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

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (6): 2326-2335.doi: 10.16431/j.cnki.1671-7236.2022.06.033

BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立

麻宝艺², 盛陈艳², 刘宏莹¹, 马青霞², 汪婷婷², 李健明¹, 时坤¹, 杜锐^1,3,4, 冷雪¹

1. 吉林农业大学中药材学院, 长春 130118;
2. 吉林农业大学动物科学技术学院, 长春 130118;
3. 动物生产及产品质量安全教育部重点实验室, 长春 130118;
4. 吉林省梅花鹿高效养殖和产品开发技术工程研究中心, 长春 130118

收稿日期:2021-11-08 出版日期:2022-06-05 发布日期:2022-05-27
通讯作者: 冷雪 E-mail:lengxue_79@163.com
作者简介:麻宝艺,E-mail:mabaoyi1997@163.com。
基金资助:
吉林省科技发展计划项目（20190304004YY、20190301004NY）；国家重点研发计划项目（2018YFC1706600）

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

MA Baoyi², SHENG Chenyan², LIU Hongying¹, MA Qingxia², WANG Tingting², LI Jianming¹, SHI Kun¹, DU Rui^1,3,4, LENG Xue¹

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

Received:2021-11-08 Online:2022-06-05 Published:2022-05-27

摘要/Abstract

摘要： 【目的】建立一种快速检测牛病毒性腹泻病毒1型（BVDV1）和2型（BVDV2）的TaqMan双重实时荧光定量PCR方法。【方法】根据GenBank上收录的95株BVDV1和BVDV2 5'-非编码区设计特异性引物，构建含有BVDV1和BVDV2目的片段的阳性质粒，优化反应条件，构建标准曲线，进行双重实时荧光定量PCR方法的特异性、敏感性和重复性检测，并利用建立的实时荧光定量PCR检测方法对采自吉林省的临床样品进行检测。【结果】建立的双重实时荧光定量PCR方法最适退火温度为57.0 ℃，最适引物浓度为0.5 μmol/L，最适探针浓度为0.3 μmol/L，BVDV1和BVDV2型的标准曲线分别为：Y=－3.54X+37.36（R²=0.990）和Y=－3.18X+35.95（R²=0.997）。对牛传染性鼻气管炎病毒（IBRV）、牛呼吸道合胞体病毒（BRSV）和牛副流感病毒3型（BPIV3）均无特异性扩增，批内和批间变异系数均<3%，检测下限为10拷贝/μL。临床样品检测结果显示，总体阳性率为23.1%（36/156），其中BVDV1阳性率为17.9%（28/156），BVDV2阳性率为5.1%（8/156）。【结论】本研究建立了可同时快速、准确鉴别BVDV1和BVDV2的TaqMan双重实时荧光定量PCR方法，为BVDV的防控与净化提供技术支撑。

关键词: 牛病毒性腹泻病毒(BVDV); TaqMan实时荧光定量PCR方法; 探针

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

中图分类号:

S825.65⁺9.6

麻宝艺, 盛陈艳, 刘宏莹, 马青霞, 汪婷婷, 李健明, 时坤, 杜锐, 冷雪. BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2022, 49(6): 2326-2335.

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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BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立

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

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