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A reverse transcription-multiplex PCR strategy devised for concomitant detection and differentiation of foot and
Jajati Keshari Mohapatra1, Manoranjan Rout1, Saravanan Subramaniam1
1ICAR-National Institute on Foot and Mouth Disease, International Centre for FMD, Arugul, Bhubaneswar 752050, Odisha, India.
Abstract:
Serotype identification occupies the central part of foot and mouth disease (FMD) diagnosis workflow and vaccination decision tree. In this study, a reverse transcription-multiplex PCR (RT-mPCR) strategy wherein three assays with unique combinations of serotype specific primers targeting the VP1 region was developed to differentiate FMD virus serotypes O, A and Asia 1 based on differential size of the PCR amplicons on agarose gel. Their diagnostic performance relative to the mPCR assay in use in India was evaluated on 169 clinical samples and 210 cell culture grown virus isolates. The relative diagnostic sensitivity was found to be 99.69%, 98.78% and 99.08% for primer combinations 1, 2 and 3, respectively. These assays proved their worth by detecting serotype in three FMD suspected specimens that went undiagnosed in the existing mPCR and also by identifying multiple serotypes in the same sample. Their detection limits varied from log10 2 to log10 4 viral RNA dilution and from 100 to 0.1 TCID50 virus depending on the serotype. The validated novel mPCR assays show promise to be included in the routine diagnostic tool-box to augment the efficiency of diagnosis of FMD virus serotypes that display extreme genetic diversity and a tendency of transboundary dispersal.
Insights
A new reverse transcription-multiplex PCR (RT-mPCR) method accurately identifies Foot and Mouth Disease virus (FMDv) serotypes O, A, and Asia 1. This advanced diagnostic tool improves FMDv detection in clinical samples and virus isolates.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Epidemiology
Background:
- Foot and Mouth Disease virus (FMDv) serotype identification is crucial for diagnosis and vaccination strategies.
- Existing diagnostic methods may have limitations in differentiating FMDv serotypes, especially those with high genetic diversity.
Purpose of the Study:
- To develop and validate a novel reverse transcription-multiplex PCR (RT-mPCR) strategy for differentiating FMDv serotypes O, A, and Asia 1.
- To evaluate the diagnostic performance of the new RT-mPCR assays compared to existing methods.
Main Methods:
- Development of three unique primer sets targeting the VP1 region of FMDv for multiplex PCR.
- Evaluation of diagnostic sensitivity and specificity using 169 clinical samples and 210 cell culture-grown virus isolates.
- Determination of detection limits for viral RNA dilution and TCID50.
Main Results:
- The novel RT-mPCR assays demonstrated high relative diagnostic sensitivity (99.69%, 98.78%, 99.08%).
- The assays successfully identified serotypes in samples missed by the existing mPCR and detected multiple serotypes in single samples.
- Detection limits ranged from log10 2 to log10 4 viral RNA dilution and 0.1 to 100 TCID50, varying by serotype.
Conclusions:
- The validated RT-mPCR assays offer a promising tool for efficient and accurate FMDv serotype diagnosis.
- These assays can enhance the diagnostic toolbox for managing FMDv, which exhibits genetic diversity and transboundary dispersal.
