IBRV TaqMan-MGB实时荧光定量PCR方法在BVDV和IBRV共感染中病毒定量检测的应用

Quantitative determination of bovine viral diarrhea virus and infectious bovine rhinotracheitis virus in coinfection by IBRV Taqman-MGB real-time fluorescent quantitative PCR

目的应用牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)Taq Man-MGB实时荧光定量PCR方法进行牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)和IBRV共感染的病毒定量检测,进一步研究BVDV和IBRV共感染后BVDV对IBRV复制的影响。方法分别用0.2和1.0 MOI的BVDV感染MDBK细胞,待细胞出现50%CPE时,再用0.1 MOI的IBRV共感染,作用24 h后收集样品,采用Taqman-MGB实时荧光定量PCR方法检测IBRV的拷贝数,并与对照组(IBRV单独感染组)进行比较。结果在先感染0.2和1.0 MOI BVDV情况下,IBRV拷贝数分别为3.0×10~6和6.47×10~6拷贝/μL,单独感染IBRV的对照组的拷贝数为1.15×10~7拷贝/μL。结论在体外情况下,BVDV对IBRV的复制影响较小,为研究BVDV和IBRV的共感染机制以及制备BVDV和IBRV感染同一细胞的二联苗奠定了基础。

Objective To quantitatively determine the bovine viral diarrhea virus（BVDV） and infectious bovine rhinotracheitis virus（IBRV） in coinfection by Taq Man-MGB real-time fluorescent quantitative PCR, and investigate the effect of BVDV on replication of IBRV after coinfection. Methods MDBK cells were infected with BVDV at MOIs of 0. 2 and 1. 0 respectively and, after appearance of 50% CPE, infected with IBRV at a MOI of 0. 1. Samples were collected 24 h after coinfection and determined for the copy number of IBRV by Taq Man-MGB real-time fluorescent quantitative PCR,and the result was compared with that of control（infection with IBRV alone）. Results The copy numbers of IBRV in cells previously infected with BVDV at MOIs of 0. 2 and 1. 0 were 3. 0 × 10^6 and 6. 47 × 10^6 copies/μL respectively,while that in cells infected with IBRV was 1. 15 × 10^7 copy/m L. Conclusion BVDV showed little effect on replication of IBRV in vitro, which laid a foundation of study on mechanism of coinfection of BVDV and IBRV and preparation of combined vaccine by infection of the same cells with the two kinds of viruses.