CAJ | 학술논문

目的开发新型二聚体突变荧光引物技术,建立一种能广泛应用于临床检测HCv的实时PCR方法.方法构建重组质粒pMD18-T-HCV 5′NCR作为标准品,设计二聚体突变荧光引物,优化定量PCR体系,并进行方法学评价.将本法应用于临床确诊的30份HCV阳性患者、30份其他病毒性肝炎患者和30份健康志愿者血清标本的检测,定量结果与商品化TaqMan HCv定量试剂盒的定量结果进行比较.结果建立了利用二聚体突变荧光引物的实时PCR方法,检测的线性范围为20～109IU/ml;批内CV在1.37%～4.59%之间,批间CV在1.58%～4.81%之间;对所有其他病毒性肝炎患者和健康志愿者血清HCV的检测结果均阴性,检测特异性为100%(60/60);对临床确诊的HCV感染患者血清标本全部检出HCV阳性,定量结果与商品化TaqMan HCV定量试剂盒的定量结果具有很好的相关性,相关系数R2=0.9501.结论建立的以二聚体突变荧光引物为平台的HCV实时PCR检测方法,具有快速、价廉、准确、结果可靠等特点,可为HCV感染的诊断、治疗监测和流行病学调查提供较好的技术支持.
목적 개발신형이취체돌변형광인물기술,건립일충능엄범응용우림상검측HCv적실시PCR방법.방법 구건중조질립pMD18-T-HCV 5′NCR작위표준품,설계이취체돌변형광인물,우화정량PCR체계,병진행방법학평개.장본법응용우림상학진적30빈HCV양성환자、30빈기타병독성간염환자화30빈건강지원자혈청표본적검측,정량결과여상품화TaqMan HCv정량시제합적정량결과진행비교.결과 건립료이용이취체돌변형광인물적실시PCR방법,검측적선성범위위20～109IU/ml;비내CV재1.37%～4.59%지간,비간CV재1.58%～4.81%지간;대소유기타병독성간염환자화건강지원자혈청HCV적검측결과균음성,검측특이성위100%(60/60);대림상학진적HCV감염환자혈청표본전부검출HCV양성,정량결과여상품화TaqMan HCV정량시제합적정량결과구유흔호적상관성,상관계수R2=0.9501.결론 건립적이이취체돌변형광인물위평태적HCV실시PCR검측방법,구유쾌속、개렴、준학、결과가고등특점,가위HCV감염적진단、치료감측화류행병학조사제공교호적기술지지.
Objective To establish a novel real-time PCR method to detect HCV RNA using Selfreporting duplex mutation primers.Methods The recombinant vector pMD18-T-HCV 5′-NCR was used as the calibrator.The Self-reporting duplex mutation primers were designed according to the gene sequence.And then the PCR reaction system was optimized and evaluated.The specificity,sensitivity and reproducibility of real-time PCR were estimated,The serum specimens from 90 cases(30 cases of HCV,30 cases of other viral hepatitis and 30 healthy volunteers) were tested with this real-time PCR; Results were compared with those obtained using a commercial TaqMan kit.Results The assay was established.It showed linearity over a wide range from 20 - 109 IU/ml.Intra-experimental coefficients of variation(CVs) were 1.37% -4.59%,and inter-experimental CVs were 1.58% -4.81%,respectively.There was no significant difference of HCV genome number tested by the two methods(R2 = 0.95) in 30 hepatitis C patients; HCV DNA was not detected in any serum samples of 30 healthy volunteers by the two methods.The specificity was 100%(60/60).All the samples in patients with clinically confirmed HCV infections showed HCV RNA positive.There wass good correlation between the quantitaive results and results obtained using the commercial TaqMan kit.Conclusions It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for HCV.The establishment of this assay provided alternative technology for clinical diagnosis or therapeutic drug monitoring in the field of HCV infection and epidemiologic survey.