应用实时荧光聚合酶链反应检测婴幼儿腹泻标本中的腺病毒

Detection of adenoviruses in feces of infants with diarrhea by Real-time PCR

目的建立实时荧光聚合酶链反应（Real-time PCR）方法检测腺病毒，并分析浙江省温州地区散发性婴幼儿腹泻患者不同型别腺病毒的感染情况。方法根据GenBank中腺病毒六邻体基因序列设计一对通用引物，建立Real-time PCR方法检测婴幼儿腹泻标本中腺病毒DNA，并与免疫层析法比较；同时对Real-time PCR方法检测阳性的标本进行PCR产物测序和病毒分离培养及酶切鉴定。结果建立快速、特异检测婴幼儿腹泻标本中腺病毒DNA的Real-time PCR技术。157份婴幼儿腹泻标本中免疫层析法检测阳性3份，阳性率为1．91％；Real-time PCR检测阳性5份，阳性率为3．18％（5／157）；154份免疫层析法检测阴性标本中，有2份Real-time PCR法检测为阳性。5份Real-time PCR检测阳性标本经测序鉴定，Ad3型占1．91％（3／157），Ad7型占1．27％（2／157）；经病毒分离培养检出阳性2例，酶切鉴定为Ad3型。结论Real-time PCR技术结合PCR产物直接测序分析具有敏感、特异等优点，适用于腹泻标本中腺病毒的检测及分型。2008年2—4月浙江省温州地区散发性婴幼儿腹泻的腺病毒主要为Ad3型和Ad7型。

Objective A Real-time PCR method was established to study the infection of adenovirus （Ad） in infants with sporadic diarrhea in Wenzhou. Methods According to hexon gene of adenovirus, one prime pair was designed as universal primes and applied to detect adenovirus DNA by Real-time PCR. It was also compared with immunochromatographic assay. 157 fecal specimens from diarrhea infants were tested while positive specimens were sequenced and identified by isolate culture and restriction endonucleases. Results A rapid and specific Real-time PCR assay for detection adenovirus was set up. The positive rates of adenovirus in fecal specimens by immunochromatographic assay and Real-time PCR were 1.91% （3/157） and 3.18% （5/157） , respectively. Out of the 154 specimens with negative result from immunochromatographic assay, 2 showed positive by Real-time PCR. 5 positive specimens, identified by Real-time PCR, were sequenced as Ad3 （3/157, 1.91% ） and Ad7 （2/157, 1.27% ）. 2 of the 5 positive specimens were proved to be Ad3 by cell culture and restriction endonucleases. Conclusion Real-time PCR combined with sequence analysis seemed more sensitive and specific so could be used for identifying types of adenovirus in clinical specimens. Ad3 and Ad7 were important pathogens which caused infant sporadic diarrhea in Wenzhou during February and April in 2008.