| Objective:1. To develop a new method for hepatitis B virus(HBV) genotyping, and detect the HBV genotypes of some patients who are infected with HBV, so that we can investigate the distribution of HBV genotypes in Fujian Province, and discuss the relationship between HBV genotypes with liver fibrosis, HBeAg positive incidence, and HBV DNA level.2. Construct plasmids of YMDD, YVDD, to establish and evaluate the method for detecting HBV YMDD motif mutation quantitatively by amplification refractory mutation system (ARMS).Methods:1. The establishment of method for HBV genotyping by PCR melt curve analysis: We designed type-specific primers according to the analysis of HBV B and C genomes in China reported in document and use HBV B and C plasmids as standard, to establish a new method for detecting HBV genotype with melt curve analysis based on real-time PCR, we could get the HBV genotype by Tm value of PCR product. Evaluate its sensitivity, specificity and repetitiveness.2. The evaluation of method for HBV genotyping by PCR melt curve analysis: Detect HBV genotypes of 51 patients who are infected with HBV by melt curve analysis and kit used for HBV genotyping on market respectively, and compare the coincidence rate of two assays to evaluate the melt curve analysis.3. The clnical application of method for HBV genotyping by PCR melt curve analysis: Detect HBV genotypes of 346 patients who is HBsAg-positive with the melt curve analysis to investigate the distribution of HBV genotypes in Fujian Province, and discuss the relationship between the HBV genotype with liver fibrosis, HBeAg positive incidence, and HBV DNA level.4. The establishment of method for detecting HBV YMDD motif mutation by RT-ARMS-qPCR: YMDD and YVDD plasmids were constructed and used as standard to establish a stand curve. The ARMS reaction system was optimized and evaluated.Results:1. The method for detecting HBV genotypes by melt curve analysis was established successfully. The Tm value of genotype B is 80.79±0.35, and genotype C is 85.75±0.33; we can get genotypes of specimen with HBV DNA≥103copies/ml; The CV values of Tm for high, medium and low concentrations were 0.15%, 0.09% and 0.16% respectively.2. The validity of method for detecting HBV genotype by melt curve analysis was evaluated. Detect HBV genotypes of 51 patients who are infected with HBV by melt curve analysis and kit used for HBV genotyping respectively, all the 14 samples which were tested as genotype B by melt curve analysis are judged as genotype B by kit on market and all the 21 samples of genotype C are judged as genotype C; but in the 16 samples of genotype B/C, 1 sample is judged as genotype B, 2 samples are judged as genotype C, and 13 samples are judged as mixed genotype B/C by kit on market. So the coincidence rates between two assays were 100%for genotype B, 100%for genotype C, 81.25%for genotype B/C, and 94.12%for all the 51 samples(Kappa=0.909,P<0.05)。3. The clinical application of method for HBV genotyping by PCR melt curve analysis.3.1 Distribution of HBV genotypes in Fujian Province: All the 346 samples'HBV genotypes were detected by melt curve analysis. HBV genotype B,C and genotype B/C were detected in 346 serum samples, accounting for 54.91%(n = 190), 23.70%(n = 82), 21.39%(n = 74) of total specimens respectively. It suggests that the main genotype is B in Fujian Province. 3.2 The relationship between HBV genotype with liver fibrosis, HBeAg positive incidence, and HBV DNA level. 346 samples are genotyped by PCR melt curve analysis, all of them are HBsAg-positive, and 91 of them are diagnosised as liver fibrosis, 142 of them are quantitied HBeAg and HBV DNA. The results of 91 samples which are diagnosised as liver fibrosis show that 39 samples are genotype C, account for 42.86%(39/91), it is higher than the proportion(23.7%) of genotype C among the 346 samples; and 38 samples are genotype B, account for 41.76%(38/91), it is lower than the propotion(54.91%) of genotype B among the 346 samples; 142 samples are genotyped by melt curve analysis, all of the HBV DNA≥10~3copies/ml, the results show that the HBV DNA of genotype C is higher significantly than that of genotype B and B/C( P<0.05), the HBeAg positive rate of genotype C(87.18%) is higher significantly than that of genotype B(68.35%) and B/C(41.67%)( P<0.05), so we get the conclution that genotype C is closely ralated with the severity of liver disease.4. The method for quantitation of HBV YMDD by ARMS has been established successfully. Its lower limif of detection is 10~1copies/μl, and the detection linear range is 10~2-10~8copies/μl, the correlation coefficient of standard curve is 0.998986. The CV values for high, medium and low concentrations were 1.06%, 1.12% and 1.71% respectively; The method for quantitation of HBV YVDD by ARMS has been established successfully. Its lower limif of detection is 101copies/μl, and the detection linear range is 101-107copies/μl, the correlation coefficient is 0.998287. The CV values for high, medium and low concentrations were 0.96%, 1.11% and 1.29% respectively.Conclusions:1. The method for genotyping HBV by melt curve analysis was established successfully, it has a good sensitivity, specificity and repetitiveness, and has high coincidence rate with the kit on market. It can be used for detecting HBV genotype B,C,and B/C mixed genotype.2. The main genotype of HBV is B in Fujian province. The genotype of HBV is related with liver fibrosis, HBeAg positive incidence, and HBV DNA level. Genotype C is closely ralated with the severity of liver disease. 3. The method for quantitation of HBV YMDD motif mutation by ARMS has been established successfully. It has a good sensitivity, specificity repetitiveness and wide detection range. |
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