论文部分内容阅读
目的运用分子生物学方法,对2013年5月河源市1所幼儿园发生的1起由CV-A6型肠道病毒引起的手足口病聚集性疫情进行肠道病毒型别快速鉴定。方法采集该起手足口病聚集性疫情的病例及密切接触者的粪便或咽拭子标本,运用荧光定量PCR方法进行总肠道病毒、EV-A71、CV-A16和CV-A6检测,并结合半巢式PCR方法进行肠道病毒VP1序列扩增,通过对目的条带序列测定及分析进行肠道病毒分型鉴定。结果经荧光定量PCR检测,26例病例35份标本中共有25例34份标本检出总肠道病毒阳性,38名密切接触者38份标本中有11人11份标本检出阳性,肠道病毒阳性率分别为96.15%和28.95%,其中分别有25例病例和7名密切接触者CV-A6阳性,阳性率分别为96.15%和18.42%,全部标本EV-A71和CV-A16检测阴性。7例病例和3名密切接触者的10份标本半巢式PCR扩增阳性,测序结果显示VP1序列均属于CV-A6,且核苷酸同源性为100.0%。结论广东省河源市此次发生于幼儿园的手足口病疫情由肠道病毒CV-A6型引起;荧光定量PCR与半巢式PCR扩增肠道病毒VP1序列相结合的方法有利于对引起手足口病的CV-A6型肠道病毒进行快速及准确鉴定。
Objective To use molecular biology method to rapidly identify enterovirus type in a cluster epidemic situation of hand, foot and mouth disease caused by CV-A6 enterovirus in a kindergarten in Heyuan City in May 2013. Methods The cases of aggregated outbreak of HFMD and the feces or throat swab specimens of close contacts were collected and detected by using the method of real-time fluorescence quantitative PCR for total enterovirus, EV-A71, CV-A16 and CV-A6 The half-nested PCR method was used to amplify the VP1 of enterovirus, and the enterovirus type was identified by sequencing and analysis of the target band. Results The results of real-time fluorescence quantitative PCR showed that in 35 of 26 cases, 34 samples were positive for total enterovirus, 25 of 11 samples of 38 closely-related samples were positive, The positive rates were 96.15% and 28.95% respectively. The positive rates of CV-A6 in 25 cases and 7 close contacts were 96.15% and 18.42% respectively, and all samples were negative for EV-A71 and CV-A16. Semi-nested PCR amplification was positive in 10 samples from 7 cases and 3 close contacts. Sequencing results showed that all the VP1 sequences belonged to CV-A6, and the nucleotide homology was 100.0%. Conclusion Heyuan City, Guangdong Province, the occurrence of hand-foot-mouth disease in kindergarten is caused by the enterovirus CV-A6 type; fluorescence quantitative PCR and semi-nested PCR amplification of VP1 sequence combination of intestinal virus is conducive to causing foot and mouth Sick CV-A6 enterovirus for rapid and accurate identification.