SYBR Green实时荧光PCR方法检测广谱人乳头瘤病毒的方法建立

Establishment of SYBR Green Real-time Fluorescence PCR Assay covering a broad range of Human Papillomavirus Genotypes

目的建立实时荧光PCR方法以检测广谱人乳头瘤病毒。方法该实时荧光PCR方法利用了degenerate PCR引物与非特异性的SYBR Green染料替代通常使用的PCR特异性引物与探针,并与市售高危13型、低危6型试剂盒以及degener-ate PCR方法进行比较。结果对于用高危13型试剂盒及低危6型试剂盒检测的100例黏膜型人乳头瘤病毒阳性标本,用SYBR Green实时荧光PCR方法检出的阳性数为69例,敏感性为69%,相反,用SYBR Green实时荧光PCR方法检测出的40例皮肤型人乳头瘤病毒阳性标本,用高危13型试剂盒以及低危6型试剂盒检测均为阴性;对于用SYBR Green实时荧光PCR方法及degenerate PCR方法检测的90例黏膜型、60例皮肤型标本,其检测结果显示两种方法有高度一致性(Kappa=0.907)。结论与市售试剂盒相比,SYBR Green实时荧光PCR方法可以覆盖广谱的HPV型别(皮肤型与黏膜型),并且比degenerate PCR方法高效、快速和简便,不失为一个可用于临床与科研的良好检测手段。

Objective To establish a novel Real-time fluorescence PCR assay for simultaneous detection of a broad range of HPV. Genotypes. Methods Instead of universal specific PCR primers and specific probe,degenerate PCR primers and SYBR Green stain were used in novel Real-time fluorescence PCR assay,positive rates were compared with the kits（13 high risk and 6 low-risk） used in clinical diagnosis,meanwhile,detected rate were compared between SYBR Green Real-time Fluorescence PCR and degenerate PCR. Results Detected rate of SYBR Green Real-time fluorescence PCR was 69 percent among the 100 positive case from cervical swab samples detected by the kits, but the kits showed negative results for 40 positive case of cutaneous warts detected by meth ods of SYBR Green Real time Fluorescence PCR. Positive rates of SYBR Green Real time Fluorescence PCR for 90 cervical type and 60 cutaneous type consist with degenerate PCR（Kappa=0. 907）. Conclusion Compared with the kits used in clinical,SYBR Green Real-time Fluorescence PCR covered a wide types of HPV genotypes including cutaneous types and cervical types. It also has high efficiency,quick and simple features than degenerate PCR. However,it is satisfactory for clinical and research.