

Quantitative research demands focus and precision from the researcher. If you need a guide in doing your research, here are 10+ Quantitative research examples you can use.

1. Free Quantitative Research Flowchart  Example

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2. Free Quantitative Research Analyst Resume  Example

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3. Quantitative Research Review Template

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4. Quantitative Research Plan Template

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5. Quantitative Research Descriptive Analysis Template

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6. Quantitative Research Checklist Template

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7. Quantitative Research Survey Template

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8. Quantitative Research Data Analysis Template

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9. Quantitative Research Guide Template

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10. Quantitative Research Proposal Template

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11. Quantitative Research Question Template

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12. Quantitative Research Literacy Template

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13. Quantitative Research Correlation Template

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14. Quantitative Research Template

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15. Quantitative Research Report Template

16. Simple Quantitative Research Template

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17. Quantitative Research Paper Template

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18. Example of Quantitative Research

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19. Quantitative Research Design Examples

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20. Quantitative Research Examples for Students

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21. Impact of Social Media Reviews on Brands Perception  Example

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In the age where likes, comments, and retweets measure the relevance of an entity online, brands make sure that their followers and customers have a positive perception of them on the web. The internet puts companies and individuals at a spot where the public eye sees reviews and comments about them. But how do these things affect the way people view a company’s branding? This quantitative study of the impact of social media reviews on brands perception answers that. Use this research as a guide in conducting your quantitative research.

22. Teacher Perceptions of Professional Learning Communities  Example

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Educators lead young minds to great success. That is why there are training programs examples  and models where teachers can collaborate and share how they can improve students’ learning. Saying this, some do question the effectiveness of models such as Professional Learning Communities. Research called “A Quantitative Study of Teacher Perceptions of Professional Learning Communities’ Context, Process, and Content,” looks into these queries. If you are conducting your quantitative research, you can use this research as an example for your study. Format your content like this investigation for a foolproof thesis paper.

23. Quantitative Research On The Level of Social Media Addiction  Example

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The worldwide web is a being of wonder and mystery. That’s what makes it fascinating to young audiences. The internet helps them connect and interact with people through various social media platforms. With features and advancements that intrigue even the unexcited, addiction does become inevitable. An investigation in 2015 titled “A Quantitative Research on the Level of Social Media Addiction among Young People in Turkey” looks into the statistics of this problem. For your quantitative research, use this study as a guide in organizing and formatting your quantitative data.

24. Course Grades and Retention Comparing Online and Face-to-face Classes

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Are you taking online classes, or are your classes held in a classroom? Do you believe there is a difference between online and face-to-face courses? There has always been a discussion between which education instructional method is more effective. Although both help students learn, some argue that the way they are taught makes an education gap. This quantitative study of course grades and retention comparing online and face-to-face classes can help answer your questions. It can also serve as a model in making your own quantitative research. Pattern your research design like this one now!

25. Free Nursing Quantitative Research Proposal  Example

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One of a nurse’s primary duties is to assure patients are taken care of and attended to. Their line of work deals with peoples’ lives and health. This also means that they still need to address patients even if they’re close to death. In Ireland, a study called “A Quantitative Study of the Attitude, Knowledge, and Experience of Staff Nurses on Prioritizing Comfort measures in Care of the Dying Patient in an Acute Hospital Setting” was conducted. If you plan on undertaking any medical  SWOT  analysis , using this study as a guide would be beneficial for you. 

26. Quantitative Research Of Consumer’s Attitude Towards Food Products Advertising

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In the corporate world, you can’t just start selling something without proper research. You first have to make sure that your products and services are relevant and marketable. The first step should be conducting marketing research. Marketing research can use either qualitative or quantitative data collection methods. But if you want to figure out how your clients react to your products and marketing strategy, this quantitative research of consumer’s attitude towards food products advertising could be your guide. You can even use this for your undergraduate research.

27. Free Effective Teacher Leadership  Example

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Research projects have to be conducted with precision and accuracy, especially if it’s quantitative research. You need to make sure you get the right numbers to get valid results. In research called “Effective Teacher Leadership: A Quantitative Study of the Relationship Between School Structures and Effective Teacher Leaders,” quantitative data analysis is conducted to look into the school’s management plans. For your research, this would be a useful guide in doing comprehensive qualitative research. You can outline your investigations and even term papers using this as a sample.

28. Quantitative Studies of Water and Sanitation Utilities  Example

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Quantitative research is a method that studies numerical values. It follows a strict process of data collection. This type of research is used by different industries and even as undergraduate research. That is why the research design should reflect the nature of your research. It should look professional and comprehensive. But that doesn’t mean that your research project plan has to look dull. This study called “Quantitative Studies of Water and Sanitation Utilities: A Literature Survey” can be used as a sample. It’s research methodology utilizes surveys as a way to collect data needed for research.

29. Free Perceptions of First Year College Students  Example

Do you want kids to be college-ready? Are you looking for a college planner to prepare high school kids for a higher level of education? The first year of college serves as an adjustment period for students. The way they cope and accustom themselves use different methods. That’s why you need a study to help you. If your research looks into college kids, this qualitative study of the perceptions of first-year college students regarding technology and college readiness could be your guide. Us it as an outline for the quantitative research you are conducting. 

30. Free Qualitative Research Paper  Example

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Like any research, you must follow a particular format. A poorly organized study might give the impression of having unreliable data and results. You need to make sure your research is detailed and understandable. This applies significantly to quantitative project analysis example . This type of investigation urges researchers to be careful and efficient when gathering and analyzing information and statistics. Getting the wrong value can mess up your whole investigation. For your research, you can make use of this qualitative research paper as an outline. It details all the right parts needed in your research.

31. Quantitative Research For Health Programmes  Example

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If you are creating health newspapers and programs, you need to make sure you have the correct data. Your program will tackle a person’s health so you need to have the correct information as not to cause further complications. That’s also why you need to conduct quantitative research to get precise data. For your research, you can make this quantitative research for health programmes your guide. The World Health Organization uses it so you can be sure it is professionally made. Follow the formats on this document to make sure your research is high-quality.

What are the Quantitative research characteristics?

• Objective and Empirical: Quantitative research is based on objective and empirical observations, focusing on measurable, observable phenomena. It aims to collect data that can be analyzed statistically.
• Numerical Data: It primarily relies on numerical data, such as counts, measurements, percentages, and statistics, to draw conclusions and make comparisons.
• Structured and Controlled: Quantitative research is highly structured and controlled, with predefined methods and data collection procedures. Researchers follow standardized processes to ensure reliability and replicability.
• Large Sample Sizes: It often involves larger sample sizes to ensure statistical significance and generalizability. Sampling techniques are used to select representative samples from the population.
• Hypothesis-Driven: Quantitative research typically begins with a clear hypothesis or research question. Researchers aim to test hypotheses and draw conclusions based on data analysis.
• Quantitative Instruments: Researchers use various quantitative instruments, such as surveys, questionnaires, experiments, and structured observations, to collect data.
• Statistical Analysis: Data collected in quantitative research are subject to statistical analysis. Common statistical techniques include descriptive statistics, inferential statistics, regression analysis, and hypothesis testing.
• Objective Measurement: Measurements are typically objective and standardized to minimize bias and subjectivity. Instruments are designed to ensure consistency and reliability.
• Generalization: Quantitative research aims to generalize findings from a sample to a larger population. The results are often used to make broader conclusions and predictions.
• Numerical Results: Research findings are presented using numerical values, charts, graphs, and tables, making the results easily interpretable and comparable.
• Structured Questioning: Surveys and questionnaires used in quantitative research have structured questions with predefined response options to facilitate data collection and analysis.
• Replicability: Quantitative studies are designed to be replicable, allowing other researchers to conduct similar studies and verify or challenge the findings.
• Causality: While correlation can be established, quantitative research is suited for investigating causal relationships between variables by controlling for extraneous factors.
• Reductionist Approach: It often involves a reductionist approach, breaking down complex phenomena into measurable variables for analysis.
• Predefined Research Design: Quantitative research typically follows a predefined research design, including experimental designs, cross-sectional or longitudinal studies, and surveys.
• Validity and Reliability: Researchers pay careful attention to the validity (the accuracy of measurements) and reliability (the consistency of measurements) of data and instruments.
• Data-Based Conclusions: Conclusions in quantitative research are based on data analysis and statistical significance, emphasizing objectivity and evidence-based decision-making.

What are the 4 types of quantitative research?

1. Descriptive Research:

Descriptive research aims to describe and analyze a phenomenon, population, or variable. It provides a detailed account of the characteristics, behaviors, or attributes of a subject without manipulating it. Surveys, observational studies, and content analysis are often used in descriptive research.

2. Correlational Research:

Correlational research examines the relationship between two or more variables. It assesses how changes in one variable are associated with changes in another. The strength and direction of the relationship are measured using correlation coefficients. This type of research doesn’t establish causation but helps identify patterns and associations.

