摘要
目的:建立丙型肝炎病毒(hepatitis C virus,HCV)逆转录-抗污染-联体聚合酶链反应(polymerase chain reaction,PCR),并优化尿苷酶(Uracil-DNA glycosylase,UDG)的抗污染剂量和抗污染时间。方法:逆转录-抗污染-联体PCR技术的第1次PCR实行逆转录-抗污染-PCR同步化;第2次PCR经分层蜡处理进行全程抗污染,并应用该技术扩增HCV cDNA,制备全含脱氧尿嘧啶核苷酸(dUTP)的DNA(dU-DNA)为模板,作为UDG实验室抗污染质控对照并验证该联体PCR检测HCV RNA灵敏度,用DNA-酶免疫测定技术检测HCV cDNA。结果:抗污染实验结果证实,0.2u的UDG在37℃和42℃40min均可达到抗污染的效果。对高浓度全dU-DNA模板也有良好的抗污染效果。灵敏度检验结果表明,用HCV RNA浓度为2.110×105copies/mL的血清,稀释到10-4倍后仍可检测出HCV RNA。结论:应用该逆转录-抗污染-联体PCR技术进行全程抗污染,减少了因程序中多次加样造成的污染,为HCV逆转录PCR(reverse transcription-PCR,RT-PCR)检测RNA提供良好的防污染和抗污染操作技术。
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∶10^4 dilution of the HCV RNA sample containing 2.110×10^5copies/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.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2007年第4期426-428,共3页
Journal of Peking University:Health Sciences
基金
国家"十五"科技攻关项目(2001BA705B06
2004BA718B)资助~~
关键词
尿苷
肝炎病毒
逆转录聚合酶链反应
Ridine
Hepacivirus
Reverse transcriptase polymerase chain reaction
