BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2022.06.033

Abstract

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.

Key words: Bovine viral diarrhea virus (BVDV); TaqMan Real-time PCR method; probes

CLC Number:

S825.65⁺9.6

MA Baoyi, SHENG Chenyan, LIU Hongying, MA Qingxia, WANG Tingting, LI Jianming, SHI Kun, DU Rui, LENG Xue. Establishment of a Duplex TaqMan Real-time PCR Method for Detection of Bovine Viral Diarrhea Virus Types 1 and 2[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2326-2335.

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Establishment of a Duplex TaqMan Real-time PCR Method for Detection of Bovine Viral Diarrhea Virus Types 1 and 2

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