含内质控的新型检测HIV-1感染的双特异性探针实时荧光RT-PCR方法的建立  

A novel real-time multiplex reverse transcriptase PCR to detect HIV-1 RNA using dual-specific armored RNA as internal control

作　　者：黄杰[1] 李金明[1] 王露楠[1] 邓巍[2] 

机构地区：[1]卫生部北京医院 卫生部临床检验中心免疫室,100730 [2]卫生部北京医院卫 生部临床检验中心免疫室,100730

出　　处：《中华检验医学杂志》2007年第8期929-933,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(30571776;30371365);国家科技部十五科技攻关项目(2004BA719A04);首都医学发展科研基金项目(2002-3041)

摘　　要：目的以包含内标 RNA 的病毒样颗粒作为内质控,建立一种新型的检测 HIV-1感染的包含双特异探针实时荧光 RT-PCR 方法(Dual-specificity probe real-time reverse transcriptase-PCR,DSPrtRT-PCR),提高HIV-1RNA 的检出率。方法采用针对 HIV-1基因组保守区域内两个不同区域的两条不同的荧光探针和两组引物,建立检测 HIV-1RNA 的实时荧光 RT-PCR 方法,并构建了内含HIV-1内标 RNA 的病毒样颗粒,将其作为本方法的内部假阴性质控,考察了所建立方法的检测灵敏度和特异性。比较了单探针和双探针检测方法在 HIV-1亚型检测能力、检测灵敏度和临床样本的检测适用性。结果所建立方法的灵敏度为125 U/ml;在检测 HIV-1阴性样本时具有100%的特异性。和单探针方法比较,双特异探针方法在HIV-1亚型检测中能检测到M族的所有亚型以及 O 族;检测灵敏度和单探针方法比较无统计学意义;在对60份临床样本的检测中,单探针方法只能检出39份,而双探针方法能检出50份(10份检测阴性的样本用 COBAS 检测证明为阴性)。结论本研究建立的 HIV-1 RNA 的 DSPrtRT-PCR 具有很高的检测灵敏度以及 HIV-1多亚型的检测能力。在对临床样本的检测中证明具有很好的临床适用性。Objective To establish a novel dual-specificity probe real-time reverse transcriptase- PCR（DSPrtRT-PCR）using dual-specific armored RNA as internal control for HIV-1 detection for increasing the positive detection rate. Methods We developed a dual-specificity probe real-time reverse transcriptase- PCR（DSPrtRT-PCR） by two different probes and two pairs of primers with dual-specific armored RNA as internal false-negative control for HIV-1 detection. We tested the sensitivity and specificity of the DSPrtRT- PCR detection And compared the detection Capability for the subtypes, sensitivity and clinical applicability. Results The of detection limit was about 125 U/ml. The specificity of DSPrtRT-PCR was 100% for the HIV-negative plasma. Compared with the real-time PCR with mono-speeifieity probe, all HIV- 1 group M and group O could be detected by DSPrtRT-PCR. There was no significant difference between mono-speeifieity probe and DSPrtRT-PCR assays for sensitivity. 50 samples were positive when the DSPrtRT- PCR detection was used and the other 10 samples were confirmed negative by COBAS AmpliScreen on 60 plasma samples, but only 39 samples were positive when the real-time PCR with mono-speeifieity probe was used. Condusious we developed a feasible and specific real-time multiplex RT-PCR to detect HIV-1 RNA. The technique showed high sensitivity and the detection for HIV-1 different groups, showing good clinical application.

关 键 词：HIV-1 核酸探针 聚合酶链反应 

分 类 号：R450[医药卫生—治疗学]