Establishment of a Duplex TaqMan Real-time PCR Method for Detection of Bovine Viral Diarrhea Virus Types 1 and 2 |
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| Authors: | MA Baoyi SHENG Chenyan LIU Hongying MA Qingxia WANG Tingting LI Jianming SHI Kun DU Rui LENG Xue |
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| Institution: | 1. College of Chinese Medicine Materials, Jilin Agricultural University, Changchun 130118, China;2. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China;3. Key Laboratory of Animal Production and Product Quality Safety of Ministry of Education, Changchun 130118, China;4. Jilin Provincial Engineering Research Center for Efficient Breeding and Product Development of Sika Deer, Changchun 130118, China |
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| Abstract: | 【Objective】 This study was aimed to establish a duplex TaqMan Real-time PCR method for rapid detection of Bovine viral diarrhea virus types 1(BVDV1) and 2(BVDV2).【Method】 Specific primers were designed based on the 5'-non-coding region of 95 strains of BVDV1 and BVDV2 in GenBank.The positive plasmids containing the target fragments of BVDV1 and BVDV2 were constructed.The reaction conditions were optimized, the standard curve was constructed, the specificity, sensitivity and reproducibility of the duplex TaqMan Real-time PCR method were tested, and the established duplex TaqMan Real-time PCR assay was used to detect the clinical samples collected from Jilin province.【Result】 The results showed that the optimal annealing temperature of the duplex TaqMan Real-time PCR was 57.0 ℃, the optimum primer concentration was 0.5 μmol/L, and the optimum probe concentration was 0.3 μmol/L.The standard curves of BVDV1 and BVDV2 were Y=-3.54X+37.36 (R2=0.990) and Y=-3.18X+35.95 (R2=0.997), respectively.There was no specific amplification of Infectious bovine rhinotracheitis virus (IBRV), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus type 3 (BPIV3), with intra- and inter-batch CV less than 3%, and the lower limit of detection was 10 copies/μL.The results of clinical samples showed that the overall positive rate was 23.1% (36/156), of which 17.9% (28/156) were positive for BVDV1 and 5.1% (8/156) were positive for BVDV2.【Conclusion】 In this study, a duplex TaqMan Real-time PCR method was established, which could identify BVDV types 1 and 2 simultaneously, quickly and accurately, and provided technical support for the prevention, control and purification of BVDV. |
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| Keywords: | Bovine viral diarrhea virus (BVDV) TaqMan Real-time PCR method probes |
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