3. Experimental Research:

Experimental research is conducted to establish cause-and-effect relationships between variables. Researchers manipulate one or more independent variables to observe their impact on a dependent variable while controlling extraneous factors. Randomized controlled trials (RCTs) and laboratory experiments are common experimental research designs.

4. Quasi-Experimental Research:

Quasi-experimental research shares similarities with experimental research but lacks the full level of control over variables. In quasi-experiments, researchers often cannot use random assignment due to ethical or practical constraints. However, they still manipulate independent variables and measure their effects on dependent variables.

What is Quantitative Research vs Qualitative Research?

Which example demonstrates quantitative research?

Example 1: A study that surveys 1,000 consumers to determine the percentage who prefer Product A over Product B for a specific feature.

Example 1 demonstrates quantitative research because it involves collecting numerical data (the percentage of consumers) and relies on surveys, which are a common quantitative data collection method. This type of research is suitable for quantifying preferences and making statistical comparisons between products.

What are the advantages of quantitative research?

• Objectivity: Quantitative research is often highly structured and relies on empirical data, reducing the potential for bias and subjectivity. This enhances the objectivity of the research.
• Measurability: It allows for precise measurement of variables, making it easier to quantify and analyze data. This facilitates the comparison of findings across studies.
• Generalizability: Large sample sizes and statistical analysis enable researchers to generalize findings to a larger population, enhancing the external validity of the results.
• Replicability: Quantitative research is designed to be replicable, allowing other researchers to conduct similar studies and validate or challenge the findings.
• Data Analysis: Statistical analysis provides robust tools for testing hypotheses, identifying patterns, and drawing conclusions from data.
• Causality: It is well-suited for investigating causal relationships, as researchers can manipulate variables and control extraneous factors to establish cause-and-effect links.
• Efficiency: Surveys and questionnaires can collect data from a large number of participants efficiently. This is particularly useful for large-scale studies.
• Quantitative Comparison: It allows for direct comparison between groups or variables, facilitating the identification of differences and relationships.
• Data Precision: The use of standardized instruments and measurements results in precise and consistent data, reducing measurement errors.
• Data Visualization: Numerical data can be presented in charts, graphs, and tables, making it visually accessible and aiding in data interpretation.
• Decision Support: Quantitative research provides empirical evidence that can inform data-driven decision-making in various fields, including business, healthcare, and policy.
• Clear Findings: The structured nature of quantitative research often leads to clear and easily interpretable findings, which can be valuable for making informed conclusions.
• Resource Efficiency: While it may require substantial resources for data collection and analysis, quantitative research can be more cost-effective than qualitative research when dealing with large sample sizes.

General FAQ’s

What is quantitative research.

Quantitative research is a systematic approach to gathering and analyzing numerical data to understand and draw conclusions about a specific phenomenon or problem, often using statistical techniques.

What is the greatest strength of quantitative research?

The greatest strength of quantitative research is its ability to provide precise, objective, and statistically reliable data, enabling researchers to identify patterns, relationships, and make generalizable conclusions.

What is a common weakness of quantitative research?

A common weakness of quantitative research is its potential for oversimplification, as it may not capture the full complexity of human behavior or phenomena and may rely on limited predefined variables.

What are the risks of quantitative research?

Risks in quantitative research include the potential for data inaccuracies, oversimplification of complex phenomena, and overlooking unmeasurable factors, which can lead to biased or incomplete conclusions.

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Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples

• Aneta Pluta 1 , 13 ,
• Juan Pablo Jaworski 2 ,
• Casey Droscha 3 ,
• Sophie VanderWeele 3 ,
• Tasia M. Taxis 4 ,
• Stephen Valas 5 ,
• Dragan Brnić 6 ,
• Andreja Jungić 6 ,
• María José Ruano 7 ,
• Azucena Sánchez 7 ,
• Kenji Murakami 8 ,
• Kurumi Nakamura 8 ,
• Rodrigo Puentes 9 ,
• MLaureana De Brun 9 ,
• Vanesa Ruiz 2 ,
• Marla Eliana Ladera Gómez 10 ,
• Pamela Lendez 10 ,
• Guillermina Dolcini 10 ,
• Marcelo Fernandes Camargos 11 ,
• Antônio Fonseca 11 ,
• Subarna Barua 12 ,
• Chengming Wang 12 ,
• Aleksandra Giza 13 &
• Jacek Kuźmak 1  

BMC Veterinary Research volume  20 , Article number:  381 ( 2024 ) Cite this article

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Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

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Introduction

Bovine leukemia virus (BLV) is a deltaretrovirus from the Orthoretrovirinae subfamily of the Retroviridae family. An essential step in the BLV replication cycle is the integration of DNA copy of its RNA genome into the DNA of a host cell [ 1 ]. Once integrated, the proviral DNA is replicated along with the host’s DNA during cellular divisions, as for any cellular gene. The BLV is the etiologic agent of enzootic bovine leukosis (EBL). BLV causes a persistent infection in cattle, and in most cases this infection is asymptomatic [ 2 ]. In one-third of infected animals the infection progresses to a state of persistent lymphocytosis, and in 1 to 10% of infected cattle it develops into lymphosarcoma [ 2 ]. BLV induces high economic losses due to trade restrictions, replacement cost, reduced milk production, immunosuppression, and increased susceptibility to pneumonia, diarrhea, mastitis, and so on [ 3 , 4 , 5 , 6 ]. BLV is globally distributed with a high prevalence, except for Western Europe and Oceania, where the virus has been successfully eradicated through detection and elimination of BLV-infected animals [ 7 , 8 ]. The agar gel immunodiffusion and ELISA for the detection of BLV-specific antibodies in sera and milk are the World Organization for Animal Health (WOAH, founded as OIE) prescribed tests for serological diagnosis but ELISA, due to its high sensitivity and ability to test many samples at a very low cost, is highly recommended [ 9 ]. Despite the advantages of serologic testing, there are some scenarios in which direct detection of the BLV genomic fragment was important to improve BLV detection. The most frequent cases is the screening of calves with maternal antibodies, acute infection, animals without persistent antibody response and animal subproducts (i.e., semen). In this regard, nucleic acid amplification tests such as real-time quantitative PCR (qPCR) allows for a rapid and highly sensitive detection of BLV proviral DNA (BLV DNA) that can be used to test infected and asymptomatic animals, before the elicitation of anti-BLV specific antibodies and when proviral load (PVL) are still low [ 10 ]. Furthermore, qPCR assays can serve as confirmatory tests for the clarification of inconclusive and discordant serological test results usually associated with these cases [ 11 ]. For these reasons, the inclusion of qPCR in combination with other screening tests might increase control programs efficiency. Additionally, qPCR allows the estimation of BLV PVL which is important for studying the dynamics of BLV infection (i.e., basic research). Further, considering that BLV PVL correlates with the risk of BLV transmission, this feature of qPCR can be exploited for developing rational segregation programs [ 12 , 13 ]. The results of Kobayashi et al. suggest that high PVL is also a significant risk factor for progression to EBL and should therefore be used as a parameter to identify cattle for culling from the herd well before EBL progression [ 14 ]. Several qPCRs have been developed globally for the quantitation of BLV DNA. Although most assays have been properly validated by each developer, a proper standardization and harmonization of such tests is currently lacking. Considering that standardization and harmonization of qPCR methods and results are essential for comparisons of data from BLV laboratories around the world, this could directly impact international surveillance programs and collaborative research. We built a global collaborative network of BLV reference laboratories to evaluate the interlaboratory variability of different qPCRs and sponsored a harmonization of assays to hopefully impact international surveillance programs and research going forward.

In 2018 we conducted the first global trial of this kind to assess the interlaboratory variability of six qPCRs for the detection of BLV DNA [ 15 ]. Since this complex process is a continuous rather than a one-time effort, we now started a second study of this type. In this follow up study, we built a more comprehensive sample panel, accounting for a broader geographical diversification. Additionally, we increased the number of participants to ten collaborating laboratories plus one WOAH reference lab and tested novel methodologies including digital PCR (ddPCR) and FRET-qPCR. Finally, we established the next steps towards the international standardization of molecular assays for the detection of BLV DNA.

Materials and methods

Participants.

