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Development of visual dye-based loop-mediated isothermal amplification assays for Mycobacterium avium subsp.
Sudu Hakuruge Madusha Pramud Wimalasena1, Sang Young Seo1, Han Gyu Lee1
1Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea.
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.
Insights
A new visual loop-mediated isothermal amplification (LAMP) method offers sensitive and rapid detection of Mycobacterium avium subsp. paratuberculosis (MAP), aiding Johne's disease control in livestock.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Livestock Disease Control
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (JD) in ruminants, posing a zoonotic risk.
- Asymptomatic animals shed MAP, silently spreading the disease, necessitating early detection for effective control in regions like the Republic of Korea.
Purpose of the Study:
- To develop a novel primer set for loop-mediated isothermal amplification (LAMP) enabling visual detection of MAP.
- To optimize LAMP conditions for enhanced diagnostic sensitivity and specificity for MAP.
Main Methods:
- Designed LAMP primers targeting the MAP-specific IS1311 gene.
- Optimized reaction conditions and incorporated a mixed dye (calcein/hydroxynaphthol blue) for visual readout.
- Validated the assay using 170 field samples (feces, intestinal tissues) from cattle and goats.
Main Results:
- The LAMP assay detected as low as 0.1 pg MAP DNA per reaction, showing superior sensitivity to conventional PCR.
- Visual results were clearly distinguishable under natural light due to the optimized mixed dye.
- The assay achieved a 100% true positive rate and 92.22% true negative rate in practical field sample analysis.
Conclusions:
- The developed mixed dye-LAMP method provides a user-friendly, sensitive, and rapid diagnostic tool for MAP.
- This method significantly enhances Johne's disease control in the livestock industry by reducing diagnostic time and eliminating the need for specialized equipment.
