Development of a SYBR Green I based duplex real-time PCR for detection of bovine herpesvirus-1 in semen

被引：10
作者：
Pawar, Sachin S. [1 ]
Meshram, Chetan D. [1 ]
Singh, Niraj K. [2 ]
Saini, Mohini [3 ]
Mishra, B. P. [1 ]
Gupta, Praveen K. [1 ]
机构：
[1] Indian Vet Res Inst, Div Vet Biotechnol, Izatnagar 243122, Uttar Pradesh, India
[2] Guru Angad Dev Vet & Anim Sci Univ, Sch Anim Biotechnol, Ludhiana, Punjab, India
[3] Indian Vet Res Inst, Ctr Wildlife, Izatnagar 243122, Uttar Pradesh, India
来源：
JOURNAL OF VIROLOGICAL METHODS | 2014年 / 208卷
关键词：
Bovine herpesvirus-1; Duplex real-time PCR; Bovine semen; SYBR Green I; POLYMERASE-CHAIN-REACTION; RT-PCR; INFECTED BULLS; WILD-TYPE; ASSAY; VIRUS; BHV-1; DIFFERENTIATION; QUANTIFICATION; AMPLIFICATION;
D O I：
10.1016/j.jviromet.2014.07.027
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
Bovine herpesvirus-1 (BoHV-1) is a viral pathogen found in infected bull semen, which is transmitted to inseminated cows by artificial insemination. BoHV-1 infection can cause reproductive disorders leading to significant economic loss to cattle industry. To detect BoHV-1 in semen, in this study, a SYBR Green I based duplex real-time PCR was developed. The assay included primers from BoHV-1 glycoprotein C (gC) and bovine growth hormone (bGH) genes for simultaneous detection in single tube. The result was interpreted by analysing melting temperature (T-m) peaks obtained after melt curve analysis of the amplified products at the end of reaction. The Tm peaks for BoHV-1-gC indicated presence of BoHV-1 while the bGH peak indicated reaction without inhibition. The sensitivity of the assay was to detect ten BoHV-1 genome copies per reaction. The analytical sensitivity was to detect 0.21 TCID50 infectious BoHV1 in spiked semen. The assay was validated with 80 semen samples collected from breeding bulls. The diagnostic sensitivity and specificity of the assay was 100% with OIE recommended TaqMan probe based real-time PCR. (C) 2014 Elsevier B.V. All rights reserved.
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收藏
页码：6 / 10
页数：5
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