The eleven laboratories that took part in the study were:(i) the Auburn University College of Veterinary Medicine (Auburn, Alabama, United States): (ii) AntelBio, a division of CentralStar Cooperative (Michigan, United States); (iii) Laboratórios Federais de Defesa Agropecuária de Minas Gerais (LFDA-MG, Pedro Leopoldo, Brasil); (iv) Centro de Investigación Veterinaria de Tandil (CIVETAN, Buenos Aires, Argentina); (v) the Faculty of Agriculture Iwate University (Iwate, Japan); (vi) Universidad de la República de Uruguay (UdelaR, Montevideo, Uruguay); (vii) the Croatian Veterinary Institute (Zagreb, Croatia); (viii) Instituto Nacional de Tecnología Agropecuaria (INTA, Buenos Aires, Argentina); (ix) Laboratorio Central de Veterinaria (LCV, Madrid, Spain); (x) the National Veterinary Research Institute (NVRI, Puławy, Poland) and (xi) the French Agency for Food, Environmental and Occupational Health and Safety (Anses, Niort, France). All European laboratories participating in this study are acting as national reference laboratories for EBL, NVRI acts as WOAH reference laboratory for EBL, while the remaining laboratories are nationally renowned entities for BLV diagnostics. The eleven participating methods are referred to below as qPCR1 – qPCR5, ddPCR6, qPCR7 – qPCR11, respectively.

Sample collection and DNA extraction

A total of 42 DNA samples obtained from blood of naturally BLV-infected dairy cattle from Poland, Moldova, Pakistan, Ukraine, Canada and United States were used for this study. Thirty-six of them were archival DNA samples obtained between 2012–2018 as described in our previous studies on samples from Poland ( n  = 21) [ 16 , 17 ], Moldova ( n  = 4) [ 18 ], Pakistan ( n  = 5) [ 19 ] and Ukraine ( n  = 6) [ 15 , 20 ]. Between 2020–2021 6 peripheral blood and serum samples from naturally BLV-infected cattle were obtained from three dairy farms of Alberta, Canada and two dairy farms of Michigan, US. Serological testing and sample processing were conducted by the laboratories from which the samples originated. The genomic DNA from Canadian and US samples was extracted from whole blood using a Quick DNA Miniprep Plus kit (Zymo Research) and a DNeasy Blood & Tissue Kit (Qiagen), respectively in University of Calgary and Michigan State University and sent to the NVRI in the form of DNA solutions. Additionally, one plasmid DNA sample (pBLV344) was kindly supplied by Luc Willems (University of Liège, Belgium) and DNA extracted from FLK-BLV cells were included as positive controls. Finally, DNA extracted from PBL of a serologically negative cattle was included as negative control. At the NVRI, the DNA concentration in all samples was estimated by spectrophotometry using a NanoPhotometer (Implen). Each sample was divided into eleven identical aliquots containing between 800 and 4,000 ng of lyophilised genomic DNA. Eleven identical sets of these samples were lyophilized (Alpha 1–4 LSC basic, Martin Christ Gefriertrocknungsanlagen GmbH) and distributed to participating laboratories. At the NVRI, all samples were coded (identification [ 21 ] run numbers 1 to 44) to perform a blinded testing. The samples, together with instructions for their preparation (Additional file 1), were shipped by air at room temperature (RT).

Examination of DNA quality/stability

Since different extraction methods and lyophilization process were employed for the preparation of the DNA samples, it was necessary to test the quality of the DNA at the NVRI laboratory. For that purpose, one complete set of samples ( n  = 44) was tested by Fragment Analyzer (Agilent Technologies), before and after freeze-drying, to assess DNA quality by calculating a Genomic Quality Number (GQN) for every sample. Low GQN value (< 2.5) represents sheared or degraded DNA. A high GQN (> 9) represents undegraded DNA. In addition, quality of DNA was assessed by determination of copy number of the histone H3 family 3A ( H3F3A ) housekeeping gene using quantitative real-time PCR (qPCR) [ 22 ]. The qPCR results were expressed as the number of H3F3A gene copies per 300 ng of DNA in each sample. Grubbs´ test was performed to determine outliers. To test the stability of DNA, samples were stored for 20 days at RT (10 days) and at + 4 °C (10 days) and were retested by Fragment Analyzer and qPCR 21 days later. A Mann–Whitney U-test was used to compare the median values between fresh and stored samples (time 0 and time 1), respectively.

Description of BLV qPCR protocols used by participating laboratories

All participating laboratories performed their qPCR or ddPCR using a variety of different equipment, reagents, and reaction conditions, which had been set up, validated, and evaluated previously and are currently used as working protocols. The specific features of each of these protocols are described below and summarized in Table  1 .

All laboratories applied standard procedures for avoiding false-positive results indicative of DNA contamination, such as the use of separate rooms for preparing reaction mixtures, adding the samples, and performing the amplification reaction. One of the ten BLV qPCRs used LTR region and the remaining nine qPCRs used the pol gene as the target sequence for amplification, while the ddPCR amplified the env gene.

Method qPCR1

The BLV qPCR amplifying a 187-bp pol gene was performed according to a previously published methods [ 23 , 24 ]. A real-time fluorescence resonance energy transfer (FRET) PCR was carried out in a 20-μl PCR mixture containing 10 μl handmade reaction master mix and 10 μl genomic DNA. The PCR buffer was 4.5 mM MgCl2, 50 mM KCl, 20 mM Tris–HCl, pH 8.4, supplemented with 0.05% each Tween20 and Non-idet P-40, and 0.03% acetylated BSA (Roche Applied Science). For each 20 μl total reaction volume, the nucleotides were used at 0.2 mM each and 1.5 U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) was used. Primers were used at 1 μM, LCRed640 probe was used at 0.2 μM, and 6-FAM probe was used at 0.1 μM. Amplification was performed in the Roche Light Cycler 480 II (Roche Molecular Biochemicals) using 10 min denaturation step at 95 °C, followed by 18 high-stringency step-down thermal cycles and 30 low-stringency fluorescence acquisition cycles.

A plasmid containing the BLV-PCR amplicon region was diluted ten-fold from 1 × 10 5 copies to 10 copies per 10 µl and was used as a standard to measure the BLV copy numbers.

Method qPCR2

A BLV proviral load qPCR assay developed by AntelBio, a division of CentralStar Cooperative Inc. on Applied Biosystems 7500 Real-Time PCR system [ 25 , 33 ]. This multiplex assay amplifies the BLV pol gene along with the bovine β-actin gene and an internal amplification control, “Spike”. A quantitative TaqMan PCR was carried out in a 25-μl PCR mixture containing 12.5 µl of 2X InhibiTaq Multiplex HotStart qPCR MasterMix (Empirical Bioscience), 16 nM each BLV primer, 16 nM each β-actin primer, 8 nM each spike primer, 8 nM BLV FAM-probe, 8 nM β-actin Cy5-probe, 4 nM spike JOE-probe, 1 µl of an internal spike-in control (10,000 copies per µl), 7.25 µl of nuclease-free water and 4 µl of DNA sample for each qPCR reaction. The thermal PCR protocol was as follows: 95 °C for 10 min, 40 × (95 °C for 15 s, 60 °C for 1 min). Copy numbers of both the BLV pol gene and bovine β-Actin were derived using a plasmid containing target sequences, quantified by ddPCR, diluted 1 × 10 6 copies per µl to 10 copies per µl in tenfold dilutions. DNA concentrations of each sample were measured using a Qubit 4 Fluorometer and used in combination with the qPCR copy numbers to calculate BLV copies per 100 ng.

Method qPCR3

The qPCR assays for the BLV LTR gene were performed according to a previously published methods [ 26 ]. Genomic DNA was amplified by TaqMan PCR with 10 μl of GoTaq Probe qPCR Master Mix × 2 (Promega), 0.6 pmol/μl each primer, 0.3 pmol/µl double-quenched probe and 100 ng genomic DNA. Amplification was performed in the CFX96 cycler (BioRad) according to the protocol: 5 min denaturation at 95°C followed by 45 cycles (60 s at 94°C and 60 s at 60°C). The efficiency of each reaction was calculated from the serial dilution of DNA extracted from BLV persistently infected fetal lamb kidney (FLK) cells, starting at a concentration of 100 ng/µl [ 21 ]. The detection limit was tested using a plasmid containing the target of the qPCRs, starting at 10 3 ng/µl.

Method qPCR4

The quantitative real-time PCR was done with the primers for the BLV pol gene as previously described [ 34 ]. The qPCR reaction mix contained 1 × PCR Master Mix with SYBR Green (FastStart Universal SYBR Green Master Rox, Roche), 0.3 μM each primer and 30 ng of extracted genomic DNA. Amplification was performed in QuantStudio 5 Real-Time PCR System (Applied Biosystems) under the following conditions: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C. A standard curve of six tenfold serial dilutions of pBLV, containing 1 × 10 6 to 10 BLV copies, was built and run 3 times for validation of the method. The number of provirus copies per reaction (100 ng) was calculated.

Method qPCR5

BLV PVLs were determined by using qPCR kit, RC202 (Takara Bio, Shiga, Japan) [ 28 , 35 ]. This qPCR assay amplifies the BLV pol gene along with the bovine RPPH1 gene as an internal control. Briefly, 100 ng genomic DNA was amplified by TaqMan PCR with four primers for pol gene and RPPH1 gene according to the manufacturer’s instructions: 30 s denaturation at 95 °C followed by 45 cycles (5 s at 95 °C and 30 s at 60 °C). The qPCR was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific K.K., Tokyo, Japan). Standard curve was generated by creating tenfold serial dilutions of the standard plasmid included in the kit. The standards for calibration ranged from 1 to 10 6 copies/reaction and were run in duplicate. The number of provirus copies per 100 ng was calculated.

