SYBR Green Ⅰ实时荧光PCR检测网状内皮增生症病毒方法的建立

根据禽网状内皮组织增生症病毒(REV)长末端重复序列LTR基因保守区域设计并合成一对引物,建立基于SYBR GreenI模式的实时荧光PCR方法(Real-timePCR)。以常规PCR产物为标准品建立标准曲线,并对方法的特异性、敏感性、可重复性以及再现性、检出率进行评价。结果表明,该方法只从REV阳性样本检出扩增信号,不与其它病原出现交叉反应;熔解温度为86.8±0.5℃,无引物二聚体;扩增产物片段大小为270bp,与预期大小相符,测序结果证实为REV靶序列;在1.01×102～1.01×109拷贝之间呈现良好的线性关系,相关系数为0.999,扩增效率为99.7%,最低可检测101拷贝/反应阳性标准品,比常规PCR敏感103倍;组内变异系数、组间变异系数分别为4.80%～5.72%、0.75%～3.87%;对16例临床疑似病例进行检测,阳性率为81.25%(13/16),随机抽取7份阳性扩增产物进行测序,证实为REV序列。本研究建立的SYBR GreenI实时荧光PCR特异性强、通量高、灵敏度高、检测速度快,为REV的快速检测、分子流行病学调查以及监测REV污染疫苗情况提供新方法。

Development of SYBR Green Ⅰ Real-time PCR for Detection of Avian Reticuloendotheliosis Virus

According to the avian reticuloendotheliosis virus(REV) LTR sequence available in GenBank,a pair of specific primers were designed which target to the conserved region of LTR,and SYBR Green I-based real-time PCR was developed.The standard curve was constructed by using the product of conventional PCR.The specificity,sensitivity,reproducibility and detection rate of the assay were evaluated.The result showed high specificity as only the REV positive samples were amplified,and no cross-reaction was found with other pathogens.Melting peak ap-peared at 86.8±0.5℃ representing the LTR gene products.The PCR product of predicted size(270bp) was observed without primer-dimer in agarose gel electrophoresis and confirmed by sequencing.The standard curve covered a lin-ear range of 1.01×102～1.01×109 copies(R2=0.999),and the amplification efficiency was 99.7%.The detection limit was 101 copies /reaction for positive standard samples,1000-folds more sensitive when compared with the conven-tional PCR using the same primer set.The CVs of intra assay and inter assay were in the range of 4.80%～5.72% and 0.75%～3.87%,respectively.13/16 positive samples were identified from clinical suspected samples using this assay and the PCR products were confirmed to be REV specific sequences.In conclusion,the assay was sensitive,specific,rapid,and could treat big amount of samples,it has provided a new way for rapid diagnosis and molecular epidemiological investigation of REV and for quantity control of vaccines.