ISSN: 0973-7510

E-ISSN: 2581-690X

Research Article | Open Access
Fateh Singh , Katherukamem Rajukumar, Dhanapal Senthilkumar, Govindarajulu Venkatesh, Gopal Sarkar, Jaswant Patel, Rohit Sahu, Nourin Khan, Atul Kumar Pateriya, Vijendra Pal Singh and Aniket Sanyal
ICAR-National Institute of High Security Animal Diseases, Bhopal, Madhya Pradesh, India.
Article Number: 10213 | © The Author(s). 2025
J Pure Appl Microbiol. 2025;19(1):699-706. https://doi.org/10.22207/JPAM.19.1.60
Received: 06 January 2025 | Accepted: 23 January 2025 | Published online: 04 March 2025
Issue online: March 2025
Abstract

The study focused on developing an indigenous, one-step reverse transcription (RT)-PCR assay for detecting the porcine transmissible gastroenteritis virus (TGEV) genome. In summary, a gene construct and two sets of primers were designed by aligning N gene sequences from various TGEV strains, which were subsequently synthesized. The gene construct was sub-cloned into the pTZ57R/T vector, enabling the synthesis of in vitro transcribed (IVT) RNA, which served as a TGEV-positive control for RT-PCR protocol optimization. The assay optimization involved systematic testing of various parameters, including primer concentrations, magnesium (Mg++) levels, RNA template quantities, annealing temperatures, and other thermal variables. The analytical sensitivity was evaluated by examining serial 10-fold dilutions of IVT-RNA, both in actual form and when recovered from swine feces after spiking with the same dilutions of IVT-RNA. The developed assay demonstrated analytical sensitivities of 47.548 × 10² and 24.629 × 10³ RNA copies at 10-7 and 10-6 dilutions of IVT-RNA and spiked fecal RNA, respectively. Specificity was confirmed by testing against porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), swine influenza virus (SIV), and known TGEV-negative swine fecal or rectal swab samples (n = 320) collected from the field. The assay exhibited specific amplification for TGEV without cross-reactivity to PEDV, PRRSV, CSFV, SIV, or field samples. This one-step RT-PCR assay proved to be both sensitive and specific for TGEV genomic detection, offering a reliable diagnostic tool for future outbreaks and subsequent monitoring of TGE.

Keywords

Diagnostic Preparedness, Disease Monitoring, Genomic Detection, Pigs, Reverse Transcription-PCR, Transmissible Gastroenteritis, TGE Virus

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© The Author(s) 2025. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.