[Detection of serum HCV RNA in patients with chronic hepatitis C by transcription mediated amplification and real-time reverse transcription polymerase chain reaction]

Abstract

Objective: To observe the stability and sensitivity of transcription mediated amplification (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 specific primers firstly, 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 °C (placed for 24 hours only), but it was stable for 6 days when stored at 4°C or within 6 months when stored at -20 °C . 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, P<0.01). Compared with the results of RT-PCR whose reagent was made by Shanghai Kehua Bio-engineering Corporation, TMA system had 34 positive samples and 36 negative samples, while the RT-PCR technology had 32 positive samples and 38 negative samples out of 70 samples. The concordance rate of the two methods was 97.1%, with no statistical difference in the positive rate of the two methods (P>0.05). 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 °C storage temperature. Both TMA and RT-PCR HCV RNA can detect serum HCV RNA well, and the two methods have good correlation and consistency.