双链置换探针实时荧光定量PCR结合融解曲线特性分析检测高危型人乳头瘤病毒16、58亚型

目的:建立一种准确、快速、实用的检测人乳头瘤病毒(HPV)16、58亚型的方法。方法:根据HPV16和HPV58亚型的序列特异性各设计一对引物和一对型特异性双链置换探针,采用实时荧光PCR扩增后,通过非高分辨的融解曲线分析在同一荧光通道内有效区分两种HPVDNA基因亚型。结果:1ng/μl、0.1ng/μl和0.01ng/μlHPV16和HPV58模板荧光PCR扩增及融解曲线分析显示,融解曲线分析较定量扩增更灵敏、特异。HPV16和HPV58融解曲线对应的Tm值分别为83.5°C和87°C。结论:所建双链置换探针实时荧光定量PCR结合融解曲线分析检测HPV16、58亚型的方法灵敏、特异,有临床应用价值。

Double-stranded displacing probe real-time fluorescence quantitative PCR in combination with dissociation curve analysis to construct a method to detect high-risk human papillomavirus HPV16, 58 subtypes

Objective: To establish an accurate, rapid and practical human papillomavirus (HPV) 16,58 subtype sequence detection method. Methods: A pair of primers and a pair of specific double-stranded displacing probes were designed by the sequence characteristics of HPV 16 and 58 subtypes; after real-time fluorescence PCR amplifies, the two subtypes in the same fluorescence channel were distinguished effectively by analysis of non-high-resolution dissociation curve of the HPV. Results: 1ng/μl, 0.1ng/μl, 0.01ng/μl HPV DNA template fluorescence PCR amplification of dissociation curve analysis showed that dissociation curve analysis was more sensitive and specific than the quantitative amplification analysis. Tms corresponding to HPV 16, 58 dissociation curve were 83.5 ℃ and 87 ℃. Conclusion: The method that human papillomavirus (HPV) 16,58 are detected by double-stranded displacing probe real-time quantitative PCRand analysed by dissociation curve is sensitive and specific .