简并PCR技术在肠道病毒血清型鉴定中的应用

目的 探讨共有序列简并杂合寡核苷酸引物PCR（CODEHOP-PCR）在肠道病毒血清型鉴定中的临床应用价值。方法 以CODEHOP-PCR检测经实时荧光RT-PCR鉴定为EV71和CA16 手足口病患者的临床样本和病毒分离培养细胞上清的肠道病毒VP1基因片段,经琼脂糖凝胶电泳并回收、测序,与Genbank提供的序列比较,确定肠道病毒的血清型。结果 CODEHOP-PCR后的序列分析表明,所有的EV71毒株和CA16毒株与近几年国内报道的部分毒株同源性在96%以上,其血清分型结果验证了实时荧光RT-PCR分型结果的准确性。结论 CODEHOP-PCR合并测序用于肠道病毒血清型的准确鉴定,与传统的病毒分离及血清中和试验相比,具有更快速、准确的优点。

The application of degenerate PCR to identify serotype of enteroviruses

【Abstract】 Objective To explore the clinical value of identifing serotype of enteroviruses by consensus degenerate hybrid oligonucleotide primer (CODEHOP) PCR. Methods VP1 gene fragments of enteroviruses were detected by CODEHOP-PCR from clinical specimens of hand, foot and mouth disease (HFMD) and their cell suspensions of virus isolation. The PCR products were purified from agarose gel and sequenced. The results were compared with sequences of enteroviruses provided by the Genbank to identify serotypes of enterovirus. Results The sequence assay after CODEHOP-PCR showed that EV71 and CA16 shared more than 96% of homology comparing with enterovirus reported by domestic studies in recent years. The serotyping results by CODEHOP PCR demonstrated the accuracy of real-time fluorescent RT-PCR. Conclusions CODEHOP-PCR and sequencing can be adopted to identify serotypes of enteroviruses. It is a more rapid and accurate identifying method than virus isolation fllowing neutralization test