基于定型肠道病毒VP1基因的不同巢式或半巢式PCR评价

目的评价三组基于肠道病毒定型的VP1基因的不同巢式或半巢式PCR方法,建立一种快速、灵敏的分子检测方法用于直接检测临床样品。方法从文献中选出目前常用三组引物,外加一对针对B群肠道病毒改良引物。对代表A群肠道病毒的CVA16毒株和代表B群肠道病毒的CVB5和ECHO20三个毒株分别进行连续10倍稀释至10^-7,对稀释度为10^-3~10^-7的病毒株进行巢式或半巢式PCR。对20份手足口病（HFMD）患儿的粪便样品和16份病毒性脑炎的脑脊液样品进行巢式或半巢式PCR。结果所选三组引物均能检测2.50CCID50/ml CA16病毒;在CVB5和ECHO20检测中,Leitch等设计的引物灵敏度最高,即能够检测含1.12CCID50/ml和1.00CCID50/ml的病毒。在粪便临床样品检测中,Iturriza-Gómara等设计的引物阳性率最高,其中A群肠道病毒检测率为55.0%（11/20）,B群为10.0%（2/20）;而Leitch等设计的引物检测率则为A群为15.0%（3/20）,B群为45.0%（9/20）,其次为Nix等设计的引物检测率为45.0%（其中A群为30.0%,B群为15.0%）,改良的引物为20.0%（4/20）。但脑脊液样品仅有Leitch等设计引物检出,而检出率仅为6.25%（1/16）。结论 Iturriza-Gómara等设计的A群肠道病毒的引物和Leitch等设计的B群的引物组合检测临床样品最佳。

Evaluation of different nested or semi-nested PCR for the detection of VP1 gene of enteroviruses

Objective To evaluate the efficiency of different nested and semi-nested PCR in the detection of VP1 gene of enteroviruses（EV）,and to develop a rapid and sensitive method for the direct identification of human enteroviruses.Methods Three groups of primers commonly used from references and an additional pair of modified primers were included to type EV-B.CVA16（representative of EV-A）,ECHO20,CVB5（representatives of EV-B）samples were prepared at 10^-fold serial dilutions,respectively,from 10^-1 to 10^-7,and the dilutions of 10^-3 to 10^-7 were tested by different PCR primers.Furthermore,20 stool samples of children suffering from hand-foot-mouth disease and 16 CSF samples of viral meningitis were detected by these primers.Results CVA16（EV-A）infected samples could be detected by all 3groups of primers as low as 2.50CCID50/ml.In the detection of ECHO20 and CVB5（EV-B）,primers used by Leitch et al.showed the highest sensitivity,with the detection limit at 1.12 and 1.00,respectively.In the detection of stool samples,primers used by Iturriza-Gómara et al.presented the best efficiency with positive incidence of detecting species A as high as55.0%（11/20）,and 10.0%（2/20）for species B,while the detection rates of primers used by Leitch were15.0%（3/20）of species A,45.0%（9/20）of species B.The detection rates of primers used by Nix et al.were 45.0%（30.0% of species A and 15.0% of species B）,while the detection rate of modified primers was20.0%（4/20）for species B.However,the VP1 gene of enteroviruses in CSF samples could only be detected by primers published by Leitch et al with the detection rate of 6.25%（1/16）.Conclusions The combination of primers for EV-A used by Iturriza-Gómara with that for EV-B used by Leitch may have the ideal performance on typing EVs directly from clinical specimens.