Establishment and application of fluorescent quantitative real-time PCR with internal amplification control for the detection of infectious bovine rhinotracheitisv virus

According to the gB gene sequence of bovine herpesvirus 1 (BHV1),specific primers and fluorescent probe were designed to amplify the nucleotide fragment containing the fluorescent quantitative real-time PCR(FQ-PCR) amplify region.Cloned the fragment into pMD18-T vector and got the target template.The internal amplification control (IAC) template of FQ-PCR was achieved by nucleic acid sequenced and amplifying with bridge-building PCR.By optimizing the quantity of the IAC template and the reaction condition,the FQ-PCR detection system with IAC was established.The detective limit was about 10 copies plasmids and 0.01TCID50 BHV1 virus each PCR reaction.Compared with conventional PCR and virus separation method,the FQ-PCR with IAC was 10 to 100 folds more sensitive.This result was almost coincident with FQ-PCR without IAC.The detection results from clinical samples indicated that the FQ-PCR was a rapid,sensitive,specific and reproducible.What is more,the IAC in the real-time PCR system can indicate and proofread false negative results.Using this method,40 bovine semen (8 were identified for positive) and 10 nasal swabs (2 were identified for positive) were detected,3 semen samples showed the present of inhibitor.After a further purification to DNA,all the samples got the expected results.It suggested that this method was useful for the rapid detection of BHV1 and lab quality control.