TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection

被引：38

作者：

Bora, D. P. ^{[1
]}

Venkatesan, G. ^{[1
]}

Bhanuprakash, V. ^{[1
,2
]}

Balarnurugan, V.

Prabhu, M. ^{[1
]}

Sankar, M. S. Siva ^{[1
]}

Yogisharadhya, R. ^{[1
]}

机构：

[1] Indian Vet Res Inst Nainital Distt, Pox Virus Lab, Div Virol, Mukteswar 263138, Uttarakhand, India

[2] Project Directorate Anim Dis Monitoring & Surveil, Bangalore 560024, Karnataka, India

来源：

JOURNAL OF VIROLOGICAL METHODS | 2011年 / 178卷 / 1-2期

关键词：

Orf virus; DNA polymerase gene; Real time PCR; TaqMan probe; Diagnosis; CHAIN-REACTION; SEQUENCE-ANALYSIS; VIRUS; GOATS; PARAPOXVIRUSES; DIAGNOSIS; STRATEGY; FUTURE; SHEEP;

D O I：

10.1016/j.jviromet.2011.09.005

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103 bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TagMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5 fg or 15 copies of ORFV genomic DNA. Both intra- (0.38-1.0%) and inter-assay (0.53-2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats. (C) 2011 Elsevier B.V. All rights reserved.

引用

页码：249 / 252

页数：4

共 17 条

[1] Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence [J].