Duplex PCR for Simultaneous Detection of Brucella and Bovine Herpesvirus – Genome in Clinical Samples

Abstract

A disease-free germplasm is a prerequisite for getting healthy progeny. Many diseases are transmitted through semen either by natural service or artificial insemination causing reproduction failure and economic losses. The intermittent excretion of Bovine Herpesvirus1 (BoHV-1) and Brucella in semen and their transmission through artificial insemination has been recorded. PCR has become an invaluable tool because of speed and simplicity with which specific DNA fragment can be amplified from a background of complex genomes. The primers specific for glycoprotein C genomic region of BoHV-1 (5′-CAACCGAGACGG AAAGCTCC-3′and 5′-GTGCACGTACAGCG GCTCG-3′) and bcsp 31 gene of Brucella (5′-GCTCGGTTGCCAATATCAATGC-3′ and 5′-GGGTAAAGCGTCGCCAG AAG-3′) has been used for simultaneous detection of Brucella and BoHV-1. The primers pairs have amplified a PCR product of ~350 bp specific for glycoprotein C region of BoHV-1; and ~150 bp specific for bcsp31 region of Brucella spp. viz.,. Brucella abortus, Brucella melitensis, Brucella suis. Out of 127 bovine semen samples screened, three semen samples of Jersey bulls have shown a ~150 bp amplicon specific to Brucella sps and none found positive for BoHV-1. Further, the duplex PCR is more sensitive, specific, and rapid as compared to other PCR/serological methods. The cost of the traditional PCR has been reduced to half by simultaneous detection of both the infecting agents. Since it could able to detect Brucella and BoHV-1 genomes in routine agarose gel electrophoresis; it can be an alternative assay tool of Real Time PCR. All these advantages make this duplex PCR a preferable detection methodology for routine screening and molecular epidemiology of brucellosis and IBR.