Development of a novel quantitative real-time assay using self-reporting duplex mutation primers for detection of HCV
10.3760/cma.j.issn.1009-9158.2011.08.014
- VernacularTitle:新型二聚体突变荧光引物定量PCR方法的建立及其在检测HCV中的应用
- Author:
Qianfeng XIA
;
Yangan WEN
;
Jinbo LIU
;
Pu LI
;
Zhiguang TU
- Publication Type:Journal Article
- Keywords:
Hepacivirus;
Polymerase chain reaction;
RNA,viral
- From:
Chinese Journal of Laboratory Medicine
2011;34(8):735-738
- CountryChina
- Language:Chinese
-
Abstract:
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.
