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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

Abstract

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.

Keywords

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

References

  1. Azizzadeh, M., Shooroki, H.F., Kamalabadi, A.S., Stevenson, M.A. (2012). Factors affecting calf mortality in Iranian Holstein dairy herds. Preventive Veterinary Medicine.104: 335-340.
  2. Baxi, M., McRae, D., Baxi, S., Greiser-Wilke, I., Vilcek, S., Amoako, K., Deregt, D., (2006). A one-step multiplex real-time RT- PCR for detection and typing of bovine viral diarrhea viruses. Veterinary Microbiology. 116: 37-44.
  3. Cao, Y. (2018). Establishment and application of BRV and BCV SYBR Green I RT qPCR detection method. Heilongjiang Bayi Agricultural University.
  4. Gomez, D.E., Arroyo, L.G., Poljak, Z., Viel, L., Weese, J.S. (2017). Detection of Bovine Coronavirus in Healthy and Diarrheic Dairy Calves. Journal of Veterinary Internal Medicine. 31: 1884-1891.
  5. Hur, T., Jung, Y.-H., Choe, C.-Y., Cho, Y.-I., Kang, S.-J., Lee, H.-J., Ki, K.-S., Baek, K.-S., Suh, G.-H. (2013). The dairy calf mortality: The causes of calf death during ten years at a large dairy farm in Korea. Korean Journal of Veterinary Research. 53: 103-108.
  6. Jiang, H.H., Hou, P.L., He, H.B., Wang, H.M. (2020). Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay. BMC Veterinary Research. 16: 1-9.
  7. Letellier, C., Kerkhofs, P. (2003). Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus. Journal of Virological Methods.114: 21-27.
  8. Ren, Y.C., Zhu, T., Chu, H.M., Cheng, K.H., Xie, X.L., Zhang, L., Sun, Y.Y., Yang, H.J. (2019). Establishment and applicaton of SYBY Green I quantitative real time PCR for detection of BVDV. Shandong Agricultural Sciences. 51: 106-110.
  9. Ryu, J.-H., Shin, S.-U., Choi, K.-S. (2020). Molecular surveillance of viral pathogens associated with diarrhea in pre-weaned Korean native calves. Tropical Animal Health and Production. 52: 1811-1820.
  10. Zhang, N., Liu, Z., Han, Q., Qiu, J., Chen, J., Zhang, G., Li, Z., Lou, S., Li, N. (2011). Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR. Virology Journal. 8: 374.
  11. Zhang, S.-Q., Tan, B., Li, P., Wang, F.-X., Guo, L., Yang, Y., Sun, N., Zhu, H.-W., Wen, Y.-J., Cheng, S.-P. (2014). Comparison of conventional RT-PCR, reverse-transcription loop- mediated isothermal amplification and SYBR green I- based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches. Journal of Virological Methods. 207: 204-209.
  12. Zhuang, X.T., Bai, J., Zhang, Z.C. (2018). Application and development of new molecular diagnosis technology in animal disease detection. Animal Husbandry and Feed Science. 39: 108-112.
  13. Zhao, G., Hou, P., Huan, Y., He, C., Wang, H., He, H. (2018). Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the mycoplasma bovis. BMC Veterinary Research. 14: 412.

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