Study Of Gene Mutations And Genotyping Of Hepatitis B Virus By PCR-reverse Dot Blot And PCR-direct DNA Sequencing

Objective:To compare the differences between two methods of PCR-reverse dot blot and PCR-direct DNA sequencing in hepatitis B gene mutations and gebotypes.Methods:102patients samples from chronic hepatitis B were received. Gene mutations and genotypes were detected by PCR-reverse dot blot and PCR-direct DNA sequencing at the same time. The correlations were analyzed between gene mutations and genotype, the index of liver function, HBV DNA level.Result:1. The accuracy of two methods were compared:102patients which had taking nucleoside drugs were detected by PCR-reverse dot blot.79specimens were mutated in102cases, and the mutation rate was77.5%. including48cases of180M and YIDD,60.8%,22of YVDD,27.8%,9of181V,11.4%. HBV genetyping showed that10type B and84type C.102patients were detected by PCR-direct DNA sequencing.84specimens were mutated in102cases, and the mutation rate was82.4%. including50cases of180M and YIDD,59.5%,24of YVDD,28.6%,9of181V,10.7%. HBV genetyping showed that10type B,84type C and1type D. There were no statistical significance between PCR-reverse dot blot and PCR-direct DNA sequencing (P>0.05)2. The sensitivity of two methods were compared:when HBV DNA levels in the serum were less than1000copies/ml, HBV gene mutantions were not detected by PCR-reverse dot blot. But five cases were detected by PCR-direct DNA sequencing. At the same HBV DNA level, PCR-reverse dot blot and PCR-direct DNA sequencing can simultaneously detect the mutations and wild-type strains. There were no statistical significance between PCR-reverse dot blot and PCR-direct DNA sequencing (P>0.05)3.The correlation analysis in clinical datas:There was no significant correlation between HBV genotypes and genotype, liver function (P>0.05). There was significant correlation between HBV genotypes and HBV DNA viral load.(P<0.05)Conclusions:1. The accuracy and sensitivity of PCR-reverse dot blot were high with PCR-direct DNA sequencing.2. PCR-reverse dot blot method is a fast, accurate detetion method of the resistance loci nucleoside drugs.3. There was no significant correlation between HBV genotypes and genotype, liver function, but there was significant correlation between HBV genotypes and HBV DNA viral load.