Reverse transcription-PCR to detect Bovine Respiratory Syncytial Virus (BRSV)

摘要

BRSV is an important cause of acute respiratory disease mostly in post weaning calves and feedlot cattle but also in adult cattle. Real-time reverse transcriptase PCR protocols were developed to detect BRSV infection in infected animals. Sensitivity of RT-PCR protocols targeting fusion gene was optimized using different Mastermixes such as Qiagen One Step RT-PCR Kit (Qiagen) for conventional RT-PCR, Superscript probe (Invitrogen) and QuantiTect probe (Qiagen) for real-time RT-PCR with and without internal control. The detection limit of different RT-PCR protocols using serial dilutions from BRSV plasmid and based on different probes was 10 RNA copies/ml. The specificity of real-time RT-PCR was evaluated using different bacterial and viral strains which could be isolated from respiratory infected animals. Real-time RT-PCR in combination with ss-actin and conventional RT-PCR showed detectable Ct-values only with BRSV strain.