鹦鹉博尔纳病毒4型SYBR GreenⅠ实时荧光定量PCR检测方法的建立

Establishment of SYBR GreenⅠreal-time qPCR method for the detection of parrot Bornavirustype 4

为建立鹦鹉博尔纳病毒4型SYBR GreenⅠ实时荧光定量PCR检测方法,通过扩增病毒M基因并与pMD19-T载体连接,构建重组质粒作为标准品,根据M基因序列设计特异性定量引物,进行了荧光定量PCR的敏感性、特异性、重复性试验。结果显示,荧光定量PCR比普通PCR检测灵敏度高100倍,可检出最低拷贝数为6.7×10^2 copies,μL的标准品,而普通PCR只能检测到最低拷贝数为6.7×10^4 copiesμL的标准品;用荧光定量PCR方法检测H5N6、H7N9、H9N2 AIV,NDV,IBV时均未见扩增;批内及批间重复变异系数均小于1%;对8只鹦鹉脑组织样品进行检测,荧光定量PCR检出阳性样品6份,普通PCR检出阳性样品4份,检出率两者相差25%。采用本方法对64份临床采集的鹦鹉粪便样本进行检测,结果筛选出阳性样品1份。以上结果表明,本研究建立的SYBR GreenⅠ实时荧光定量PCR方法具有良好的灵敏性、特异性和重复性,可用于鹦鹉博尔纳病毒4型的检测。

In order to establish a SYBR GreenⅠreal-time qP CR detection method for parrot bornavirus type 4,the M gene fragment was cloned into the pM D19-T vector to construct a recombinant plasmid which was used as positive standard,and according to M gene sequence,the quantitative specific primers were designed for sensitivity assay,specificity assay and repetitive assay.The results showed that the detection sensitivity was 100 times higher than the conventional PCR method,in particular to the limitation of real-time qP CR detecting for the positive standard is 6.7×10^2 copies/L,while the conventional PCR only 6.7×10^4 copies/μL;carrying out the specific assay of H5 N6,H7 N9,H9 N2,NDV and IBV shows only positive standard was detected;the variation coefficients of intra-assay and inter-as-say were both less than 1%.Among the 8 samples from parrot,6 samples were positive based on the real-time qP CR,but based on conventional PCR only 4 samples which less 25%positive detection rate.64 field samples were detected using both the real-time qP CR and conventional PCR,1 sample were determined.The above-mentioned results showed the detection method have good sensitivity,specificity and repeatability,anditcan be used for the detection of parrot bornavirus type 4 infection.