Objective: To develop a new method for the detection of TPMT gene mutations and determine the frequencies of four TPMT alleles, TPMT *1, *3A , *3B and *3C in a healthy Chinese population. Methods: A TDI-FP assay syste...Objective: To develop a new method for the detection of TPMT gene mutations and determine the frequencies of four TPMT alleles, TPMT *1, *3A , *3B and *3C in a healthy Chinese population. Methods: A TDI-FP assay system was set up in out lab. To evaluate this system, 220 healthy individuals were analyzed for the polymorphic sites at positions 460(G→A)and 719(A→G)of the TPMP gene using our new TDI–FP method. Results: Three TPMP*3C(G 460→G 719) heterozygotes were identified, TPMP *3A and TPMP *3B were not found. All mutations were confirmed by conventional DNA sequencing analysis. Conclusion: TDI-FP method has proven to be very efficient as a rapid and accurate approach for TPMP genotyping. TPMP *3C was the only polymorphism identified in this clinical samples we have registered.展开更多
AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients.METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a mod...AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients.METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system.The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and R110 or TAMRA labeled acyclo-terminator was added on the 3' end of TDIprimer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed.RESULTS: The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9%), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDD phenotype (7.1%).CONCLUSION: This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.展开更多
文摘Objective: To develop a new method for the detection of TPMT gene mutations and determine the frequencies of four TPMT alleles, TPMT *1, *3A , *3B and *3C in a healthy Chinese population. Methods: A TDI-FP assay system was set up in out lab. To evaluate this system, 220 healthy individuals were analyzed for the polymorphic sites at positions 460(G→A)and 719(A→G)of the TPMP gene using our new TDI–FP method. Results: Three TPMP*3C(G 460→G 719) heterozygotes were identified, TPMP *3A and TPMP *3B were not found. All mutations were confirmed by conventional DNA sequencing analysis. Conclusion: TDI-FP method has proven to be very efficient as a rapid and accurate approach for TPMP genotyping. TPMP *3C was the only polymorphism identified in this clinical samples we have registered.
文摘AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients.METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system.The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and R110 or TAMRA labeled acyclo-terminator was added on the 3' end of TDIprimer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed.RESULTS: The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9%), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDD phenotype (7.1%).CONCLUSION: This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.
