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.
ABSTRACT
Objective To obtain purified ClpP produced by prokaryotic expression system,and to evaluate the protection effect elicited by ClpP in animal protection test.Methods The template DNA was isolated from the culture of TIGR4 Streptococcus pneumoniae.The complete ClpP open reading frame(ORF)was cloned into pET-32a expression vector by gene recombination technology in vitro.After prokaryotic expression,purification and sequence identification,the recombinant ClpP were innoculated into mice,and at the same time a group of mice were inoculated with the antibody to ClpP.We monitored the survival time of the innoculated mice after being challenged intraperitoneally with,TIGR4.Results We obtained high-expressed recombinant antigen protein which was then identified by Western blot,and we have got the recombinant antigen protein with a purity of more than 90%after being purified through Ni2+ affinity chromatography and processed by dialysis.The sunrvival time of mice immunized with ClpP or ClpP antibody were significantly langer than that of the mice received PBS.Conclusion The recombinant ClpP antigen protein can elicit protection to the invasive S.pneumoniae infection in mice,which might make ClpP as a candidate of S.pneumoniae vaccine.