聚合酶链反应及微板杂交法对人乳头瘤病毒分型检测  

Detecting and Typing Human Papillomaviruses By PCR Enzyme-immunoassay

作　　者：魏军[1] 李国富[2] 王波[3] 吕怀盛[2] 

机构地区：[1]宁夏医学院附属医院中心实验室,银川750004 [2]宁夏医学院附属医院病理科,银川750004 [3]宁夏医学院附属医院检验科,银川750004

出　　处：《宁夏医学院学报》2000年第2期90-92,共3页Journal of Ningxia Medical College

摘　　要：目的 :为适应临床人乳头瘤病毒分型检测 ,建立了通用引物扩增 (GP -PCR)及微孔板杂交 -ELISA检测法。方法 :先进行GP -PCR ,其中一条引物 5’端标记有地高辛 ,探针标记有生物素 ,通过包被在微孔板上的亲和素预先固定 ;将扩增产物加入预先包被有HPV型特异寡核苷酸探针和HPV基因组混合探针的聚苯乙烯微孔板中 ,42℃杂交仅需 2 0～30min ;最后 ,用酶底物与所标记的酶进行显色反应 ,通过测定吸光度来判断结果。结果 :本法HPV各型之间无交叉杂交 ;杂交灵敏度比PCR -琼脂糖凝胶电泳检测高 10 0倍 ,最佳杂交时间为 2 0～ 30min ,重复性好 ,CV为 8 75 % ;6 0例子宫颈癌标本的总检出率为 92 5 % ,2 0例正常宫颈石蜡标本本法测定均为阴性。结论 :该法特异性强 ,灵敏度高 ,操作简单 ,无放射性和E .B .污染 ,杂交时间短 ,检测成本低 ,便于临床常规应用。Objective: To develop a novel easy method of detecting and typing human papillomaviruses in clinical laboratory.Methods:This procedure involved two steps: first, general primer mediated PCR(GP-PCR) was applied successfully to detect a broad spectrum of HPV in specimens diagnosed pathologically as squamous cell carcinoma of the uterine cervix And second, in order to facilitate HPV typing, a colorimetric microplate base hybridization assay was developed. One of the primer 5' ends was labeled by digoxin and all probes were labeled by biotin. The microplate was previously covered with avidin which can bind a biotinylated HPV-probe, fix with the biotinylated HPV genomic probes mix and type specific probes respectively. Hybridization was completed in a short time and the hybridized signal was detected by ELISA-like assay. Results: This assay was a high specific, no cross-reaction was noted within all types of HPVs. The sensitivity was one hundred times higher than that of agrose-electrophic technigue. The time to hybrid was very short and it just needed 20 to 30 minutes only. The repetitiveness very well and the variability was 8.75%.Conclusion: The method seems to be of the potential for rapid, specific and sensitive HPVs examination of clinical specimens.

关 键 词：人乳头瘤病毒 ELISA 聚合酶链反应 微板杂交法 

分 类 号：R446.5[医药卫生—诊断学]