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

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

本文旨在建立一种快速、高效的检测肠道病毒71型(EV71)和柯萨奇病毒A16型(CA16)的方法,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、EV71的型特异性引物,建立以不同引物浓度配比及两阶段退火温度提高检测敏感度和特异度的多重反转录-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5.32pg/ml和0.64pg/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77.4%,其中CA16与EV71的检测阳性率分别为31.8%和35.4%,两者检测阳性比为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。

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 were used at different concentrations in one single reaction tube to develop a multiplex reverse transcriptase-polymerase chain reaction（RT-PCR）which was carried out with two annealing temperatures.After the sensitivity and specificity of the multiplex RT-PCR were evaluated,the technique was applied to detect EV71 and CA16 from clinical specimens collected from pediatric patients with HFMD who visited the Children＇s Hospital Affiliated to Capital Institute of Pediatrics during the period of March to October in 2010.All specimens were also inoculated into the Vero cells for virus isolation,which was used as a gold standard for this study.The minimum detectable concentrations for CA16 and EV71 were 5.32 pg/ml and 0.64 pg/ml,respectively.The specificity of multiplex RT-PCR was 100%.The application of multiplex RT-PCR in 381 clinical samples collected from 371 patients with HFMD showed that the total positive rate was 77.4%,of which the detection positive rates of CA16 and EV71 were 31.8% and 35.4%,respectively（the tested positive ratio of CA16∶EV71 was 1∶1.1）.Comparative analysis with virus isolation demonstrated that the detection accuracy of this method was up to 95.2% for CA16 and 98.6% for EV71.The results indicate that this novel multiplex RT-PCR offers a rapid,sensitive and time-saving method to detect EV71 and CA16 from clinical specimens and can be used for the etiological surveillance of HFMD.Both CA16 and EV71 were still the major pathogens of HFMD in Beijing in 2010.