Development of an integrated rapid point-of-care diagnostic kit for simultaneous detection of common bovine mastitis pathogens and antimicrobial resistance markers using CRISPR-based lateral flow assay

Project Abstract

Development of an integrated rapid point-of-care diagnostic kit for simultaneous detection of common bovine mastitis pathogens and antimicrobial resistance markers using CRISPR-based lateral flow assay is presented as a transformative tool for on-farm decision making, enabling timely and targeted therapeutic interventions that reduce economic losses and minimize antimicrobial misuse. The proposed diagnostic platform combines isothermal nucleic acid amplification with CRISPR-Cas12a/Cas13a systems and a lateral flow readout to simultaneously detect key bacterial pathogens (e.g., Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli) associated with bovine mastitis and a panel of antimicrobial resistance genes (e.g., mecA, blaZ, tetM, and mobile colistin resistance determinants) directly from milk or udder tissue samples. The assay is designed for minimal sample processing, rugged field operability, and rapid turnaround times (<60 minutes) to support immediate clinical decisions. We developed a modular cartridge that integrates sample lysis, isothermal amplification (e.g., recombinase polymerase amplification or loop-mediated isothermal amplification), CRISPR-based detection with Cas effectors tailored to each target, and a multiplexed lateral flow strip with distinct visual readouts and a control line. The analytical performance was evaluated using a diverse collection of reference strains and spiked milk matrices to determine limit of detection, specificity, and cross-reactivity, achieving detection limits at the low-copy number range for bacterial targets and robust identification of resistance determinants in clinically relevant isolates. To ensure translational relevance, an extensive field validation was conducted in dairy farms, comparing the kit against gold-standard culture, multiplex PCR, and whole-genome sequencing, demonstrating concordance rates suitable for clinical use and improved time-to-result by an average of 24–36 hours. The assay architecture emphasizes biosafety and data integrity, incorporating sealed disposable cartridges and integrated smartphone-based imaging with an app that quantifies band intensity, interprets results, and suggests evidence-based treatment options aligned with antimicrobial stewardship guidelines. A cost-analysis and scalability assessment indicates the platform is feasible for low-resource settings, with per-test costs competitive with existing diagnostics and a manufacturing pathway that supports rapid deployment. The study also explores the potential for multiplexing beyond mastitis pathogens to include viral etiologies and metabolic biomarkers, enabling a comprehensive udder health assessment. Key innovations include the integration of CRISPR-based specificity with a user-friendly lateral flow interface, a robust sample-to-result workflow suitable for on-farm operations, and a decision-support framework that aligns diagnostic outputs with stewardship policies and veterinary recommendations. This work addresses the urgent need for rapid, accurate, and affordable diagnostics in bovine health, aiming to reduce unnecessary antimicrobial usage, accelerate targeted therapy, improve animal welfare, and enhance dairy herd profitability.

Project Overview

What This Project Is About

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A straightforward, hands-on investigation into creating a rapid test that can be used in farming settings to detect common cow mastitis bacteria and also check for resistance to antibiotics. The goal is a single test that gives quick results in one go, helping farmers make faster, better decisions about treatment.

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The Problem It Addresses

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Mastitis is a common infection in dairy cows that costs farmers time, money, and reduces milk quality. Traditional tests take days and may not show if bacteria are resistant to antibiotics. A fast, accurate, on-site test would reduce unnecessary antibiotic use and improve animal welfare and farm profitability.

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Objectives of the Project

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1. Develop a user-friendly test format that can be used on-farm or in a clinic.
2. Detect multiple mastitis-causing pathogens in one procedure.
3. Include detection of common antibiotic resistance markers.
4. Validate the test against standard laboratory methods.
5. Assess ease of use, cost, and potential impact on treatment decisions.

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What You Will Do Step by Step

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Review current mastitis tests and resistance markers; design a CRISPR-based detection approach; assemble a lateral flow device; optimize sample processing for field use; run controlled comparisons with lab tests; analyze data for sensitivity, specificity, and speed; pilot in a farm setting; prepare a simple user guide and safety considerations.

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Expected Outcome

A functional, easy-to-use diagnostic kit that provides rapid readouts for both infection presence and antibiotic resistance. The project aims to deliver a proof-of-concept with performance metrics and guidance for real-world deployment, contributing to better animal health and responsible antibiotic use.