简并PCR在肠道病毒血清型鉴定中的应用

The application of consensus degenerate hybrid oliogonucleotide primer PCR in identification of enterovirus serotype.

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。

Objective To study the clinical significance of consensus degenerate hybrid oliogonucleotide primer （CODEHOP） PCR in identification of serotype of enteroviruses. Methods VP1 gene fragments of enterovirus were detected by CODEHOP- PCR in clinical specimens of patients with hand, foot and mouth disease （HFMD） and suspensions of virus isolation, respectively. The PCR products were purified with agarose gel and sequenced for serotyping of enterovirus with comparison with sequences provided by the Genbank. Results The sequence assay after CODEHOP - PCR showed that EVT1 and CA16 traits shared more than 96% of homology with enterovirus reported by domestic studies in recent years. The serotyping results with CODEHOP - PCR demonstrated the accuracy of real - time fluorescent RT - PCR. Conclusion CODEHOP - PCR combined sequencing can be adopted to identify serotypes of enteroviruses, providing a more rapid and accurate method than virus isolation following neutralization test.