Development and validation of a point-of-care diagnostic tool for rapid detection of major bovine reproductive pathogens (e.g., Brucella spp., Campylobacter fetus, Neospora caninum) using CRISPR-based assays in field settings

Project Abstract

This study reports the development and validation of a point-of-care diagnostic tool for rapid detection of major bovine reproductive pathogens, including Brucella spp., Campylobacter fetus, and Neospora caninum, employing CRISPR-based assays adapted for field use. The core objective was to create a portable, user-friendly diagnostic platform capable of delivering accurate,asiaic results within 30–60 minutes at farm or clinic settings, thereby enabling timely interventions to reduce reproductive losses and curb zoonotic risk. We integrated CRISPR-Cas12a and Cas13a systems with isothermal amplification strategies to obviate the need for complex thermal cycling, utilizing a simple, battery-powered device and lateral-flow readouts for visual interpretation. The assay design targeted species- and serovar-specific genomic regions with rigorous in silico validation against a diverse panel of reproductive pathogens and non-target organisms to ensure high analytical specificity. To assess diagnostic performance, we conducted multi-site field evaluations across dairy and beef operations, incorporating clinically informed sampling (uterine, vaginal swabs, aborted tissue, and blood) from animals with varying reproductive statuses. Analytical sensitivity was established using quantified synthetic targets and well-characterized reference strains, while diagnostic sensitivity and specificity were determined against gold-standard culture, serology, and PCR benchmarks. The detection platform demonstrated limits of detection in the low copy-number range for Brucella spp., robust detection of Campylobacter fetus under field conditions, and reliable identification of Neospora caninum DNA in fetal tissues. We optimized sample preparation workflows to balance rapid processing with biosafety requirements, incorporating simplified lysis, filtration, and inhibitor-tolerant CRISPR reagents compatible with non-laboratory environments. A key innovation was the development of a modular microfluidic cartridge that houses pre-aliquoted reagents and enables automated mixing, incubation, and readout, reducing operator-induced variability. Field performance metrics indicated diagnostic accuracy exceeding 95% for Brucella detection, with comparable performance for Campylobacter and Neospora, and minimal cross-reactivity with common bovine pathogens. The assay demonstrated resilience to environmental fluctuations (temperature 20–37°C, humidity variations) and required minimal training, making it suitable for extension services and smallholder contexts. Economic analyses highlighted a favorable cost-per-test and a short turnaround time that supports rapid decision-making, such as culling, vaccination, movement controls, or targeted treatment. We also conducted user-centered evaluations, collecting feedback on usability, interpretation, and workflow integration from veterinarians and farm staff, which guided iterative refinements. Potential limitations include occasional false positives in complex matrices and the need for confirmatory testing in epidemiologically atypical outbreaks. Nonetheless, the CRISPR-based field-deployable diagnostic tool shows strong promise to transform bovine reproductive health management by enabling timely, accurate detection of key pathogens, reducing spontaneous abortion events, improving herd fertility, and mitigating zoonotic transmission. Further work will focus on expanding pathogen panels, integrating data capture with farm management systems, and pursuing regulatory pathways for widespread adoption.

Project Overview

What This Project Is About

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A straightforward, beginner-friendly overview of developing a quick, on-site test to detect key cattle reproductive diseases. The project combines science methods with practical field use to identify Brucella, Campylobacter fetus, and Neospora caninum quickly outside a traditional lab.

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The Problem It Addresses

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Farmers and veterinarians need fast, reliable tests to confirm infections that can harm cow fertility and livestock production. Traditional tests take longer, require lab access, or are expensive. This project aims to fill that gap with a simple, portable testing approach.

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Objectives of the Project

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1. Learn how a point-of-care test works in real farm settings.
2. Investigate how to detect three major pathogens with a rapid assay.
3. Assess the test’s accuracy, speed, and ease of use.
4. Evaluate cost and practicality for field adoption.
5. Prepare guidelines for safe, ethical field testing.

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What You Will Do Step by Step

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1. Review background material on the three pathogens and existing tests.
2. Learn basic CRISPR-based detection concepts in simple terms.
3. Design a small, field-friendly testing workflow (sample, process, readout).
4. Test the workflow with mock samples, then real field samples.
5. Record results and compare with standard lab tests.
6. Analyze accuracy, time to result, and user feedback.
7. Refine the procedure for clarity and reliability.
8. Discuss practical challenges and ethical considerations.

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Expected Outcome

A usable blueprint for a field-ready diagnostic tool that provides quick, reliable results for the three pathogens, with guidance on how to deploy it on farms and interpret results to support timely disease management.