Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus
Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus
复制标题
DOI：
10.1016/j.jviromet.2003.08.004
复制
发表时间：
2003-12-01
影响因子：
3.1
通讯作者：
Kerkhofs, P
中科院分区：
文献类型：
--
作者：
Letellier, C;Kerkhofs, P
由于比利时牛群中存在牛病毒性腹泻病毒（BVDV）的两种基因型，因此区分它们对于疾病监测非常重要。建立了一种实时荧光定量PCR方法，用于牛病毒性腹泻病毒基因型I和II的检测和分类。设计了一对特异性引物和两个TaqMan探针。FAM和VIC-labeled探针序列相差三个核苷酸，允许区分基因型I和II。基因型I和11实时荧光定量PCR检测灵敏度分别为1000和100拷贝。由于运行间阈值循环值的变异系数小于2.2%，因此获得了高度可重复的数据。通过使用两种基因型的参考菌株和特征性田间分离株评估基因型I和II病毒的正确分类。通过运行后测量FAM和维克相关荧光，对合并血液样本的临床诊断应用进行了评价。与传统的RT-PCR方法的100%的一致性证实，这种新技术可以用于持续感染免疫耐受动物的常规检测。(C)2003 Elsevier B. V.保留所有权利。
Since two genotypes of bovine viral diarrhea viruses (BVDV) occur in Belgian herds, their differentiation is important for disease surveillance. A quantitative real-time PCR assay was developed to detect and classify bovine viral diarrhea viruses in genotype I and II. A pair of primers specific for highly conserved regions of the 5'UTR and two TaqMan probes were designed. The FAM and VIC-labeled probe sequences differed by three nucleotides, allowing the differentiation between genotype I and II. The assay delectability of genotype I and 11 real-time PCR assay was 1000 and 100 copies, respectively. Highly reproducible data were obtained as the coefficients of variation of threshold cycle values in inter-runs were less than 2.2%. The correct classification of genotype I and II viruses was assessed by using reference strains and characterized field isolates of both genotypes. The application to clinical diagnosis was evaluated on pooled blood samples by post run measurement of the FAM- and VIC- associated fluorescence. The 100% agreement with the conventional RT-PCR method confirmed that this new technique could be used for routine detection of persistently infected immunotolerant animals. (C) 2003 Elsevier B.V. All rights reserved.