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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.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 BlandAltman 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.
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Objective:To observe the stability and sensitivity of transcription mediated ampliifcation (TMA) system, and to compare it with real-time reverse transcription polymerase chain reaction (RT-PCR) in amplifying serum HCV RNA in HCV infected patients. Methods: TMA system was established by moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase and 2 speciifc primers ifrstly,and then its stability and repeatability were compared at different storage temperatures by the correlation change of HCV RNA amplification curve. The sensitivity difference between TMA and RT-PCR was evaluated by amplifying a group of 10-fold diluted HCV RNA samples which were transcribed in vitro. A total of 101 serums of chronic HCV infected patients were measured by TMA system and RT-PCR to observe the positive rate and their correlation. Linear correlation and linear regression were used to observe the correlation of the two methods. Results:TMA system was successfully established. TMA system was not stable when stored at 20℃ (placed for 24 hours only), but it was stable for 6 days when stored at 4℃ or within 6 months when stored at-20 ℃. Compared with RT-PCR whose reagent was made by Hunan Sansure Biotechology Corporation, TMA system showed 20 positive samples and 11 negative samples in a total of 31 samples. So was the RT-PCR kit of the Sansure Biotechology Corporation, and the concordance rate of the two methods was 100%. Advanced quantitative study of the 20 positive samples found that the two methods had good correlation and consistency (r=0.91,P0.05). Advanced quantitative study of 29 positive samples found that the two methods had good correlation and consistency (r=0.96,P<0.01). Conclusion:The stability and repeatability of TMA system are good within 6 months when stored at-20 ℃ storage temperature. Both TMA and RT-PCR HCV RNA can detect serum HCV RNA well, and the two methods have good correlation and consistency.
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Background:Present scenario of tuberculosis (TB) demands a reliable method for rapid diagnosis of TB. Several newer methodologies have been introduced over last 2 decades. However, clinical evaluation of all these methods is essential before bringing them into routine diagnostic practice. Aim: In the present study, we have evaluated 3 most promising methodologies viz. BACTEC 460 TB system (TB BACTEC), Transcription Mediated Amplification (TMA) and Phage assay for rapid detection of M. tuberculosis complex directly from respiratory and non-respiratory specimens. Material and Methods: Total 139 AFB smear positive and 41 AFB smear negative respiratory and non-respiratory specimens were tested by these methods and results were compared with conventional LJ method. Results and Conclusions: TMA is found to be most rapid and reliable method for detection of M. Tuberculosis complex from respiratory specimens with 93.8% sensitivity. However, for non-respiratory samples, TB BACTEC can be the method of choice. An average detection time for TB BACTEC is found to be 13 days and 15 days, compared to 31 days 35 days by LJ method in cases of smear positive respiratory and non-respiratory specimens respectively. Phage assay is highly specific for detecting M. tuberculosis complex and can be used as a rapid screening method for TB in cases of respiratory specimens collected from patients not receiving anti-TB therapy. As only viable TB bacilli are detected by phage assay, it can be used as a sensitive tool to monitor treatment success.