杂交探针技术结合熔解曲线快速鉴别水痘-带状疱疹病毒野毒株/疫苗株

Application of Fluorophore-labeled Hybridization Probes With Melting Curve Analysis for Rapid Identification of Varicella-zoster Virus Vaccine and Wild-Type Strains

目的利用杂交探针结合熔解曲线分析技术，建立一种快速、可靠的适用于鉴别水痘一带状疱疹病毒（Varicella—zosterVirus，VZV）野毒株与疫苗株的方法。方法采集临床诊断为水痘患者的水疱液并进行病毒分离，抽提阳性分离物和部分阳性水疱液的基因组脱氧核糖核酸（DeoxyribonucleicAcid，DNA）。以荧光素Cy5．5标记检测探针5端，相应的锚定探针在3端用FAM羧基荧光索标记，再从研究对象基因组DNA中用聚合酶链反应（PolymeraseChainReac—tion，PCR）特异引物扩增ORF62基因中301碱基对（basepair，bp）片段，将其与特异探针杂交后，引发荧光共振能量转移，通过罗氏LightCycler实时PCR仪描绘扩增的目标DNA片段的熔解曲线，根据熔解温度（MeltingTemperature，Tm）峰值对待测标本进行鉴别。最后，用限制性片段长度多态性（RestrictionFragmentLengthPolymorphism，RFLP）方法验证杂交探针结合熔解曲线鉴别野毒株／疫苗株的准确性。结果VZVOka疫苗株的扩增产物与检测探针的Tm为52．47℃，而水痘原始临床标本／临床分离株与检测探针的Tm在57．33℃～60．50℃。通过检测DNA扩增产物与VzVORF62探针Tm的差异可以明确鉴别VZV野毒株和疫苗株。利用杂交探针技术结合熔解曲线的方法对水痘原始临床标本及分离株进行野毒株／疫苗株的鉴别结果与传统的RFLP方法进行鉴别的结果完全一致。结论杂交探针技术结合熔解曲线操作简便省时，不易交叉污染；获得的病毒野毒株／疫苗株特征性强，适用于VZV野毒株／疫苗株在临床和流行病学调查中的快速鉴别诊断。

Objective To establish a rapid, reliable meth- od to identify varicella - zoster virus （VZV） vaccine and wild - type strains by using fluorophore - labeled hy- bridization probes with melting curve analysis. Methods Based on the principle of fluorescence resonance energy transfer （FRET） , the differentiation of polymorphic site between VZV vaccine and wild - type strains was ana- lyzed. Vesicle fluid or skin swab of the rash were collected from patients which were clinically diagnosed as varicella and then inoculated in human embryonic tung fibroblast （MRC -5） cell lines. Genomic deoxyribo- nucleic acid （DNA） was extracted from a cell culture positive for VZV. Further evaluation was carried out u- sing DNA samples from original clinical samples. A 301 base pair （bp） fragment of VZV open reading frame （ORF） 62, which included the polymorphic site at position 106 262, was amplified. The detection probe was labeled at the 5 ~nd with CyS. 5 and the anchor probe was labeled at the 3 ~nd with FAM. Polymerase chain re- action （PCR） was performed and melting curves for each sample （ F versus T） were produced by the fluores- cence signal （F） plotted in real time against the temperature （T）. Melting curves were then converted to melting peaks. To evaluate the Fluorescence Resonance Energy Transfer （FRET） method, DNAs from VZV specimens that had analyzed by the new method were genotyped by restriction fragment length polymorphism （RFLP） analysis of ORF 62 amplicons. Results Heteroduplex annealing of ORF 62 DNA from the Oka vac- cine strain with the ORF 62 detection probe occurred at 52.47 ~C. In contrast, annealing of the detection probe with DNA from any of the wild - type strains took place at between 57.33 - 60. 50 ~C. The genotypes de- termined by FRET -based strain discrimination and RFLP methods were in complete concordance. Conclusion The FRET method is a rapid and reliable method to genotype VZV wild/vaccine strains and that is also practical for the processing of large numbers of specimens.