肠道病毒71型和柯萨奇病毒A16型多重反转录聚合酶链反应的建立及初步应用

本文旨在建立一种快速、高效的方法检测肠道病毒71型（EV71）和柯萨奇病毒A16型(CA16)的方法，以用于儿童手足口病的病原学监测。通过设计肠道病毒通用引物和CA16与EV71的型特异性引物，建立不同引物浓度配比及两阶段退火温度以提高检测敏感性和特异性的多重反转录聚合酶链反应（RT-PCR）方法，并对首都儿科研究所附属儿童医院2010年3~10月收集的371例手足口病患儿共381份临床标本同时进行病毒分离和核酸检测。结果显示，本研究建立的多重RT-PCR方法对CA16和EV71的最低模板检测浓度分别为5.32 pg/ml和0.64 pg/ml，反应特异度为100%。应用该方法检测381份手足口病临床标本的总阳性率为78.4%，其中CA16与EV71的检测阳性率分别为32.6%和35.8%，二者检测阳性比为1:1.1。以病毒分离为标准，多重RT-PCR对CA16及EV71检测的准确率分别为95.2%和98.6%。因此，本研究新建立的多重RT-PCR方法准确、简便，适用于较大量样本的手足口病病原学监测。2010年引起北京地区儿童手足口病的主要病原为CA16和EV71。

Development and preliminary application of a novel multiplex reverse transcriptase-polymerase chain reaction assay for simultaneous detection for enterovirus 71 and coxsackievirus A16

The current paper aims to develop a rapid,accurate,and efficient technique to simultaneously detect enterovirus 71(EV71)and coxsackievirus A16(CA16)for the etiological investigation of hand,foot and mouth disease(HFMD)in children during epidemic seasons in Beijing.Primers were designed to include one pair of universal enterovirus primers based on a highly conserved region of 5'UTR of enteroviruses and two pairs of type-specific primers based on highly conserved VP1 regions of EV71 and CA16.These primers wer...