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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。
Objective To evaluate three different nested or semi-nested PCR methods based on enterovirus-typed VP1 gene and establish a rapid and sensitive molecular detection method for direct detection of clinical samples. Methods Three commonly used primers were selected from the literature, plus a pair of modified primers for group B enterovirus. The CVA16 strain representing group A enterovirus and the CVB5 and ECHO20 group representing group B enterovirus were respectively serially diluted to 10-7 in succession to 10-7 for virus with dilution of 10-3 to 10-7 Strains were subjected to nested or semi-nested PCR. Nested or semi-nested PCR was performed on fecal samples from 20 children with HFMD and from 16 CSF samples of viral encephalitis. Results The three selected primers could detect 2.50CCID50 / ml CA16 virus. The highest sensitivity of Leitch and other primers was detected in CVB5 and ECHO20 assays, ie, 1.12CCID50 / ml and 1.00CCID50 / ml were detectable. The positive rate of primers designed by Iturriza-Gómara was the highest in clinical stool samples. The detection rate of group A enterovirus was 55.0% (11/20) and that of group B was 10.0% (2/20) The detection rate of primers was 15.0% (3/20) in group A, 45.0% (9/20) in group B, followed by 45.0% for primers designed by Nix (30.0% in group A, group B 15.0%), the modified primer was 20.0% (4/20). However, only samples of cerebrospinal fluid were detected by Leitch and other primers, but the detection rate was only 6.25% (1/16). Conclusion The primers designed by Iturriza-Gómara et al. For group A enterovirus and the primer set designed by Leitch et al.