Development of visual dye-based loop-mediated isothermal amplification assays for Mycobacterium avium subsp. paratuberculosis detection in ruminants.

Abstract

Importance: Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) in ruminants, is a potential zoogenic pathogen. At the asymptomatic subclinical stage, infected animals periodically shed the pathogen through feces and milk, serving as silent carriers for disease transmission. In Republic of Korea, where treatment for JD is not pursued and culling is the primary management strategy for infected animals, early detection is crucial for effective disease control.

Objective: In this study, we aimed to develop a new primer set for the loop-mediated isothermal amplification (LAMP) method for facilitating visual-based detection of MAP.

Methods: We designed LAMP primers for the MAP-specific gene IS1311 and optimized the reaction conditions to enhance diagnostic sensitivity and specificity. Practical application was evaluated using 170 field samples of feces and intestinal tissues from cattle and goats.

Results: The LAMP method demonstrated sensitivity superior to that of the conventional polymerase chain reaction (PCR) method and equal to that of real-time-PCR targeting the F57 gene, detecting as low as 0.1 pg MAP DNA per reaction. Notably, we integrated a mixed dye composed of calcein and hydroxynaphthol blue at an optimal ratio, enabling clear visual distinction of results under natural light. In practical application, the LAMP assay demonstrated a true positive rate of 100% and true-negative rate of 92.22%.

Conclusions and relevance: The developed mixed dye-LAMP method facilitates immediate, sensitive diagnosis by providing a user-friendly readout. By reducing diagnostic time and enhancing visual clarity without specialized equipment, it significantly improves JD control in the livestock industry.