巢式多重聚合酶链反应在腺病毒检测及分型中的应用

Identification and typing for adenovirus by multiplex nest-PCR

目的建立一种快速、敏感、特异的腺病毒检测及分型方法。方法根据编码腺病毒六邻体基因的核苷酸序列设计一对外引物(即通用引物),扩增产物为1278bp;再分别设计针对腺病毒六邻体基因3、7、11和21型特异性序列的4对内引物,扩增产物分别为502、311、880和237 bp。应用多重巢式聚合酶链反应(nest-PCR)技术将这4对引物用于同一聚合酶链反应管中,根据扩增产物在琼脂糖凝胶电泳上的大小,即可鉴别所测毒株的型别。结果3、7、11和21型腺病毒标准株和分离株分别产生预期的特异性扩增片段,与其他常见的呼吸道病毒等无交叉反应。118份经病毒分离和/或间接免疫荧光法确定为腺病毒阳性的急性呼吸道感染患儿临床标本中,腺病毒3型占64.4%(76/ 118),7型占31.4%(37/118),11型占2.5%(3/118),未检测到21型;2份病毒分离和间接免疫荧光均为腺病毒阳性的标本中,用该方法未能鉴定出型别,可能是3、7、11、21型以外的腺病毒,占1.7%(2/ 118)。33份经病毒分离和/或间接免疫荧光法均未检测到腺病毒的标本中,有3份为PCR阳性(包括1份7型,2份3型)。结论该方法鉴别3、7、11、21型腺病毒具有快速、简便、特异等特点,适用于腺病毒型别鉴定,可以为急性呼吸道感染的临床或群体公共卫生事件提供病原学诊断依据。

Objective To develop a rapid,sensitive and specific method in identifying and typing on adenovirus from clinical specimens. Methods Primers were designed using hexon gene of adenovirus as target. One primer pair was designed as universal primers for amplifying a 1278 bp gene fragment located at the hexon gene of adenovirus from all types. Four primer pairs located within the region of this 1278 bp were specifically designed for amplifying types 3, 7, 11 and 21 of adenoviruses, which were used for multiplex nest-PCR in a single tube. The products from this multiplex nest-PCR were 502 bp （for type 3）, 311 bp （for type 7） ,880 bp （for type 11） and 237 bp （for type 21） ,respectively. Type of the adenovirus tested could then be determined after agarose electrophoresis analysis of the PCR products. Results PCR products with predicted sizes were visualized in the agarose gel for prototype strains of adenovirus types 3,7, 11 and 21, but not for other respiratory viruses, indicating that the technique was specific without cross reaction with other viruses. Out of the 118 clinical specimens which had been proved to be adenovirus positive by tissue culture and/or immunofluerescence assay,76 belonged to adenovirus type 3 （76/118,64.4%）,37 to adenovirus type 7 （37/118,31.4%）,3 to adenovirus type 11 （3/118,2.5%） but no adenovirus type 21 was detected. Two of the 118 positive specimens which were positive by both tissue culture and immunofluerescence could not be identified, suggesting that these 2 strains （ 1.7 % ） were with the types other than types 3,7,11 and 21. Out of the 33 specimens which were negative by both tissue culture and immunof[uereseenee,3 showed positive by this multiplex PCR （2 of type 3 and 1 of type 7）, suggesting this method was more sensitive than tissue culture and immunofluerescence. Conclusion This multiplex nest-PCR method had the benefit of rapid,sensitive and specific nature so could be used for identifying types of adenoviruses in the clinical specimens.