Utilization of Uracil-DNA glycosylase for combining reverse transcription and anti-contamination with polymerase chain reaction in hepatitis C virus
- VernacularTitle:尿苷酶在丙型肝炎病毒逆转录-抗污染-联体聚合酶链反应中的应用
- Author:
Shaocai DU
;
Rui ZHANG
;
Junqiang LI
;
Lai WEI
- Publication Type:Journal Article
- Keywords:
Ridine;
Hepacivirus;
Reverse transcriptase polymerase chain reaction
- From:
Journal of Peking University(Health Sciences)
2003;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To develop a hepatitis C virus(HCV) reverse transcription-polymerase chain reaction (RT-PCR) assay using Uracil-DNA glycosylase (UDG) for amplicon contamination control and evaluate the temperature and UDG concentrations for anti-contamination. Methods: In this new HCV RT-PCR assay, reverse transcription, UDG anti-contamination and the first PCR were carried out at the same time. The layer candles were used to prevent the contamination in the second PCR. dU-DNA was used as quality control for UDG anti-contamination and templates to determine the sensitivity of the new HCV RT-PCR assay. HCV cDNA was detected by DNA enzyme immunoassay (DNA-EIA). Results: Complete degradation of amplicon DNA was observed on the conditions of 0.2 u UDG per reaction volume respectively at 37 ℃ and 42 ℃ for 40 min. The anti-contamination condition also could eliminate all detectible dU-DNA, including the highest concentration of amplicon DNA.The 1∶104 dilution of the HCV RNA sample containing 2.110?105copies/mL copies of RNA could be detected. Conclusion: Our results indicate that this new RT-PCR assay can control the contamination stringently and is sensitive as well.
