ABSTRACT
Objective To develop a clinically useful assay for detecting the mutations of HBV pre-C/BCP based on the pyrosequencing and accuracy, reproducibility and reliability of this assay was evaluated. Methods The pyrosequencing primers for HBV pre-C/BCP mutation were designed through the cluster analysis among one hundred HBV gene sequences. After the amplification of the fragment of pre-C/BCP with the template of pre-C/BCP mutation plasmids, the pyrosequencing method for pre-C/BCP detection was initially set up with this standard sample. The accuracy, reliability and reproducibility of the pre-C/BCP pyrosequencing were confirmed through the pre-C/BCP plasmids as a standard sample when compared with Sanger/genechip sequencing method pre-C/BCP pyrosequencing assay was applied for detecting pre-C/BCP mutation types of 60 chnical serum samples in HBV patients. Results The pre-C/BCP mutation detection assay based on pyrosequencing has been established in our study. The coincidence rate between pyrosequencing and Sanger squencing was 100%. The coincidence rate between the result of pyrosequencing and of genechip method was 91.7%. The reproducibility of this assay was 97. 8%. It indicates the pre-C/BCP pyrosequencing is a high-accurate method with, good-reproducibility and high-reliability. And multi-site detection can be achieved by pyrosequencing one time. A rare mutation T1758C was also detected. Conclusion Pyrosequencing for pre-C/BCP mutations assay is high-throughout method for simultaneous detection of multi-site mutation.
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Objective To study the development a clinical useful microarray which can detect 4 most commonly and well documented sites for detection of lamivudine-resistance mutants in HBV lamivudine therapy. Methods (1) The selected 16 oligonucleotide probes to screen 4 mutated sites in YMDD region are immoblized in a specific treatment slice by GMS 417 Arrayer. The target 308 bp nucleic acids segment of HBV were amplificated and labeled with Cy5-dCTP fluorescent dye by PCR. After hybridization of target DNA with microarray, the microchips were scanned with Gene TAC LS IV scanner and the data were obtained after processed in computer. (2) To evaluated the specialty and reproducibility of the microarray, the plasmid with Leu515Met, Met539Ile, Met539Val,V542I mutations were condstructed and serve as positive sample. Another 50 sera of lamivudine treated Hepatitis B patients for 6 to 12 months as well as 50 sera without lamivudine administration were assayed by this microarray to evaluated the rate and genetype in lamivudine related resistance mutation. Results The microarray can identify a single base change of selected lamivudine resistance-related mutation and multiple mutation detection by a single assay as well. The specialty are well in agreement with sequencing for 98%. The reproducibility rate of repeat examination is range from 96%-100%. Conclusion The microarray of lamivuine resistance related mutation can identify a single base change as multiple mutations well. And its hight reproducible results may be useful for clinical monitoring lamivudine resistance related HBV mutation.
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Objective The newly developing gene therapy method and dominant negative mutants were bein g used as new promising HBV therapy method, and a dominant negative mutant of HB V X g ene we have reported in our previous report has some effects both on HBV replica tion and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG 2 2.2.15 cell line in the experiment. To mak e sure the effects of dominant negative mutant of HBx gene, we established a HBx DN stably expressing cell clone, and evaluated the effects of HBx dominant negat ive mutant on HBV gene expression. Methods The prev HBx-GFP dominant mutant and the plasmids pRev Xwt, pRev GFP which contain the wild type X gene or GFP gene then transfected into HepG 2 2.2.15 cells by liposome. The HBsAg, HBeAg by in media were as sayed by RIA and HBV-related RNA were assayed by Northern blot. Results The pRev HBx-GFP, GFP and wild type X constructs can be effectively expressed in HepG 2 2.2.15 cells. The stable expressed HBx -GFP can significantly reduce HBeAg, HBeAg in media and the HBV-related RNA in HepG 2 2.2.15 cells, but not for pRev Xwt and pRev GFP. Conclusions The dominant negative mutant pRev HBx-GFP can significantly inhibit the HBV gen e expression. It also suggested that X gene may be one promising target for HBV gene therapy.
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Objective To discuss the clinical values of 18 F-FDG imaging in screening of the recipients with liver transplantation.Methods By using positron emission tomography, 16 case of hepatocellular carcinoma and 21 case of hepatic cirrhosis with discompensation were subjected to 18 F-FDG imaging. The obtained images were fused with CT images. According to the result of the abnormal 18 F-FDG uptake in 18 F-FDG imaging and image fusion with CT imaging, extrahepatic malignant tumor was judged. The initial routine examinations showed no extrahepatic malignant tumor in these 37 cases , including primary extrahepatic carcinoma and extrahepatic metastasis of liver carcinoma.Results Among the 21 case of hepatic cirrhosis with discompensation, there were 5 cases of extrahepatic primary carcinoma and metastasis. In 16 cases of primary hepatocellular carcinoma, there were 7 cases of extrahepatic metastasis.Conclusion 18 F-FDG positron emission tomography imaging can find the extrahepatic carcinomas which can not be discovered by other examinations, which can provide more information for screening of the recipients undergoing liver transplantation.