多重PCR技术检测儿童急性呼吸道感染DNA病毒方法的建立与应用

Detection and identification of DNA viruses in the specimens of children with acute respiratory infection by multiplex PCR assay

目的建立多重PCR技术检测儿童急性呼吸道感染DNA病毒的方法,以便及时监测并快速诊断急性病毒性呼吸道感染。方法参照NCBI数据库病毒核酸序列设计多重PCR引物,优化反应条件,并对392份儿童呼吸道标本进行检测,验证多重PCR反应的敏感度及特异性。结果采用多重PCR引物对人博卡病毒（h BOV）、KI多瘤病毒（KI）、腺病毒（Ad V）、WU多瘤病毒（WUPy V）、人细小病毒B19（HPVB19）5种DNA病毒进行扩增,分别获得404、324、248、77、128 bp片段,均无非特异性扩增条带,与设计相符;392份儿童呼吸道标本多重PCR扩增出190阳性标本,阳性率为48.46%（190/392）,6个月~3岁以内婴幼儿阳性率70.00%（112/160）为最高。结论本研究初步建立了应用多重PCR技术快速检测儿童呼吸道标本中DNA病毒敏感性、特异性好的方法,6个月~3岁以内婴幼儿检出率高。

Objective To develop a multiplex PCR assay for detection of DNA viruses in specimens of children with acute res- piratory infection so as to timely monitor and rapidly diagnose acute viral respiratory infection. Methods The primers were designed based on the NCBI sequence genomes analysis of the viruses, and then the reaction conditions were optimized. 392 chil- dren＇s respiratory specimens were subjected to detection of DNA virus to validate the sensitivity and specificity of the multiplex PCR assay. Results Specific segments of 404, 324, 248, 77 and 128bp could be seen in the multiplex PCR products of res- piratory viruses hBOV, KI, AdV, WUPyV and HPVB19, which matched the designs. 190 of the 392 respiratory specimens showed positive results, with the positive rate of 48.46 % （190/392）. The positive rate in children aged between 6 months and three years was the highest （70.00 %, 112/160）. Conclusions A multiplex PCR assay to detect respiratory DNA viruses has preliminarily developed in this study. It is highly sensitive and specific. The detection rate is high in children aged 6 months to three years.