实时荧光定量PCR检测幼儿腹泻腺病毒

Real-Time Quantitative PCR Detecting Infant Diarrhea Associated with Adenovirus Infection

目的:建立实时荧光定量PCR方法,并检测腺病毒Ad40或Ad41感染的粪便标本。方法:用T-A克隆技术构建含腺病毒基因的载体作为标准模板,采用Taqman探针标记技术,建立实时荧光定量PCR方法。采集幼儿腹泻标本248例,分别用直接免疫荧光法和实时荧光定量PCR检测腺病毒Ad40和Ad41,比较两种方法的阳性检出率。结果:实时荧光定量PCR灵敏度和准确度(95.60%和96.80%)均高于直接免疫荧光法(78.5%和82.40%)(P<0.05),而两者特异度差异无统计学意义(97.30%vs 96.70%,P>0.05)。248例待检样本,直接免疫荧光法检出率为2.42%(6/248),实时荧光定量PCR检出率为7.66%(19/248),明显高于直接免疫荧光法(χ2=3.675,P<0.05)。结论:成功建立实时荧光定量PCR检测腺病毒Ad40或Ad41方法。其灵敏度、准确度和阳性检出率经均优于直接免疫荧光法,且简便快速。

Objective:To establish real-time quantitative PCR for detecting the feces samples infected by adenovirus types(Ad40or Ad41).Method:The vector containing adenovirus gene as standard template was constructed with T-A cloning technique.The Taqman probe was used to a real-time quantitative PCR method.248infant diarrhea samples were collected.Ad40or Ad41was determined using direct immune fluorescence methods and real-time quantitative PCR respectively,and their relevance ratios were compared.Results:The comparison experiment revealed that six positive samples of adenovirus DNA molecules were test by direct immune fluorescence methods,and it's relevance ratio was 2.42%(6/248).Besides of above six positive samples,13positive samples of above having been tested as negative amples were detected by real-time quantitative PCR method,and the relevance ratio was 7.66%(19/248)that was higher than that of direct immune fluorescence methods.Conclusion:Succeed in the foundation of real-time quantitative PCR for detecting adenovirus Ad40or Ad41.And it's sensitivity was better than that of the direct immune fluorescence method,and it was feasible and fast.