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(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。
Objective： 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, q-he 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 ofHIV 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.
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2017年第7期776-782,共7页
Journal of Central South University ：Medical Science
基金
湖南省科技厅科技计划项目(2013SK3025)~~
关键词
转录介导的扩增技术
人类免疫缺陷病毒
实时荧光定量反转录聚合酶链反应
线性回归
transcription mediated amplification
human immunodeficiency virus
real-time reversetranscription polymerase chain reaction
linear regression
