鸡包涵体肝炎病毒荧光定量PCR滴度测定方法的建立及与不同滴定方法间的比较

Development of a real-time PCR method for determination of chicken inclusion body hepatitis virus titer and comparative analysis between different methods

本试验根据Gen Bank中I群禽腺病毒(FAV-Ⅰ)代表株AAU46933(CELO株)的Hexon基因序列设计一对通用引物,摸索了合适的扩增条件,建立了SYBR Green I荧光定量PCR方法,并对9株鸡包涵体肝炎毒株的病毒滴度进行了测定,并与OD260法、鸡胚病变法以及细胞病变法测定的结果进行比较。试验结果表明,本方法的检测下限为0.98×102拷贝/μL。以细胞病变法为标准测定方法,经统计学分析发现,鸡胚病变法与细胞病变法之间无统计学差异(P=0.591);荧光定量PCR法与细胞病变法测得结果之间存在线性相关性(相关系数r=0.965,P<0.05);OD260法与细胞病变法测得的结果间无相关性(P>0.05)。以上结果说明,荧光定量PCR法测定结果能够间接反映病毒感染力,与传统方法相比,该方法操作简单、耗时短、成本低,具有较高的应用价值。

To establish a simple and convenient method for determination of chicken inclusion body hepatitis virus,the real-time PCR was developed with a pair of universal primers referencing Hexon gene of the CELO strain,and the amplifying conditionwas groped. Nine chicken inclusion body hepatitis virus strains were titrated by both the established real-time PCR method,andother three determination methods（optical absorbance,mbryonated infectious dose,and cytopathic effect）.Our results showed thatthe detection limit of this method was 0.98×102copies/μL.There was no statistical differences（P=0.591）between embryonated in-fectious dose and cytopathic effect.However,linear relations existed,between the determination results of real-time PCR and cyto-pathic effect（coefficient of association r=0.965,P〈0.05）.These results indicate that the determination using real-time PCR meth-od can reflect the virus infectivity indirectly,has the advantage of simple operation,timesaving,and low cost compared with tradi-tional method,and has a high application value.