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Establishment of Multiplex PCR for Detection of Calf Diarrhea Associated Virus and Analysis of its Clinical Infection Status

Establishment of Multiplex PCR for Detection of Calf Diarrhea Associated Virus and Analysis of its Clinical Infection Status

Liyun Chang*, Yazi Li, Yumei Cai and Chenghui Li

Faculty of Life Science, Tangshan Normal University, Thangshan, Hebei, 064001, China
 
Liyun Chang and Yazi Li contributed equally to this work.
 
* Corresponding author: changliyun2006@163.com

ABSTRACT

Outbreaks of calf infectious diarrhea caused by bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV), and bovine coronavirus (BCV) have increased calves morbidity and mortality in Hebei Province. To detect these three pathogens simultaneously, we designed specific primers based on the conserved gene sequences of the three pathogens available in GenBank. After optimization of the reaction conditions and system, we successfully established a novel multiplex PCR method for detection of the aforementioned three pathogens. The results show that the amplified fragments of interest were 280 bp, 151 bp, and 111 bp for BVDV, BRV, and BCV, respectively. The method had no cross-reaction to Escherichia coli, Salmonella, and infectious bovine rhinotracheitis virus. Moreover, it detected the minimum limit of 1.19 × 103 copies/μL for BVDV, 3.89 × 102 copies/μL for BRV, and 3.74 × 102 copies/μL for BCV, indicating its high specificity and sensitivity. The results of the clinical detection of 150 samples, collected form calves in Hebei Province, by multiplex PCR were the same as those obtained by colloidal gold test paper detection. We discovered that the co-infection rate of BRV and BCV was 41.3% (62/150), of BVDV and BRV 8.0% (12/150), of BVDV and BCV 6.0% (9/150), and of BVDV, BRV, and BCV 10.0% (15/150). In our clinical samples, mixed infection of BRV and BCV was the main pathogen causing calf diarrhea. The developed multiplex PCR assay is a fast, sensitive, and specific, novel detection method for disease diagnosis, clinical monitoring, and treatment of BVDV, BRV, and BCV infections.

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Pakistan Journal of Zoology

June

Pakistan J. Zool., Vol. 56, Iss. 3, pp. 1001-1500

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