Method ddPCR6

The digital droplet PCR (ddPCR) assay for the env gene of the BLV was performed using the protocol previously described by [ 28 , 29 ]. An absolute quantification by TaqMan ddPCR was performed in a typical 20-μl assay, 1 μl of DNA sample was mixed with 1 μl of each primer (10 μM), 0.5 μl of probe (10 μM), and 2 × Supermix emulsified with oil (Bio-Rad). The droplets were transferred to a 96-well plate (Eppendorf). The PCR assay was performed in a thermocycler (C1000 touch cycler; Bio-Rad) with the following parameters: initial denaturation of 10 min at 95 °C, then 40 cycles of 30 s at 94 °C, and 1 min at 58 °C, with final deactivation of the enzyme for 10 min at 98 °C. The presence of fluorescent droplets determined the number of resulting positive events that were analyzed in the software (QuantaSoft v.1.7.4; Bio-Rad), using dot charts. The number of provirus copies per 100 ng were calculated. Each sample was run in duplicate, and results were averaged.

Method qPCR7

This qPCR method for the BLV pol gene is a modified option of widely available quantitative TaqMan qPCR described by Rola-Łuszczak et al. [ 11 ], using the same primers and standards. A quantitative TaqMan PCR was performed in a 20 μl PCR mix containing 10 μl of 2 × ORA qPCR Probe ROX L Mix (highQu, Kraichtal, Germany), 2 μl primer/probe mix (final concentration 400 nM of each of the primers, 200 nM of BLV probe), and 3 μl extracted genomic DNA. Amplification was performed in the Rotor-Gene Q system (Qiagen) with an initial denaturation step and polymerase activation at 95 °C for 3 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were made from 1 × 10 10 copies to 1 × 10 1 copies per reaction and used to generate the standard curve and estimate BLV copy number per 100 ng.

Method qPCR8

Proviral load quantification was assessed by SYBR Green real-time quantitative PCR (qPCR) using the pol gene as the target sequence [ 36 ]. Briefly, 12-μl PCR mixture contained Fast Start Universal SYBR Green Master Mix (Roche), 800 nM each BLV pol primers and 1 µl DNA as template. The reactions were incubated at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, 55 °C for 15 s and 60 °C for 1 min. All samples were tested in duplicate on a StepOne Plus machine (Applied Biosystems). A positive and negative control, as well as a no-template control, were included in each plate. After the reaction was completed, the specificity of the amplicons was checked by analyzing the individual dissociation curves. As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were made from 1 × 10 6 to 10 copies per µl and used to generate the standard curve and estimate BLV copy number per 100 ng.

Method qPCR9

This qPCR method is a modified option of widely available quantitative TaqMan qPCR described by Rola-Łuszczak et al. [ 11 ], using the same primers and standards. The detection of BLV genome was combined with an endogenous control system (Toussaint 2007) in a duplex assay. Briefly, 20-µl qPCR reaction contained AhPath ID™ One-Step RT-PCR Reagents with ROX (Applied Biosystems, CA, USA) – 10 µl of 2 × RT-PCR buffer and 0.8 µl of 25 × RT-PCR enzyme mix, 400 nM each primer for pol gene, 100 nM BLV specific probe, 40 nM each β-actin primer, 40 nM β-actin specific probe and 2 µl DNA sample. All samples were tested in ABI7500 Real-Time PCR System (Applied Biosystems) according to the following protocol: 10 min at 48 °C (reverse transcription), 10 min at 95 °C (inactivation reverse transcriptase / activation Taq polymerase) followed by 45 cycles (15 s at 95 °C and 60 s at 60 °C). As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were made from 1 × 10 4 copies to 0.1 copies per μl and used to generate the standard curve and estimate BLV copy number per 100 ng.

Method qPCR10

The BLV qPCR was performed as published previously [ 11 ]. A quantitative TaqMan PCR was carried out in a 25-μl PCR mixture containing 12.5 μl of 2 × QuantiTect Multiplex PCR NoROX master mix (Qiagen), 0.4 μM each primer, 0.2 μM specific BLV probe, and 500 ng of extracted genomic DNA. Amplification was performed in the Rotor-Gene Q system (Qiagen) using an initial denaturation step and polymerase activation at 95 °C for 15 min, followed by 50 cycles of 94 °C for 60 s and 60 °C for 60 s. All samples were amplified in duplicate. As a standard, the pBLV1 plasmid (NVRI, Pulawy, PL), containing a 120-bp BLV pol fragment, was used. Tenfold dilutions of this standard were made from 1 × 10 6 copies per μl to 100 copies per μl and were used to estimate the BLV copy numbers per 100 ng.

Method qPCR11

This qPCR method for the BLV pol gene is a modified option of widely available quantitative TaqMan qPCR described by Rola-Łuszczak et al. [ 11 ], using the same primers and standards. The reaction mixture contained 400 nM of each primer, 200 nM of probe, 10 µl of 2 × SsoFast probes supermix (Bio-Rad), 5 µl of DNA sample and H 2 O up to 20 µl of the final volume. PCR assays were carried out on a CFX96 thermocycler (Bio-Rad) under the following amplification profile: 98 °C for 3 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were used to generate the standard curve and estimate BLV copy number per 100 ng.

Analysis of BLV pol, env and LTR sequences targeted by particular qPCR/ddPCR assays

In order to assess full-length pol , env and LTR sequence variability among BLV genotypes, all BLV sequences ( n  = 2191) available on 30 September 2023 in GenBank ( https://www.ncbi.nlm.nih.gov/GenBank/ ) repository were retrieved. From the collected sequences, 100 pol , env and LTR sequences, which were characterized by the highest level of sequence variability and divergence, were selected for the further analysis. A pol -based, env -based and LTR-based maximum likelihood (ML) phylogenetic trees (see Additional file 6) was constructed to assign genotypes to the unassigned BLV genomes [ 37 , 38 , 39 ]. For all genes and LTR region the Tamura-Nei model and Bootstrap replications (1,000) were applied. In this analysis, pol sequences were assigned to 7 BLV genotypes (G1, G2, G3, G4, G6, G9, and G10), while env and LTR sequences were assigned to 10 BLV genotypes (G1, G2, G3, G4, G5, G6, G7, G8, G9, and G10). Phylogeny of the same isolates assigned to particular genotypes by ML method was confirmed by Mr. Bayes analysis [ 40 , 41 , 42 ] (data not shown). From this analysis, a total of 100 full-length pol, env and LTR sequences were used for multiple-sequence alignment (MSA) using ClustalW algorithm, implemented in MEGA X. For all sequences, nucleotide diversity (π), defined as the average number of nucleotide differences per site between two DNA sequences in all possible pairs in the sample population, was estimated using MEGA X. To measure the relative variation in different positions of aligned genes and LTR region the Shannon’s entropy (a quantitative measure of diversity in the alignment, where H = 0 indicates complete conservation) was estimated using BioEdit v. 7.2.5 software 64. The statistical analyses were performed using DATAtab e.U. Graz, Austria and GraphPad Software by Dotmatics, Boston.

Examination of the quality and stability of DNA samples

To test the quality of DNA samples, the H3F3A copy number of each individual sample was assessed by qPCR at the NVRI. Copy numbers were normalized to DNA mass input and results were expressed as copy numbers per 300 ng of total DNA. The respective values were tested by Grubbs' test. The results for 43 DNA samples (sample ID: 42 with BLV genome plasmid was excluded) followed a normal distribution (Shapiro–Wilk 0.97; P  = 0.286), with a mean value of 35,626 copies (95% confidence interval [ 43 ] 33,843 to 37,408 copies), a minimum value of 19,848 copies and a maximum value of 46,951 copies (see Additional file 2). Despite a low value for sample ID: 40 no significant outlier was detected in the dataset ( P  > 0.05). Therefore, it can be assumed that the DNA quality was acceptable for all samples present in the panel. Next, DNA stability was assessed by retesting the H3F3A copy numbers in each sample ( n  = 43) after a combined storage consisting in 10 days at RT and 10 days at + 4°C. A Mann–Whitney U-test was used to compare the median values between fresh and stored samples (time 0 and time 1, respectively), and no significant difference was observed at the 5% level ( P  = 0.187) (Fig.  1 A).

