转录介导的扩增技术与real-time RT-PCR在人类免疫缺陷病毒检测中的应用

发布时间：2018-03-22 15:09

本文选题：转录介导的扩增技术　切入点：人类免疫缺陷病毒　出处：《中南大学学报(医学版)》2017年07期 　论文类型：期刊论文

【摘要】：目的:利用转录介导的扩增技术(transcription mediated amplification,TMA)扩增HIV RNA,探讨TMA技术扩增HIV RNA的敏感性及其与实时荧光定量反转录聚合酶链反应的比较。方法:利用Taq Man探针、特异性引物、鼠白血病反转录酶、T7-RNA聚合酶及PCR底物等建立TMA扩增体系。通过扩增一组10倍梯度稀释的HIV RNA转录标准品,评价TMA体系的灵敏性。收集60例HIV感染患者的血浆,同时采用TMA试剂与Cobas Amplicor HIV-1 Monitor 1.5版试剂进行检测,比较两种方法的阳性检出率,并利用线性回归和Bland-Altman法分析两种技术的相关性和一致性。结果:成功建立了TMA扩增体系,这种技术可以检测低至10 copies/m L的HIV转录标准品。60份HIV感染者血浆样本中,TMA及Cobas均检测到阳性46份,均阴性12份,TMA检测阴性而Cobas检测阳性样本2份,检测一致率为96.7%。两种技术阳性检出率差异无统计学意义(P0.05)。对其中46份TMA检测和Cobas检测均有定量结果的血浆进行线性回归分析,两种技术有非常好的相关性(r=0.997,P0.001)。Bland-Altman分析显示两种检测方法定量Lg差值平均值为0.02,44份(95.7%)样本在95%的一致性界限内。结论:TMA技术具有高灵敏性潜能。TMA与real-time RT-PCR检测血浆中HIV RNA具有非常好的相关性与一致性。
[Abstract]:Objective: to investigate the sensitivity of TMA to HIV RNA amplification and its comparison with real-time fluorescence quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using transcription mediated amplification technique (TMA). Methods: Taq Man probes, specific primers were used to amplify HIV RNAs. Mouse leukemia reverse transcriptase T7-RNA polymerase and PCR substrate were used to establish TMA amplification system. The sensitivity of TMA system was evaluated by amplification of a 10-fold gradient diluted HIV RNA transcriptional standard. The plasma samples of 60 patients with HIV infection were collected. At the same time, TMA reagent and Cobas Amplicor HIV-1 Monitor 1.5 reagent were used to compare the positive rate of the two methods, and the correlation and consistency between the two methods were analyzed by linear regression and Bland-Altman. Results: the TMA amplification system was successfully established. This technique can be used to detect as low as 10 copies/m L of HIV transcription standard. Among 60 plasma samples of HIV infected patients, 46 samples were positive for TMA and Cobas, 12 samples were negative for TMA and 2 samples were positive for Cobas. There was no significant difference in positive detection rate between the two techniques (P 0.05). A linear regression analysis was performed on 46 plasma samples with quantitative results of TMA and Cobas. There was a very good correlation between the two methods. Bland-Altman analysis showed that the average value of quantitative LG difference between the two methods was 0.02n.44 samples were within the 95% consistency limit. Conclusion the ratio TMA technique has high sensitivity potential. TMA and real-time RT-PCR can be used to detect HIV in plasma. RNA has very good correlation and consistency.
【作者单位】：中南大学湘雅医院感染病科病毒性肝炎湖南省重点实验室;传染病诊治国家重点实验室感染病诊治协同创新中心;中南大学基础医学院微生物学系;
【基金】：湖南省科技厅科技计划项目(2013SK3025)~~
【分类号】：R512.91

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