诺如病毒RT-PCR-反向斑点杂交检测方法的建立

旨在建立诺如病毒RT-PCR-反向斑点杂交检测方法。选取诺如病毒较为保守的RdRp基因作为扩增对象,经RT-PCR扩增后将目的片段克隆到pGEM-T载体中。以重组质粒为模版,选择合成寡核苷酸探针及生物素标记引物。生物素标记引物的扩增产物经热变性后与固定在硝酸纤维素膜上的探针进行杂交反应,经显色后判定结果。出现明显的蓝紫色斑点为诺如病毒阳性,如无斑点则为阴性。对5份临床样品进行检测,并以RT-PCR对比验证。结果显示,利用反向斑点杂交法对重组质粒的检测限为100拷贝/μL,在5例实际样品检测中有1例为阳性,与RT-PCR判定结果一致。建立了诺如病毒的RT-PCR-反向斑点杂交检测方法,该方法特异性好,灵敏度高,操作简便,具有重要的应用价值。

Detection of Norovirus by RT-PCR-Reverse Blot Hybridization

It was to develop a RT-PCR-Reverse blot hybridization method for detecting Norovirus.Oligonucleotide primers targeting the RNA-dependent RNA polymerase conserved RdRp gene were used to amplify viral RNA.The amplicon was cloned into vecter pGEM-T to form the recombinant plasmid,based on which oligonucleotide probes and biotinylated primers were designed.After thermal denaturation,the recombinant plasmid was hybridized with the single-stranded DNA probe.When royal purple blot formed,the detection result was p...