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Comparative evaluation of various in-house protocols on diagnostic performance for paratuberculosis IS900 PCR
Harshita Gandhi1, Mukta Jain2, Saurabh Gupta3
1Amity Institute of Microbial Technology, Amity University Rajasthan, Kant-Kalwar, NH11C, Delhi- Jaipur, 303 002, Jaipur, Rajasthan, India.
Molecular Biology Reports
|November 13, 2022
Summary
An optimized DNA isolation and PCR protocol effectively detects paratuberculosis (Johne's disease) in ruminant feces. This cost-effective method is suitable for resource-limited settings, revealing a high prevalence of 23% in field samples.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Animal Health Economics
Background:
- Paratuberculosis (Johne's disease) is a significant global ruminant infection causing economic losses.
- Control strategies like 'Test and Cull' require accurate diagnostic methods.
- Human health concerns link paratuberculosis to Crohn's disease, increasing control urgency.
Purpose of the Study:
- To optimize an in-house DNA isolation and PCR protocol for detecting paratuberculosis bacilli in feces.
- To evaluate cost-effective methods for paratuberculosis diagnosis in resource-limited areas.
Main Methods:
- Compared five fecal DNA isolation methods and three PCR master mixes in nine combinations.
- Utilized spiked, reference, and field fecal samples for IS900 PCR analysis.
- Assessed performance based on limit of detection, sensitivity, and specificity against fecal culture.
Main Results:
- A combination of a physical DNA isolation method and a Genei-based PCR master mix with additives (betaine, DMSO, BSA) showed the lowest limit of detection.
- The optimized protocol achieved 83% sensitivity and 100% specificity compared to fecal culture.
- A high prevalence of 23% for paratuberculosis was identified in the tested field samples.
Conclusions:
- The developed protocol offers acceptable sensitivity and is easily adoptable in laboratories with limited resources.
- The high prevalence underscores the need for prompt implementation of paratuberculosis control strategies.