Assessment of the stability of DNA samples. A Shown are copy numbers of the H3F3A housekeeping gene in 43 DNA samples that were stored in 10 days at RT and 10 days at + 4°C and tested twice with a 21-day interval. A Mann–Whitney U-test was used to compare the median values between two groups ( P  = 0.187); B Shown are GQN values ( n  = 43) tested twice with a 21-day interval: `before freeze-drying` and `after freeze-drying`. A Mann–Whitney U-test results between two groups ( P  = 0.236)

In addition, the quality of DNA samples after lyophilization was analyzed. DNA from individual samples ( n  = 43) was assessed with the genomic DNA quality number on the Fragment Analyzer system. The GQN from all lyophilized samples ranged from 4.0 to 9.7—that represented undegraded DNA. There was no significant difference in GQN values between `before freeze-drying` and `after freeze-drying` groups with respect to the corresponding DNA samples ( P  = 0.236) (Fig.  1 B). Altogether, these results suggested that sample storage, lyophilization and shipping has a minimal impact in DNA stability and further testing during the interlaboratory trial.

Detection of BLV proviral DNA by different qPCR assays

A total of 44 DNA samples, including two positive (ID: 42 and 43) and one negative (ID: 32) controls, were blinded and independently tested by eleven laboratories using their own qPCR methods (Table  2 ). All laboratories measured the concentration of DNA in samples (Additional file 3). BLV provirus copy number was normalized to DNA concentration and expressed per 100 ng of genomic DNA for each test.

Except for the positive (pBLV344 and FLK cell line) and the negative controls, all samples had previously shown detectable levels of BLV-specific antibodies (BLV-Abs) by enzyme-linked immunosorbent assays (ELISA). During the current interlaboratory study, both the positive and negative controls were assessed adequately by all eleven PCR tests. Of all 43 positive samples, 43, 35, 37, 36, 40, 32, 40, 42, 42, 42 and 41 samples were detected as positive by the qPCR1, qPCR2, qPCR3, qPCR4, qPCR5, ddPCR6, qPCR7, qPCR8, qPCR9, qPCR10 and qPCR11 methods, respectively. Based on these observations, the most sensitive method was the qPCR1, and the method with the lowest sensitivity was the ddPCR6. Twenty-nine out of 44 samples were identified correctly by all qPCRs. The remaining 15 samples gave discordant results. Comparison of qualitative results (positive versus negative) from all eleven methods revealed 87.33% overall agreement and a kappa value of 0.396 (Cohen's kappa method adapted by Fleiss) [ 44 , 45 ]. The levels of agreement among the results from the eleven methods are represented in Table  3 . The maximum agreement was seen between two methods (qPCR9 and qPCR10 [100% agreement and a Cohen's kappa value of 1.000]) that used similar protocols and targeted the same region of BLV pol .

Analysis of BLV pol, env and LTR sequences targeted by particular PCR assays

Due to differences in performance observed among the pol -based qPCR assays (the qPCR1, qPCR2, qPCR4, qPCR5 and qPCR7- qPCR11 methods), and considering that the env -based ddPCR6 and LTR-based qPCR3 assay showed the lowest sensitivity and the poorest agreement with the other assays, the degree of sequence variability between the pol , env and LTR genes was addressed. From the MSAs for pol , env and LTR, the nucleotide diversity (π) was calculated. The π value for pol gene was lower than that for LTR and env gene (π pol , 0.023 [standard deviation {SD}, 0.018]; π LTR , 0.024 [SD, 0.011]; π env , 0.037 [SD, 0.013]). From this analysis, pol sequences appeared to be less variable than env and LTR sequences. In addition, we performed a Shannon entropy-based per-site variability profile of the pol , env and LTR sequences used in this study (Fig.  2 A-C).

Sequence variability measured as per-site entropy. A Multiple alignment of the pol gene showing the locations of qPCR fragments in regions of the pol gene for the qPCR1 (highlighted in pink), qPCR4 (highlighted in yellow) and for the qPCR7, qPCR8, qPCR9, qPCR10 and qPCR11 assays (highlighted in orange). B Multiple alignment of the env gene targeted by ddPCR6 (highlighted by blue rectangle). C Multiple alignment of the LTR region by qPCR3 (highlighted in mint)

The all-observed entropy plots were homogeneous along the whole sequences. Considering the three regions of pol gene, the highest entropy (4.67) occurred in the region targeted by the qPCR1 primers, whereas the entropy for qPCR7—qPCR11 and qPCR4 primers were 1.57 and 0.38, respectively. For the LTR region targeted by qPCR3 primers and for env gene targeted by ddPCR6, the total entropy was equal to 4.46 and 7.85, respectively. This analysis showed a marked region of variability for LTR and env fragments. Interestingly, we noted that the qPCR7—qPCR11 targeted the most conserved regions of reverse transcriptase and qPCR4 primers targeted the most-conserved region of virus integrase (Fig.  2 A-C; see also Additional file 7).

Quantitation of BLV proviral DNA by different qPCR/ddPCR assays

To analyze whether the range of copy numbers detected by each qPCR was comparable to those of the others, Kruskal–Wallis one-way analysis of variance (ANOVA) was used. The violin plots were used to visualize the ANOVA results (Fig.  3 A-B).

Comparison of detection of BLV proviral DNA copy numbers by eleven testing methods. Shown is a box plot of data from Kruskal–Wallis ANOVA, a rank test. The DNA copy numbers for 41 samples, determined independently by each of the 11 qPCRs, were used for the variance analysis. In this analysis, the positive controls (sample ID 42 and ID 43) and negative control (sample ID 32) were excluded. A Violin plot for graphical presentation of the ANOVA of proviral copy number values. B Violin plot for ANOVA analysis of variance, copy number values are presented on a logarithmic scale (Log1.2) for better illustration of copy number differences between PCR methods

The grouping variable revealed significant differences among the distributions of proviral DNA copy numbers with the various qPCRs ( P  < 0.001). These results showed that the abilities of qPCRs/ddPCR to determine the proviral DNA copy number differed. A Dunn-Bonferroni test was used to compare the groups in pairs to find out which was significantly different. The Dunn-Bonferroni test revealed that the pairwise group comparisons of qPCR2—qPCR4, qPCR3—ddPCR6, qPCR4—qPCR5, qPCR4—ddPCR6, qPCR4—qPCR9, qPCR4—qPCR10, qPCR5—qPCR11, ddPCR6—qPCR11 and qPCR9—qPCR11 have an adjusted P value less than 0.05 and thus, it can be assumed that these groups were significantly different in each pair (see Additional file 4). The Pareto chart was used to show the average copy number values of all methods in descending order. These Pareto charts were prepared based on 80–20 rule, which states that 80% of effects come from 20% of the various causes [ 46 ]. The methods that generated the highest copy numbers was qPCR3 and qPCR4, on the other hand the lowest copy numbers and/or highest negative results were generated by ddPCR6 (Fig.  4 ).

A Pareto chart with the proviral BLV copy mean values for eleven PCR assay arranged in descending order. Pareto charts was prepared based on 80–20 rule, which states that 80% of effects come from 20% of the various causes

The correlations between copy numbers detected by different qPCRs and ddPCR assays were calculated. The Kendall's Tau correlation coefficient measured between each pair of the assays was shown in the Additional file 5 and in Fig.  5 as a correlation heatmap. The average correlation for all qPCRs and ddPCR assays was strong (Kendall's tau = 0.748; P  < 0.001).

The heatmap of Kendall’s tau correlation coefficients between copy numbers detected by ten qPCRs and one ddPCR. Statistically significant differences in the distribution of copy numbers, a moderate, strong and very strong correlation between particular qPCRs/ddPCR was observed. The strength of the association, for absolute values of r, 0–0.19 is regarded as very weak, 0.2–0.39 as weak, 0.40–0.59 as moderate, 0.6–0.79 as strong and 0.8–1 as very strong correlation

Since the differences between PCR tests may be influenced by the number of BLV proviral copies present in each sample, we compared the average number of BLV copies between a group of genomic DNA samples that gave concordant results (group I [ n  = 28]) and a group that gave discordant results (group II [ n  = 15]). The mean number of copies was 73,907 (minimum, 0; maximum, 4,286,730) in group I, and 3,479 (minimum, 0; maximum, 218,583) in group II, and this difference was statistically significant ( P  < 0.001 by a Mann–Whitney U- test) (Fig.  6 ).

Impact of BLV proviral copy numbers on the level of agreement. Violin plot for graphical presentation of Mann–Whitney U test. The test was performed to compare BLV provirus copy number in two groups of samples: 28 samples with fully concordant results from all eleven qPCR/ddPCR assays (left) and 15 samples with discordant results from different qPCR/ddPCR assays (right) ( P  < 0.001). Sample ID 42 was excluded from the statistical analysis

The results show that the concordant results group had considerably higher copy numbers (median, 5,549.0) than the discordant results group (median, 6.3).

BLV control and eradication programs consist of correct identification and subsequent segregation/elimination of BLV-infected animals [ 47 ]. Detection of BLV- infected cows by testing for BLV-specific antibodies in serum by agar gel immunodiffusion and ELISA is the key step and standard to be implemented of EBL eradication programs according to WOAH ( https://www.woah.org/en/disease/enzootic-bovine-leukosis/) [ 9 ]. Despite the low cost and high throughput of serological tests, there are several scenarios where highly specific and sensitive molecular assays for the detection of BLV DNA might improve detection and program efficiency.

