1型HIV感染多重PCR检测方法的建立及临床应用

目的建立检测人免疫缺陷病毒1型(HIV-1)的多重巢式PCR(nPCR)和多重反转录PCR(RT-PCR)方法。将其与已经上市的NASBA法试剂盒进行检测敏感性比较;探讨血浆RNA水平对PCR检测敏感性的影响;评价该方法用于我国HIV-1感染者诊断的价值。方法针对HIV-1的gag、pol和gp41区设计3套引物,建立检测HIV DNA的多重nPCR和检测HIVRNA的多重RT-PCR方法;分别以建立的PCR方法和NASBA法对119例HIV阳性患者进行检测,比较2者的检测敏感性;将患者分为不同的病毒载量组,比较PCR方法在各组患者中的检测敏感性差异;使用建立的PCR方法对10例可疑急性感染者进行检测;扩增HIV-1膜区C2-C3段,对nPCR检测阳性的43例DNA样本进行亚型鉴定。结果多重nPCR的检测敏感度为97.5%(116/119),多重RT-PCR的敏感度为78.2%(93/119),2者的特异度均为100%(50/50),多重nPCR和多重RT-PCR的阳性预测值分别为97.5%(116/119)和78.2%(93/119),阴性预测值均为100%(50/50),准确性分别为98.2%和84.6%。经比较,多重nPCR和多重RT-PCR的检测敏感度都高于NASBA法。在病毒载量<103copy/mL的患者,nPCR的检测敏感性高于RT-PCR,在病毒载量在(103～104)copy/mL的患者及病毒载量≥104copy/mL的患者,2者的敏感性比较相近,均接近100%;检测的10例可疑急性感染者中,有5例患者的nPCR和RT-PCR检测结果阳性,抗体在随访过程中阳转,证实为HIV急性感染者;检测的43例DNA样本分属于B’亚型(37例),AE亚型(5例)和BC亚型(1例)。结论本课题组建立的PCR检测方法可以对我国主要流行的B’、AE和BC亚型病毒株得到很好的扩增效果;nPCR的检测敏感性受血浆RNA水平的影响相对较小,RT-PCR的检测敏感性受血浆RNA水平的影响相对较大;PCR方法可以用于HIV急性感染者的早期诊断。

Establishment and Clinical Application of a Multiple Nested Polymerase Chain Reaction for Diagnosis of HIV-1 Infection

Objective To establish a multiple nested polymerase chain reaction(nPCR) and RT-PCR diagnosis assay system for human immunodeficiency virus-1(HIV-1) and to compare the sensitivity,specificity and other features of the method with those of a marketed assay,nucleic acid sequence-based amplification(NASBA).Methods The authors established an nPCR for detection of HIV DNA and RT-PCR for HIV RNA detection system with three sets of primers which target gag,pol and gp41 districts in HIV-1 gene.A total of 119 HIV seropositive patients were tested by the PCR methods and NASBA separately,the sensitivity was compared.The sensitivity of PCR detection in patients with different viral load were compared.Ten cases of suspected acute infection patients were tested by using the established PCR methods.The C2-C3 district in HIV-1 membrane was amplified in 43 positive DNA to identify their subtype.Results Sensitivity of nPCR was 97.5%(116/119).Sensitivity of RT-PCR was 78.2%(93/119).Specificities of both assay systems were 100%(50/50).The positive predictive values are 97.5%(116/119) and 78.2%(93/119) respectively,and the negative predictive values of both were 100%(50/50).The accuracies were 98.2% and 84.6%.The nPCR assay systems showed higher sensitivity than NASBA(97.5% vs 73.1%).The sensitivity of nPCR was markedly higher than that of RT-PCT in patients with viral load<10³ copy/mL,but the sensitivities of nPCR and RT-PCT were close to each other(nearly 100%) in patients with viral load 10³～10⁴ copy/mL and ≥10⁴ copy/mL viral load.Five of the 10 cases of suspected acute infection were tested positive by the PCR,and had a seropositive conversion afterwards during the follow up,therefore they were confirmed to have acute HIV infection.The 43 DNA samples belong to subtype B'(37cases),AE subtype(5 cases) and BC subtype(1 case).Conclusion The established PCR detective methods were highly effective in detection of HIV subtypes B',AE and BC,and showed a higher detective sensitivity than NASBA.The plasma RNA level has smaller influence on the sensitivity of nPCR than on RT-PCR.The HIV PCR diagnostic assay can be used for early diagnosis of HIV acute infection.