高通量人乳头瘤病毒分型基因诊断方法的研究

目的 建立一种可靠、简便经济、快速、高通量的人乳头瘤病毒(HPV)分型检测新方法，并推向临床应用。方法 以各型HPV通用引物行聚合酶链反应(PCR)扩增127例患者的尖锐湿疣组织和宫颈刮片，将荧光偏振检测技术与模板指导的末端延伸反应(TDI—FP)结合，应用探针杂交延伸反应对临床样本扩增产物进行HPV分型：HPV6，11，16，18，31，33，35，58检测，并以DNA序列测定结果为参照。结果 在78例患者的尖锐湿疣组织块中HPV检出率100％，其中两型混合感染14例，占18％，三型混合感染5例，占7％，三型以上混合感染1例。49例患者的宫颈刮片HPV检出率为77％，其中两型混合感染6例，占感染者的17％，三型混合感染3例，占感染者的8％，三型以上混合感染1例。但DNA序列测定方法仅能检出单独一型感染，未检出多重混合感染，两法对单一型检出符合率100％。结论 本研究初步建立了通用、特异、便捷，无需标记探针，可自动化高通量用于临床的人乳头瘤病春分型基因诊断方法。

Development of a high throughout gene diagnosis assay for genotyping human papillomavirus

Objective To developed a simple,inexpensive,quick,accurate and practical method for genotyping of human papillomavirus(HPV) DNA that allowing the mass detection of HPV-DNA. Methods Common conservative primers were used to amplified 127 samples of condylomata acuminatum(CA) and cervical scrapes HPV DNA by PCR, then the PCR product were identified and genotyped by TDI -FP:type-specific probes(HPV6,11,16,18,31,33,35,58 )hybridization and special fluorogenic label ddNTP incorporation. A fluorogenic ddNTP was directly added to the probe under direction of template: the specific PCR products. The results were measured by a fluorometric-polarization microplate reader and compared with the results of DNA sequence. Results Comparing with the results of DNA sequence, the results measured by fluorometric-polarization microplate reader were all correct. The positive rate of HPV was 100% in CA and 77% in cervical scrapes respectively. The proposed method can detect more than one type infection, but DNA sequencing method cannot. There were 14 HPV double infection, 5 HPV triple infection and 1 more than HPV triple infection in CA, 6 HPV double infection, 3 HPV triple infection and 1 more than HPV triple infection in cervical cancer scrapes respectively. Conclusions The proposed method allowed a high throughout, special, simple, rapid, automatism and economical detection of HPV-DNA genotyping without any use of labeling primers or probes. It is expected to be an extremely useful tool in clinical human papillomavirus gene diagnosis.