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目的建立一种快速、特异的实时荧光定量PCR(FQ-PCR)检测方法,用于高危型人乳头状瘤病毒感染的快速检测。方法根据GenBank公布的人乳头状瘤病毒16的E7基因序列设计一对特异性引物和TaqMan探针,提取组织DNA,优化反应体系与条件,建立基于TaqMan探针的FQ-PCR检测HPV E7基因的方法,对方法的敏感性、特异性和重复性进行评价。结果所设计的引物与探针能特异性地扩增出HPV16 E7基因,熔解曲线峰值单一,对应温度值与预期值相同,产物经琼脂糖凝胶电泳显示PCR产物分子量与预期一致,最低检测下限可达10 fg,且扩增效率达到96.0%,扩增产物测序后经Blast比较具有很高的特异性,设计的反应体系与条件具有稳定的可重复性。结论建立的FQ-PCR方法能特异、敏感地检测高危型人乳头状瘤病毒E7基因,可应用于临床检测及宫颈癌筛查工作。
Objective To establish a rapid and specific real-time fluorescence quantitative PCR (FQ-PCR) detection method for the rapid detection of high-risk human papillomavirus infection. Methods According to the E7 gene sequence of human papillomavirus 16 published in GenBank, a pair of specific primers and TaqMan probe were designed to extract the tissue DNA and optimize the reaction system and conditions. The detection of HPV E7 gene by FQ-PCR based on TaqMan probe Method to assess the sensitivity, specificity and reproducibility of the method. Results The primers and probes were designed to amplify the HPV16 E7 gene. The peak melting curve was single and the corresponding temperature was the same as the expected value. The product was confirmed by agarose gel electrophoresis. The molecular weight of PCR product was as expected. The minimum detection limit Up to 10 fg, and the amplification efficiency reached 96.0%. The amplified product was highly specific by Blast sequencing, and the designed reaction system was stable and reproducible. Conclusion The established FQ-PCR method can detect E7 gene of high-risk human papilloma virus specifically and sensitively and can be used in clinical detection and screening of cervical cancer.