Detection of serum HCV RNA in patients with chronic hepatitis C by transcription mediated amplification and real-time reverse transcription polymerase chain reaction
10.11817/j.issn.1672-7347.2014.07.003
- VernacularTitle:转录介导扩增技术检测慢性丙型肝炎患者血清中HCV RNA及与RT-PCR的比较
- Author:
Daxian WU
;
Fei LIU
;
Hongbo LIU
;
Lizhong DAI
;
Deming TAN
- Publication Type:Journal Article
- Keywords:
transcription mediated amplification;
hepatitis C;
real-time reverse transcription polymerase chain reaction;
hepatitis C virus ribonucleic acid
- From:
Journal of Central South University(Medical Sciences)
2014;(7):664-672
- CountryChina
- Language:Chinese
-
Abstract:
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,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. hTe 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 ℃ storage temperature. Both TMA and RT-PCR HCV RNA can detect serum HCV RNA well, and the two methods have good correlation and consistency.
