鸡传染性法氏囊病毒实时qRT-PCR检测方法的建立及初步应用

根据高效培养鸡传染性法氏囊病毒（IBDV）时对病毒滴度检测的需要，本文针对BDV的VP4基因的保守序列设计并合成了一对引物，以所构建的重组质粒作为阳性标准品，建立了检测IBDV核酸载量的SYBR Green I荧光定量实时RT-PCR(qRT-PCR)方法，结果表明，其Ct值与标准品模板在4.03×1E1～1E9 拷贝/μL范围内呈良好的线性关系，对IBDV核酸的最低检出量为40拷贝/μL，灵敏度是常规RT-PCR检测方法的1000倍；该方法不与其它禽源病毒发生交叉反应，批内变异系数小于0.05%。应用本方法对DF-1细胞中IBDV的增殖滴度进行了测定，并与经典的TCID50测定方法进行了比较。结果显示两种方法测定的IBDV在微载体悬浮培养和方瓶静态培养条件下DF-1细胞上的增殖曲线都具有一定的平行关系，且qRT-PCR方法比TCID50方法更加快速和敏感，更适合于对IBDV增殖滴度的实时快速测定。

The Development and Application of A SYBR Green Ⅰ Real-Time PCR Assay for Detection of Infectious Bursal Disease Virus

To meet the needs of detection of infectious bursal disease virus(IBDV) under high efficient culture,a SYBR Green Ⅰ real-time RT-PCR(qRT-PCR) was developed using a pair of primers specific to the conserved region of VP4 gene of IBDV and compared with TCID50 method by monitoring the proliferation dynamics of IBDV in DF-1 cell line adherent to micro carrier in tubular reactor.The results showed that the RT-PCRassay was linear in the range of 4.03×101～109copies/μL.The IBDV RNA detection limit was 40 copies/μL,which was 1000 times more sensitive than conventional PCR.No cross-reactions with other viruses was observed.The intra-assay coefficient of variation was less than 0.05%.There was a parallel correlation of IBDV proliferation dynamics in DF-1 cell under Micro carrier suspension and static adherent culture by the qRT-PCR assay and TCID50 method.The detection results of the IBDV samples from tubular and flask culture showed the differences of the micro carrier and adherent culture by both methods.In conclusion,the qRT-PCR assay is more rapid and sensitive than the TCID50 method,which is more appropriate for the real time detection of IBDV.