To observe the sensitivity of transcription mediated amplification (TMA), and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA). Methods: TMA system was established with TaqMan probes, specific primers, moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase, and reaction substrates. The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro. A total of 60 plasma of HIV infected patients were measured by TMA and Cobas Amplicor HIV-1 Monitor test to observe the positive rate. The correlation and concordance of the above two technologies were investigated by linear regression and Bland-Altman analysis. Results: TMA system was established successfully and HIV RNA transcribed standards at concentration of equal or more than 10 copies/mL could be detected by TMA technology. Among 60 samples of plasma from HIV infected patients, 46 were positively detected and 12 were negatively amplified by both TMA and Cobas reagents; 2 samples were positively tested by Cobas reagent but negatively tested by TMA system. The concordance rate of the two methods was 97.1% and the difference of positive detection rate between the two methods was not statistically significant (P>0.05). Linear regression was used for 46 samples which were positively detected by both TMA and Cobas reagents and showed an excellent correlation between the two reagents (r=0.997, P<0.001). Bland-Altma analysis revealed that the mean different value of HIV RNA levels for denary logarithm was 0.02. Forty-four samples were included in 95% of credibility interval of concordance. Conclusion: TMA system has the potential of high sensitivity. TMA and real-time RT-PCR keep an excellent correlation and consistency in detecting HIV RNA.
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TaqMan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/mL 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(P>0.05)。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，P<0.001)。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。.