Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

DOI: 10.18805/IJAR.B-1402    | Article Id: B-1402 | Page : 1141-1149
Citation :- Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV.Indian Journal of Animal Research.2021.(55):1141-1149
Liyun Chang, Zhiyong Liu, Yuelan Zhao, Yan Li, Jianhua Qin qjhqqq@126.com
Address : College of Veterinary Medicine, Hebei Agricultural University, Baoding, Hebei, 071001, China.
Submitted Date : 21-06-2021
Accepted Date : 6-08-2021


Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV).
Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR.
Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×10copies/μL, 3.89×10copies/μL and 3.74×10copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.


Bovine viral diarrhea virus (BVDV) Bovine rotavirus (BRV) Bovine coronavirus (BCV) Multiplex fluorescent quantitative PCR


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