In this perspective, qPCR assays can detect small quantities of proviral DNA during acute infection, in which animals show very low levels of anti-BLV antibodies [ 43 , 48 , 49 , 50 ]. qPCR methods can also work as confirmatory tests to clarify ambiguous and inconsistent serological test results [ 11 ]. Such quantitative features of qPCRs are crucial when eradication programs progress and prevalence decreases. Moreover, qPCR allows not only the detection of BLV infection but also estimation of the BLV PVL, which directly correlates with the risk of disease transmission [ 51 , 52 ]. This feature of qPCR allows for a rational segregation of animals based on the stratified risk of transmission. These considerations allow for greater precision in the management of BLV within large herds with a high prevalence of BLV ELISA-positive animals to effectively reduce herd prevalence [ 13 , 53 ]. BLV is a global burden and the lack of technical standardization of molecular detection systems remains a huge obstacle to compare surveillance data globally based on the first interlaboratory trial performed in 2018 [ 15 ]. In the 2018 study we observed an adjusted level of agreement of 70% comparing qualitative qPCR results; however, inconsistencies amongst methods were larger when low number of copies of BLV DNA were compared. Samples with low copies of BLV DNA (< 20 copies per 100 ng) accounted for the higher variability and discrepancies amongst tests. We concluded from the first interlaboratory trial that standardizing protocols to improve sensitivity of assays with lower detection rates was necessary.

In this follow up study, we re-tested the TaqMan BLV qPCR developed and validated by NVRI (acting as reference WOAH laboratory) and the one adapted from this original protocol to be used with SYBR Green dye, allowing a significant reduction in costs [ 11 ]. Another 3 laboratories also performed NVRI´s qPCR with slight modifications (i.e., Spain performed a multiplex assay for internal normalization). The remaining 6 labs introduced novel methodologies to the trial including one ddPCR (UY).

To compare different qPCR methods, a more comprehensive sample panel, accounting for a more geographical diversification was used in this trial. The amounts of BLV DNA in these samples were representative of the different BLV proviral loads found in field samples (from 1 to > 10,000 copies of BLV proviral DNA). Of note, 34% of reference samples had less than 100 copies of BLV DNA per 100 ng; samples were lyophilized to grant better preservation and reduced variability during distribution to participants around the globe.

The panel included a single negative control and two positive controls. Diagnostic sensitivity (DxSn) was estimated for each qPCR. Considering the 43 positive samples, the DxSn for the different qPCRs were: qPCR1 = 100%, qPCR2 = 82%, qPCR3 = 86%, qPCR4 = 84%, qPCR5 = 93%, ddPCR6 = 74%, qPCR7 = 93%, qPCR8 = 98%, qPCR9 = 98%, qPCR10 = 98% and qPCR11 = 95%. The most sensitive method was the qPCR1, and the method with the lowest sensitivity was the ddPCR6 method. Twenty-nine out of 44 samples were identified correctly by all qPCRs. The remaining 15 samples gave discordant results. The comparison of qualitative qPCR results among all raters revealed an overall observed agreement of 87%, indicating strong interrater reliability (Cohen´s kappa = 0.396) [ 54 , 55 ].

There are several factors that contribute to variability in qPCR results (i.e., number of copies of target input, sample acquisition, processing, storage and shipping, DNA purification, target selection, assay design, calibrator, data analysis, etc.). For that reason and as expected, the level of agreement among sister qPCRs (qPCR7, qPCR9-11) sharing similar protocols was higher compared to the rest of assays; this was also true for qPCR8 which targets the same region of BLV pol gene (shares same primers) but has a particular set-up to be used with SYBR Green chemistry. Oppositely, lower sensitivity and larger discrepancy against other tests was observed for the ddPCR6 and qPCR2-4.

Based on these observations we investigated which factors might have accounted for larger assessment variability amongst tests. In the first place, we observed that the use of different chemistries was not detrimental for the sensitivity and agreement among tests; similar DxSn and comparable level of agreement were obtained comparing TaqMan (qPCR7, 10, 11) vs SYBR Green (qPCR8) chemistries while targeting identical BLV sequence and using same standards. Also, when a multiplex qPCR (TaqMan) targeting the same BLV sequence and using the same standard was compared to previous ones, agreement was kept high, indicating that the lower sensitivity described for some multiplex qPCRs did not take place in this comparison. The use of an international calibrator and the efficiency estimation (standard curve) might inform variability associated with different chemistries. In contrast, another multiplex assay targeting another region of BLV pol (qPCR2) showed much lower sensitivity and agreement. As qPCR2 is performed as service by private company and oligonucleotide sequences were not available, we were not able to investigate in which proportion each of these two variables contributed to the lower performance of this assay, but we note the addition of 4 µl genomic DNA to this assay that would have an impact the DxSn. In this regard, there is substantial evidence showing that the variability of target sequence among strains from different geographical areas, might affect the sensitivity of BLV qPCRs. Previous studies comparing the pol , gag , tax and env genes reported that the pol gene was the most suitable region to target for diagnostic purposes, since it provided the most-sensitive assays [ 11 , 15 , 56 , 57 , 58 , 59 ]. This might be due in part to higher sequence conservation of pol among strains from different geographical areas. Supporting this observation, it is noticeable how JPN qPCR improved their performance in the current trial, by targeting pol in place of tax , as it did in the previous interlaboratory trial. Since it is a commercial test, we cannot exclude other factors contributing for the performance upgrade observed for this qPCR. In the current study, qPCR3 and ddPCR6 targeting LTR and env sequences, showed lower performances than other assays. Standardization of DNA input into each qPCR would have likely resulted in higher concordance in results. For instance, qPCR1 added 10 µl of genomic DNA per reaction and ddPCR6 added 1 µl of genomic DNA, impacting the resulting sensitivity differences.

Since the sensitivity of each assay and, consequently, the level of agreement among assays might also be influenced by the number of BLV DNA copies present in each sample [ 48 ], we compared the average number of BLV DNA copies between a group of genomic DNA samples that gave concordant results and a group that gave discordant results, and observed that samples that gave discordant results had significantly lower numbers of BLV DNA copies than samples that gave concordant results. Related to this point, the degradation of target DNA during lyophilization, shipment and resuspension, could have been more significant in low-copy compared to high-copy samples. Consequently, the degradation of target DNA in samples with low copies of BLV DNA might have accounted for the greater level of discrepancy within this subset of samples. The rational of adding a large proportion of such samples (34% samples with less than 100 BLV copies per 100 ng of total DNA) was to mimic what is frequently observed in surveillance programs (i.e., hyperacute infection, chronic asymptomatic infection, etc.).

Quantitative methods for the detection of BLV DNA copies are important for segregation programs based on animal level of BLV PVL, as well as for scientific research and the study of BLV dynamics. When the numbers of copies of BLV DNA detected by different assays were compared, in the present study, we observed that although the ability to quantify BLV DNA differed among qPCRs/ddPCR and there were statistically significant differences in the distribution of copy numbers among assays, a strong average correlation was found for the eleven qPCRs/ddPCR. In this regard, the lack of an international calibrator (standard curve) could be a major contributor to the increment of quantitative variation amongst laboratories. For that reason, plasmid pBLV1 containing pol 120 bp sequence was originally constructed for use as standard for quantification and shared with some collaborators (i.e., qPCR7, qPCR8, qPCR 9, qPCR10 and qPCR11). Remarkably, the laboratories used pBLV1 standard in the current trial obtained the most comparable results, indicating that the use of an international standard may have significant impact on the convergence of results; such standard reference material should be prepared under identical conditions. To avoid further variability a detailed protocol for lyophilized DNA sample resuspension, quantitation and template input into each qPCR should be shared with all participants.

Conclusions

BLV DNA was detected with different level of sensitivity in serologically positive samples from different origin and classified into different BLV genotypes. Overall agreement was high; however, we found significant differences in results for the samples with low BLV DNA copy numbers. This second interlaboratory study demonstrated that differences in target sequence, DNA input and calibration curve standards can increase interlaboratory variability considerably. Next steps should focus on (i) standard unification (international gold standard) to estimate individual test efficiency and improve quantitative accuracy amongst tests; (ii) building a new panel of samples with low BLV DNA copy numbers to re-evaluate sensitivity and quantitation of molecular methods. Since no variation was observed in samples from different genotypes, all samples will be collected in Poland to standardize the collection, purification, lyophilization and shipping steps with precise instructions for suspension and constant input volume for the PCR reaction. Finally, we believe that following this standardization approach we will be able to improve overall agreement amongst tests, improving the diagnostic of BLV around the world.

