[1]张贵刚?,王艳杰?,陈君彦,等. 牛病毒性腹泻病毒和牛传染性鼻气管炎病毒双重荧光定量PCR检测方法的建立[J].中国预防兽医学报,2018,(10):922-925.[doi:10.3969/j.issn.1008-0589.201712040]
 ZHANG Gui-gang?,WANG Yan-jie?,CHEN Jun-yan,et al. Establishment of the duplex TaqMan real-time PCR method of bovine viral diarrhea virus and infectious bovine rhinotracheitis virus[J].Chinese journal of preventive veterinary medicine,2018,(10):922-925.[doi:10.3969/j.issn.1008-0589.201712040]
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牛病毒性腹泻病毒和牛传染性鼻气管炎病毒双重荧光定量PCR检测方法的建立

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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年10
页码:
922-925
栏目:
诊断和检测技术
出版日期:
2018-11-15

文章信息/Info

Title:
 

Establishment of the duplex TaqMan real-time PCR method of bovine viral diarrhea virus and infectious bovine rhinotracheitis virus

文章编号:
1008-0589(2018)10-0922-04
作者:
 

张贵刚12?王艳杰12?陈君彦12范秀丽12魏学峰12刘国英12*

 

(1. 金宇保灵生物药品有限公司,内蒙古 呼和浩特 010030;2. 国家工程实验室,内蒙古 呼和浩特 010030)

Author(s):
 

ZHANG Gui-gang12? WANG Yan-jie12? CHEN Jun-yan12 FAN Xiu-li12 WEI Xue-feng12 LIU Guo-ying12*

 

(The Spirit JinyuBiological Pharmaceutical Co., Ltd,Hohhot 010030, China; 2.National Engineering Laboratory, Hohhot 010030,China)

关键词:
牛病毒性腹泻病毒牛传染性鼻气管炎病毒TaqMan荧光定量PCR
Keywords:
 

bovine viral diarrhea virus infectious bovine rhinotracheitis virus TaqMan real-time quantitative PCR

分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.201712040
文献标志码:
A
摘要:
 

为建立牛病毒性腹泻病毒(BVDV)和牛传染性鼻气管炎病毒(IBRV) TaqMan双重荧光定量PCR检测方法,本研究根据GenBank中已登录的BVDV 5’-UTR基因序列和IBRV gB基因序列,设计了2对特异性引物和2条TaqMan探针。结果显示以含有BVDV 5’-UTR基因和IBRV gB基因的重组质粒为模板绘制的标准曲线,其相关系数(R2)均大于 0.990,线性关系较好。特异性试验结果显示,该方法仅对BVDV和IBRV 检测为阳性,而对口蹄疫病毒、牛流行热病毒、牛副流感病毒、牛轮状病毒、牛巴氏杆菌、猪瘟病毒、牛支原体、牛、羊布鲁氏菌检测结果均为阴性,表明其特异性强。该方法的检测下限约为10拷贝/μL,重复性试验结果显示该方法的组内和组间变异系数均小于2 %,利用OIE采纳的BVDV、IBRV荧光定量 PCR 方法与本实验建立的方法分别对47份牛病料样品进行检测,二者对BVDV和IBRV检测的符合率分别可达97.87 %和100 %。研究表明所建立的荧光定量PCR方法具有快速、敏感、准确等优点,可以用于BVDV和IBRV的双重定量检测。

Abstract:
 

The aim of this study was to develop a duplex TaqMan real-time PCR (qPCR) assay for the detection of bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV). The TaqMan qPCR was established with 2 pairs of primers and two specific probes of BVDV and IBRV designed according to the 5’ noncoding region (5’ UTR) of BVDV and gB gene of IBRV. The result showed that the established assay possessed a linear relationship and the correlation coefficients of standard curve were both above 0.990. The assay showed a strong-specific that only amplified the DNA or cDNA from BVDV and IBRV, and had no any amplification of other virus, such as classic swine fever, bovine parainfluenza virus type 3, foot and mouth disease etc. The sensitivity of the real-time PCR method approximated 10copies/μL, and the coefficient of variation of this duplex qPCR was less than 2% for both inter or intra-groups. The coincidence rate of the BVDV, IBRV real-time PCR method adopted by OIE and the BVDV and IBRV duplex TaqMan qPCR established in this study were 97.87% and 100% for detecting 47 bovine clinical samples, respectively. In conclusion, this duplex TaqMan qPCR assay was convenient, strong-specific and high-sensitive, which could conduct a rapid detection of BVDV and IBRV.

参考文献/References:

 

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[6]康晓冬,王建东,马小明,等. 牛病毒性黏膜/腹泻病毒与牛传染性鼻气管炎病毒双重PCR检测方法的建立[J]. 黑龙江畜牧兽医,2014,(15):147-149.

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(本文编辑:李 爽)

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更新日期/Last Update: 2018-11-28