1型HIV感染多重PCR检测方法的建立  

Establishment of multiplex PCR assay for detection of HIV-1

作　　者：代丽丽[1] 陈德喜[1] 石英[1] 魏飞力[1] 绳波[1] 吴亚松[1] 柳雅立[1] 张洪海[1] 梁连春[1] 张彤[1] 吴昊[1] 

机构地区：[1]首都医科大学附属北京佑安医院感染科,100069

出　　处：《中华检验医学杂志》2009年第8期866-872,共7页Chinese Journal of Laboratory Medicine

基　　金：国家重点基础研究发展计划（“973”计划）资助项目（2006CB504201）;北京市科委科技计划重大项目资助课题（D0906003040591）

摘　　要：目的建立检测人免疫缺陷病毒1型（HIV-1）的多重巢式PCR（nPCR）和多重逆转录PCR（RT-PCR）方法。方法针对HIV-1的gag、pol和gp41区设计3套引物，分别建立检测HIV DNA的多重nPCR和检测HIV RNA的多重RT—PCR方法；以建立的PCR方法对150例HIV抗体阳性患者及50例阴性患者进行检测，以ELISA／免疫印迹实验（Westen blot，WB）相结合的抗体检测结果（ELISA／WB）为金标准，计算所建立的PCR方法的检测敏感度、特异度、阳性预测值、阴性预测值和准确性、可重复性；分别用建立的多重nPCR和多重RT-PCR检测73份阳性标本，并与HIV-1核酸序列扩增技术（NASBA法）进行检测敏感度的比较；对43份检测阳性的DNA标本进行亚型鉴定。结果多重nPCR的检测敏感度为98．0％（147／150），多重RT—PCR的敏感度为91．3％（137／150），2种方法的特异度均为100％（50／50），多重nPCR和多重RT—PCR的阳性预测值分别为98．0％（147／150）和91．3％（137／150），阴性预测值均为100％（50／50），准确性分别为98．5％和93．5％。可重复性分别为96．7％（87／90）和93．3％（84／90）；多重nPCR的检测敏感度（97．3％）高于NASBA法（72．6％），差异有统计学意义（X^2=17．34，P〈0．01），多重RT—PCR的检测敏感度（79．5％）与NASBA法相比，差异无统计学意Y-（X。=0．94，P=0．332）；检测的43份HIV DNA阳性标本分别为37份B’亚型、5份AE亚型和1份BC亚型。结论成功地建立了多重nPCR和多重RT—PCR方法，操作简便、价格低廉，具有较高的敏感度和特异度和可重复性，能覆盖我国最主要的流行病毒株。Objective To establish an HIV-1 diagnostic assay using multiplex nested polymerase chain reaction （nPCR） and reverse transcription RT-PCR. Methods Three sets of primers targeting HIV-1 gene of gag, pol and gp41 were designed respectively to establish an nPCR to detect HIV DNA and HIV RNA with RT-PCR. The established multiplex PCR detection system was conducted on 150 HIV seropositive and 50 seronegative patients. The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and reproducibility were calculated with ELISA with results of WB as the golden standard. Seventy-three positive samples were measured by the established multiplex nPCR and RT-PCR methods, and the sensitivity was compared with HIV-1 nucleic acid sequence based amplification （NASBA） method. The subtypes of 43 positive DNA samples was identified. Results The sensitivities of multiplex nPCR and RT-PCR assays were respectively 98.0% （147/150）and 91.3% （137/150）, while the specificities of both methods were 100%. The sensitivity of multiplex nPCR （97.3%） was higher than that of NASBA （72.6%） significantly （X^2 = 17.34, P =0. 01 ）. The sensitivity of multiplex RT-PCR （79. 5% ） compared with that of NASBA （72.6%）, with no significant differences （X^2 = 0.94, P = 0. 332）. Forty- three DNA samples were identified as 37 subtype B＇, 5 AE subtype and 1 BC subtype. Conclusions An HIV-1 multiplex nPCR and RT-PCR diagnostic assay system has been developed. The system is easy to operate, economical, highly sensitive and specific, and reproducible, arid may cover the major circulating strains in China.

关 键 词：HIV感染 HIV-1 聚合酶链反应 逆转录聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]