Availability of data and materials

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

One-way analysis of variance

Bovine leukemia virus

BLV-specific antibodies

Digital PCR

Diagnostic sensitivity

Enzootic bovine leukosis

Enzyme-linked immunosorbent assays

Real-time fluorescence resonance energy transfer PCR

Genomic quality number

Histone H3 family 3A housekeeping gene

Maximum likelihood phylogenetic tree

Multiple-sequence alignment

Peripheral blood leukocytes

Phosphate-buffered saline

Proviral load

Quantitative real-time PCR

Room temperature

World Organisation for Animal Health

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Acknowledgements

The authors thank Luc Willems (University of Liège, Belgium) for plasmid DNA sample pBLV344; Marlena Smagacz and Eliza Czarnecka (National Veterinary Research Institute, Poland) for lyophilizing DNA samples and DNA analysis, respectively; Ali Sakhawat (Animal Quarantine Department, Pakistan), Vitaliy Bolotin (National Scientific Center IECVM, Ukraine), Frank van der Meer and Sulav Shrestha (University of Calgary, Canada) for sharing material.

The APC was funded by the National Veterinary Research Institute, Puławy, Poland.

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Department of Biochemistry, National Veterinary Research Institute, Puławy, 24-100, Poland

Aneta Pluta & Jacek Kuźmak

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Contributions

Proposed the conception and design of the study, A.P.; data curation, A.P., J.P.J., C.D., S.V., D.B., A.S., K.M., R.P., G.D., M.F.C. and CH.W.; investigation, A.P., V.R., S.VW., S.V., A.J., M.J.R., K.N., M.L.B., M.L.G., P.L., A.F., A.G. and S.B., formal analysis, A.P.; statistical analysis, A.P.; database analysis, A.P., visualization of the results, A.P.; resources, A.P., T.M.T. and J.K; writing—original draft preparation, A.P., J.P.J.; writing—review and editing, A.P., J.P.J., C.D., S.VW., T.M.T. and J.K; project administration, A.P. All authors read and approved the submitted version.

Corresponding author

Correspondence to Aneta Pluta .

Ethics declarations

Ethics approval and consent to participate.

The study was approved by the Veterinary Sciences Animal Care Committee No. AC21-0210, Canada; the Institutional Animal Care and Use Committee No. PROTO202000096 from 4/13/2020 to 4/14/2023, Michigan State University, United States and the Ethics Review Board, COMSATS Institute of Information Technology, Islamabad, Pakistan, no. CIIT/Bio/ERB/17/26. Blood samples from Polish, Moldovan and Ukrainian cattle, naturally infected with BLV, were selected from collections at local diagnostic laboratories as part of the Enzootic bovine leukosis (EBL) monitoring program between 2012 and 2018 and sent to the National Veterinary Research Institute (NVRI) in Pulawy for confirmation study. The approval for collection of these samples from ethics committee was not required according to Polish regulation (“Act on the Protection of Animals Used for Scientific or Educational Purposes”, Journal of Laws of 2015). All methods were carried out in accordance with relevant guidelines and regulations. The owners of the cattle herds from which the DNA samples originated, the district veterinarians caring for these farms and the ministries of agriculture were informed and consented to the collection of blood from the animals for scientific purposes and the sending of samples to NVRI.

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Supplementary Information

12917_2024_4228_moesm1_esm.pdf.

Additional file 1. Copy of the instruction included with the panel of 44 DNA samples sent to participating laboratories for dilution of the lyophilisates

12917_2024_4228_MOESM2_ESM.png

Additional file 2. Detection of the H3F3A gene copy number in 43 DNA samples; no outlier was found for any samples ( P <0.05) (two-sided).

12917_2024_4228_MOESM3_ESM.docx

Additional file 3. Concentration values of 44 DNA samples measured by the 11 participating laboratories (given in ng per µl)

12917_2024_4228_MOESM4_ESM.pdf

Additional file 4. Post hoc - Dunn-Bonferroni-Tests. The Dunn-Bonferroni test revealed that the pairwise group comparisons of qPCR2 - qPCR4, qPCR3 - ddPCR6, qPCR4 - qPCR5, qPCR4 - ddPCR6, qPCR4 - qPCR9, qPCR4 - qPCR10, qPCR5 - qPCR11, ddPCR6 - qPCR11 and qPCR9 - qPCR11 have an adjusted p-value less than 0,05

12917_2024_4228_MOESM5_ESM.docx

Additional file 5. Kendall's Tau correlation coefficient values measured between each pair of assays. The numbers 1 to 11 in the first column and last row of the table indicate the names of the assays qPCR1-qPCR5, ddPCR6, qPCR7-qPCR11 respectively

12917_2024_4228_MOESM6_ESM.png

Additional file 6. Maximum-likelihood phylogenetic analysis of full-length BLV-pol gene sequences representing 7 BLV genotypes (G1, G2, G3, G4, G6, G9, and G10) (A); (B) env-based sequences assigned to 10 BLV genotypes (G1, G2, G3, G4, G5, G6, G7, G8, G9, and G10); (C) LTR-based sequences representing 10 BLV genotypes (G1-G10). For all genes and LTR region the Tamura-Nei model and Bootstrap replications (1,000) were applied in MEGA X

12917_2024_4228_MOESM7_ESM.pdf

Additional file 7. Multiple sequence alignment of reverse transcriptase, integrase, envelope and LTR sequences in the context of the specific primers used by different qPCR assays. (A) Multiple sequence alignment of reverse transcriptase (pol gene) sequences in the context of qPCR7, qPCR8, qPCR9, qPCR10 and qPCR11 assay primers. (B) Multiple sequence alignment of integrase (pol gene) sequences in the context of qPCR4 assay primers. (C) Multiple sequence alignment of env gene sequences in the context of ddPCR6. (D) Sequence alignment of LTR region sequences in the context of qPCR3 method primers

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Pluta, A., Jaworski, J.P., Droscha, C. et al. Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples. BMC Vet Res 20 , 381 (2024). https://doi.org/10.1186/s12917-024-04228-z

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Received : 24 November 2023

Accepted : 09 August 2024

Published : 26 August 2024

DOI : https://doi.org/10.1186/s12917-024-04228-z

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100+ Quantitative Research Topics For Students

Quantitative research is a research strategy focusing on quantified data collection and analysis processes. This research strategy emphasizes testing theories on various subjects. It also includes collecting and analyzing non-numerical data.

Quantitative research is a common approach in the natural and social sciences , like marketing, business, sociology, chemistry, biology, economics, and psychology. So, if you are fond of statistics and figures, a quantitative research title would be an excellent option for your research proposal or project.

How to Get a Title of Quantitative Research

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Finding a great title is the key to writing a great quantitative research proposal or paper. A title for quantitative research prepares you for success, failure, or mediocre grades. This post features examples of quantitative research titles for all students.

Putting together a research title and quantitative research design is not as easy as some students assume. So, an example topic of quantitative research can help you craft your own. However, even with the examples, you may need some guidelines for personalizing your research project or proposal topics.

So, here are some tips for getting a title for quantitative research:

• Consider your area of studies
• Look out for relevant subjects in the area
• Expert advice may come in handy
• Check out some sample quantitative research titles

Making a quantitative research title is easy if you know the qualities of a good title in quantitative research. Reading about how to make a quantitative research title may not help as much as looking at some samples. Looking at a quantitative research example title will give you an idea of where to start.

However, let’s look at some tips for how to make a quantitative research title:

• The title should seem interesting to readers
• Ensure that the title represents the content of the research paper
• Reflect on the tone of the writing in the title
• The title should contain important keywords in your chosen subject to help readers find your paper
• The title should not be too lengthy
• It should be grammatically correct and creative
• It must generate curiosity

An excellent quantitative title should be clear, which implies that it should effectively explain the paper and what readers can expect. A research title for quantitative research is the gateway to your article or proposal. So, it should be well thought out. Additionally, it should give you room for extensive topic research.

A sample of quantitative research titles will give you an idea of what a good title for quantitative research looks like. Here are some examples:

• What is the correlation between inflation rates and unemployment rates?
• Has climate adaptation influenced the mitigation of funds allocation?
• Job satisfaction and employee turnover: What is the link?
• A look at the relationship between poor households and the development of entrepreneurship skills
• Urbanization and economic growth: What is the link between these elements?
• Does education achievement influence people’s economic status?
• What is the impact of solar electricity on the wholesale energy market?
• Debt accumulation and retirement: What is the relationship between these concepts?
• Can people with psychiatric disorders develop independent living skills?
• Children’s nutrition and its impact on cognitive development

Quantitative research applies to various subjects in the natural and social sciences. Therefore, depending on your intended subject, you have numerous options. Below are some good quantitative research topics for students:

• The difference between the colorific intake of men and women in your country
• Top strategies used to measure customer satisfaction and how they work
• Black Friday sales: are they profitable?
• The correlation between estimated target market and practical competitive risk assignment
• Are smartphones making us brighter or dumber?
• Nuclear families Vs. Joint families: Is there a difference?
• What will society look like in the absence of organized religion?
• A comparison between carbohydrate weight loss benefits and high carbohydrate diets?
• How does emotional stability influence your overall well-being?
• The extent of the impact of technology in the communications sector

Creativity is the key to creating a good research topic in quantitative research. Find a good quantitative research topic below:

• How much exercise is good for lasting physical well-being?
• A comparison of the nutritional therapy uses and contemporary medical approaches
• Does sugar intake have a direct impact on diabetes diagnosis?
• Education attainment: Does it influence crime rates in society?
• Is there an actual link between obesity and cancer rates?
• Do kids with siblings have better social skills than those without?
• Computer games and their impact on the young generation
• Has social media marketing taken over conventional marketing strategies?
• The impact of technology development on human relationships and communication
• What is the link between drug addiction and age?

