title: Development of a SYBR Green I based duplex real-time PCR for detection of bovine herpesvirus-1 in semen.
abstract: 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 (Tm) 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 BoHV-1 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.
authors: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, India.
authors: Sachin S
authors: SS
authors: Pawar
authors: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, India.
authors: Chetan D
authors: CD
authors: Meshram
authors: School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India.
authors: Niraj K
authors: NK
authors: Singh
authors: Center for Wildlife, Indian Veterinary Research Institute, Izatnagar, India.
authors: Mohini
authors: M
authors: Saini
authors: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, India.
authors: B P
authors: BP
authors: Mishra
authors: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, India. Electronic address: praveen.indian@gmail.com.
authors: Praveen K
authors: PK
authors: Gupta
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D013006:Growth Hormone / Q000235:genetics; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012661:Semen / Q000821:virology*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D044366:Transition Temperature; D014764:Viral Proteins / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins; C098022:SYBR Green I; D013006:Growth Hormone
keywords: Bovine herpesvirus-1; Bovine semen; Duplex real-time PCR; SYBR Green I