Development of TaqMan Probe-Based One-Step RT-qPCR Assay Targeting 2B-NSP Coding Region for Diagnosis of Foot-and-Mouth Disease in India.

Autor: Biswal JK; ICAR-Directorate of Foot-and-Mouth Disease, ICFMD, Arugul, Bhubaneswar, Odisha, India. Jitendra.Biswal@icar.gov.in., Ranjan R; ICAR-Directorate of Foot-and-Mouth Disease, ICFMD, Arugul, Bhubaneswar, Odisha, India., Mohapatra JK; ICAR-Directorate of Foot-and-Mouth Disease, ICFMD, Arugul, Bhubaneswar, Odisha, India., Rout M; ICAR-Directorate of Foot-and-Mouth Disease, ICFMD, Arugul, Bhubaneswar, Odisha, India., Joshi HR; Wildlife Conservation Trust, Mumbai, 4000001, India., Singh RP; ICAR-Directorate of Foot-and-Mouth Disease, ICFMD, Arugul, Bhubaneswar, Odisha, India.
Jazyk: angličtina
Zdroj: Current microbiology [Curr Microbiol] 2023 Jun 16; Vol. 80 (8), pp. 245. Date of Electronic Publication: 2023 Jun 16.
DOI: 10.1007/s00284-023-03369-y
Abstrakt: A one-step TaqMan probe-based RT-qPCR assay in the duplex format simultaneously targeting FMD Virus (FMDV) 2B NSP-coding region and 18S rRNA housekeeping gene was developed and evaluated. The duplex RT-qPCR assay specifically detected FMDV genome in both infected cell culture suspensions and a variety of clinical samples such as FMD-affected tongue/feet epithelium, oral/nasal swabs, milk and oro-pharyngeal fluids. The RT-qPCR assay was found to be highly sensitive, since the assay was 10 5 -fold more sensitive than the traditional FMDV detecting antigen-ELISA (Ag-ELISA) and 10 2 -fold better sensitive than both virus isolation and agarose gel-based RT-multiplex PCR. In addition, the assay could detect up to 100 copies of FMDV genome per reaction. In the epithelial samples (n = 582) collected from the FMD-affected animals, the diagnostic sensitivity was 100% (95% CI 99-100%). Similarly, all the FMDV-negative samples (n = 65) tested were confirmed negative by the new RT-qPCR assay, corresponding to 100% diagnostic specificity (95% CI = 94-100%). Further, the duplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.4 to 3.56% for FMDV-2B gene target, and from 2 to 4.12% for 18S rRNA gene target. While analyzing FMDV-infected cell culture suspension, a fairly strong positive correlation (correlation coefficient = 0.85) was observed between 2B-based RT-qPCR and WOAH-approved 5'UTR RT-qPCR assays. Therefore, the one-step RT-qPCR assay developed here with an internal control could be used for rapid, effective, and reliable detection of FMDV in pan-serotypic manner, and has the potential for routine diagnosis of FMDV in high throughput manner.
(© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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