摘要
目的建立人乳头瘤病毒(Human papillomavirus,HPV)线性探针反向杂交(1ine probe assay,LiPA)基因分型技术,通过基因测序技术对LiPA分型检测的效果进行评价。方法利用基因工具软件比对HPV L1基因,设计HPV型别的基因探针,手工将探针点样于条形尼龙膜上,再进行LiPA杂交分型。LiPA检测的样本结果与HPV基因测序的结果进行比较,以评价LiPA检测的效果。结果74例利用LiPA成功地作出了基因分型,其中单独一型HPV感染为40例,涉及13种不同基因型HPV,最常见的型别为HPV 16,占单独感染的37.5%(15/40);另34例为不同型别HPV混合感染,二、三重感染占主导地位,同时也检测出其它多重感染。6例HPV阳性的标本应用LiPA未能作出基因分型,DNA序列分析显示这6例分别为HPV 83、84、535、9感染,这几种型别不包括在LiPA所标记的探针内。LiPA检测与基因测序结果分型的符合率高达90.9%(20/22),可以检测常见的高危型和导致性传播疾病的HPV型别,结果可通过肉眼直接判读。结论HPV LiPA分型技术具有较高的敏感性和特异性,与基因测序的结果符合率高,具有临床推广使用潜力。
Objective To establish and evaluate line probe assay（LiPA）for HPV detection and typing. Methods Primers for amplifying HPV L1 gene and probes for differentiating HPV genotypes were designed with Prime Primer 5.0. Probes were immobilized as parallel lines on membrane strips. HPV L1 fragments were cloned and transinfected into E coli DH5a. HPV L1 was sequenced to evaluate the HPV LiPA capability. Results 74 HPV positive cases were genotyped by LiPA, of which 40 cases were single HPV infect- ed concerning 13 different HPV types and the commonest type was HPV-16, occurring in 15 （37.5 % ） of all single HPV infected eas- es. The other 34 specimens contained multiple HPV types comprising 16 different HPV types. Although double, triple infections prevailed, quadruple and quintuple infections were also found. PCR products of six HPV positive cases failed to be classified by LiPA. Sequencing analysis revealed that these cases were HPV-53,59,83,84 which was not included in LiPA probes. The agreement rate between LiPA determination and sequencing was 90.9% （20/22）. The results of hybridization can be visually interpreted. Conclusion The LiPA for HPV genotyping is easy to perform. The LiPA was sensitive and specific for HPV typing and had high accordance rate with sequencing results. It will be useful tool to determine HPV early in clinical laboratories.
出处
《中国实验诊断学》
北大核心
2010年第3期389-392,共4页
Chinese Journal of Laboratory Diagnosis
基金
海南省自然科学基金资助项目(30647)
