Research Of PCR-reverse Dot Blot Hybridization Technique For Detection Of Hepatitis B Virus Genotypes And Drug-resistance Mutation

ObjectiveAgainst hepatitis B virus(HBV) genotypes(B type、C type、D type), and four kinds of nucleotides drug-resistance mutations (Wild:rt180L, rt204M, rt207M, rt207V, rt181A, rt236N;Mutant:rt180M, rt204V, rt204I, rt207I, rt181V, rt236T)wer w detected. To investigate distribution of the HBV parient’s genotypes, HBV resistance mutations, HBV patients or carriers of drug resistance, HBV genotype relationship with HBV DNA level, and the method performance of PCR-reverse dot blot hybridization technique of detection of Hepatitis B virus genotypes and drug-resistance mutation.MethodsUsing PCR-reverse dot blot hybridization technique detect424cases of October2012-September2013in our hospital outpatient and inpatient acceptance nucleos (t) ide analogues treatment HBsAg-postive patients or Carriers of Hepatitis B virus genotypes and drug-resistance mutation, and randomly selected50cases by PCR-reverse dot blot hybridization for DNA sequencing of positive samples to assess the PCR-reverse dot blot hybridization HBV genotypes and drug-resistant mutations methodology performance. Data were statistically analyzed using SPSS19.0software. ResultsIn424cases of hepatitis B patients or carriers, genotypes detected364cases,60cases were not detected genotype, of which5cases were not detected genotypes, but the detection of wild-type loci. HBV B type250cases (58.96%), HBV C type92cases (21.07%), HBV D type1cases (0.24%), HBV B/C hybrid13cases (3.07%), HBV B/D hybrid8patients (1.89%). There are drug-resistant mutations detected in76cases, rt180M detection rate of21.30%, rt204V detection rate of24.07%, rt204I detection rate of33.33%, rt181V detection rate of11.11%, rt236T detection rate of8.33%. Of drug resistance nucleos (t) drug class, B type resistance in37cases, the resistance rate was40.22%, C type resistance in17cases, the resistance rate was18.48%; B/C mixed resistant nine cases, the resistance rate of69.23%; B/D hybrid resistant to four cases, the resistance rate was80.00%, after the drug resistance χ2test groups B and C cases (χ2=9.694, P=0.0021, P<0.05), a statistically significant difference. B, C type of HBV DNA load (Log10) were5.691.70and5.79±1.51, no significant difference (t=-0.449, P>0.05). Compared with DNA sequencing, PCR-reverse dot hybridization assay for detecting HBV genotypes and drug-resistant mutations sensitivity, specificity and accuracy of98.19%,100%,99.56%, positive predictive value of100%, negative predictive value of99.41%, a positive likelihood ratio of∞, negative likelihood ratio was0.018, excellent consistency of results of two methods (Kappa value of0.958), the rate was98.12%(109/111), the difference was not statistically significant (χ2=45.97, P=0.000).Conclusions1.HBV infection in the region to B and C type based, while the presence of a certain proportion of B/C, B/D hybrid, resistant mutation detection rate from high to low were rt204I, rt204V, rt180M, rt181V, rt236T, rt207I.2. HBV B, C type differences of drug resistance nucleos (t) class of drugs, the former higher than the latter. For two kinds of drug-resistant high incidence of joint, but not many triple-drug.3. HBV B and C type for HBV DNA load in patients with no significant difference.4. PCR-reverse dot hybridization assay for detecting HBV genotype and drug-resistant mutant gene for sensitivity, specificity and accuracy of locus can meet the requirements of clinical testing, and the operation is simple, fast and suitable for clinical laboratory use.