Need more quantitative research title examples to inspire you? Here are some quantitative research title examples to look at:

• Habitation fragmentation and biodiversity loss: What is the link?
• Radiation has affected biodiversity: Assessing its effects
• An assessment of the impact of the CORONA virus on global population growth
• Is the pandemic truly over, or have human bodies built resistance against the virus?
• The ozone hole and its impact on the environment
• The greenhouse gas effect: What is it and how has it impacted the atmosphere
• GMO crops: are they good or bad for your health?
• Is there a direct link between education quality and job attainment?
• How have education systems changed from traditional to modern times?
• The good and bad impacts of technology on education qualities

Your examiner will give you excellent grades if you come up with a unique title and outstanding content. Here are some quantitative research examples titles.

• Online classes: are they helpful or not?
• What changes has the global CORONA pandemic had on the population growth curve?
• Daily habits influenced by the global pandemic
• An analysis of the impact of culture on people’s personalities
• How has feminism influenced the education system’s approach to the girl child’s education?
• Academic competition: what are its benefits and downsides for students?
• Is there a link between education and student integrity?
• An analysis of how the education sector can influence a country’s economy
• An overview of the link between crime rates and concern for crime
• Is there a link between education and obesity?

Research title example quantitative topics when well-thought guarantees a paper that is a good read. Look at the examples below to get started.

• What are the impacts of online games on students?
• Sex education in schools: how important is it?
• Should schools be teaching about safe sex in their sex education classes?
• The correlation between extreme parent interference on student academic performance
• Is there a real link between academic marks and intelligence?
• Teacher feedback: How necessary is it, and how does it help students?
• An analysis of modern education systems and their impact on student performance
• An overview of the link between academic performance/marks and intelligence
• Are grading systems helpful or harmful to students?
• What was the impact of the pandemic on students?

Irrespective of the course you take, here are some titles that can fit diverse subjects pretty well. Here are some creative quantitative research title ideas:

• A look at the pre-corona and post-corona economy
• How are conventional retail businesses fairing against eCommerce sites like Amazon and Shopify?
• An evaluation of mortality rates of heart attacks
• Effective treatments for cardiovascular issues and their prevention
• A comparison of the effectiveness of home care and nursing home care
• Strategies for managing effective dissemination of information to modern students
• How does educational discrimination influence students’ futures?
• The impacts of unfavorable classroom environment and bullying on students and teachers
• An overview of the implementation of STEM education to K-12 students
• How effective is digital learning?

If your paper addresses a problem, you must present facts that solve the question or tell more about the question. Here are examples of quantitative research titles that will inspire you.

• An elaborate study of the influence of telemedicine in healthcare practices
• How has scientific innovation influenced the defense or military system?
• The link between technology and people’s mental health
• Has social media helped create awareness or worsened people’s mental health?
• How do engineers promote green technology?
• How can engineers raise sustainability in building and structural infrastructures?
• An analysis of how decision-making is dependent on someone’s sub-conscious
• A comprehensive study of ADHD and its impact on students’ capabilities
• The impact of racism on people’s mental health and overall wellbeing
• How has the current surge in social activism helped shape people’s relationships?

Are you looking for an example of a quantitative research title? These ten examples below will get you started.

• The prevalence of nonverbal communication in social control and people’s interactions
• The impacts of stress on people’s behavior in society
• A study of the connection between capital structures and corporate strategies
• How do changes in credit ratings impact equality returns?
• A quantitative analysis of the effect of bond rating changes on stock prices
• The impact of semantics on web technology
• An analysis of persuasion, propaganda, and marketing impact on individuals
• The dominant-firm model: what is it, and how does it apply to your country’s retail sector?
• The role of income inequality in economy growth
• An examination of juvenile delinquents’ treatment in your country

Excellent Topics For Quantitative Research

Here are some titles for quantitative research you should consider:

• Does studying mathematics help implement data safety for businesses
• How are art-related subjects interdependent with mathematics?
• How do eco-friendly practices in the hospitality industry influence tourism rates?
• A deep insight into how people view eco-tourisms
• Religion vs. hospitality: Details on their correlation
• Has your country’s tourist sector revived after the pandemic?
• How effective is non-verbal communication in conveying emotions?
• Are there similarities between the English and French vocabulary?
• How do politicians use persuasive language in political speeches?
• The correlation between popular culture and translation

Here are some quantitative research titles examples for your consideration:

• How do world leaders use language to change the emotional climate in their nations?
• Extensive research on how linguistics cultivate political buzzwords
• The impact of globalization on the global tourism sector
• An analysis of the effects of the pandemic on the worldwide hospitality sector
• The influence of social media platforms on people’s choice of tourism destinations
• Educational tourism: What is it and what you should know about it
• Why do college students experience math anxiety?
• Is math anxiety a phenomenon?
• A guide on effective ways to fight cultural bias in modern society
• Creative ways to solve the overpopulation issue

An example of quantitative research topics for 12 th -grade students will come in handy if you want to score a good grade. Here are some of the best ones:

• The link between global warming and climate change
• What is the greenhouse gas impact on biodiversity and the atmosphere
• Has the internet successfully influenced literacy rates in society
• The value and downsides of competition for students
• A comparison of the education system in first-world and third-world countries
• The impact of alcohol addiction on the younger generation
• How has social media influenced human relationships?
• Has education helped boost feminism among men and women?
• Are computers in classrooms beneficial or detrimental to students?
• How has social media improved bullying rates among teenagers?

High school students can apply research titles on social issues  or other elements, depending on the subject. Let’s look at some quantitative topics for students:

• What is the right age to introduce sex education for students
• Can extreme punishment help reduce alcohol consumption among teenagers?
• Should the government increase the age of sexual consent?
• The link between globalization and the local economy collapses
• How are global companies influencing local economies?

There are numerous possible quantitative research topics you can write about. Here are some great quantitative research topics examples:

• The correlation between video games and crime rates
• Do college studies impact future job satisfaction?
• What can the education sector do to encourage more college enrollment?
• The impact of education on self-esteem
• The relationship between income and occupation

You can find inspiration for your research topic from trending affairs on social media or in the news. Such topics will make your research enticing. Find a trending topic for quantitative research example from the list below:

• How the country’s economy is fairing after the pandemic
• An analysis of the riots by women in Iran and what the women gain to achieve
• Is the current US government living up to the voter’s expectations?
• How is the war in Ukraine affecting the global economy?
• Can social media riots affect political decisions?

A proposal is a paper you write proposing the subject you would like to cover for your research and the research techniques you will apply. If the proposal is approved, it turns to your research topic. Here are some quantitative titles you should consider for your research proposal:

• Military support and economic development: What is the impact in developing nations?
• How does gun ownership influence crime rates in developed countries?
• How can the US government reduce gun violence without influencing people’s rights?
• What is the link between school prestige and academic standards?
• Is there a scientific link between abortion and the definition of viability?

You can never have too many sample titles. The samples allow you to find a unique title you’re your research or proposal. Find a sample quantitative research title here:

• Does weight loss indicate good or poor health?
• Should schools do away with grading systems?
• The impact of culture on student interactions and personalities
• How can parents successfully protect their kids from the dangers of the internet?
• Is the US education system better or worse than Europe’s?

If you’re a business major, then you must choose a research title quantitative about business. Let’s look at some research title examples quantitative in business:

• Creating shareholder value in business: How important is it?
• The changes in credit ratings and their impact on equity returns
• The importance of data privacy laws in business operations
• How do businesses benefit from e-waste and carbon footprint reduction?
• Organizational culture in business: what is its importance?

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Interesting, creative, unique, and easy quantitative research topics allow you to explain your paper and make research easy. Therefore, you should not take choosing a research paper or proposal topic lightly. With your topic ready, reach out to us today for excellent research paper writing services .

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Choose the Appropriate Research Design Directions: Determine the quantitative research design appropriate for the sample research tillos. Make sure to explain your choice into two to three sentences. 1. Relationship between Academic Stressors and Learning Preferences of Senior High School Students

Explanation