[Utilization of Uracil-DNA glycosylase for combining reverse transcription and anti-contamination with polymerase chain reaction in hepatitis C virus].
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 0.2au UDG per reaction volume respectively at 37 degrees C and 42 degrees C for 40 min. The anti-contamination condition also could eliminate all detectible dU-DNA, including the highest concentration of amplicon DNA.The 1:10(4) dilution of the HCV RNA sample containing 2.110x 10(5)copies /mL copies of RNA could be detected.


CONCLUSIONS
Our results indicate that this new RT-PCR assay can control the contamination stringently and is sensitive as well.
DU
Shao Cai
SC
Hepatology Institute, Peking University People's Hospital, Beijing 100044, China. dushaocai@sina.com
Zhang
Rui
R
Li
Jun Qiang
JQ
Wei
Lai
L
D016174:Hepacivirus / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D051981:Uracil-DNA Glycosidase
D012367:RNA, Viral; D051981:Uracil-DNA Glycosidase