| The clinical outcomes and response to therapy of hepatitie B virus infection differ depending upon viral genotypes. HBV genotype B and C are common in China mainland,where genotype C is more common in the north and genotype B is more common in the south.In addition, there are a small number of genotypes A and D in some areas. A number of methods have been developed for HBV genotyping, but most of these methods are relatively time consuming, laber intensive and costly.Objective: To establish a rapid ,inexpensive and accurate method for detection of HBV genotypes. Using the method realized detection of HBV genotypes A~D simultaneously. To investigate the feasibility of the method useing for clinical detection of HBV genotypes.Methods: 1. Designed primers and type specific probes of HBV genotypes A,B,C and D, synthesized by the Corporation.Upstream primer 5'end labled with digxin. Polymers was added to the ends of Probes.Two chronic Hepatitis B patients were included in our study, one was HBV genotype B, the other was HBV genotype C. Extracting virus DNA, TA cloning was used to get the standard strain plasmids of HBV genotypes B and C. According to the HBV genotypes A,D,E~H sequences included in NCBI, its type specific conserved regins were synthesized by the invitrogen Corporation, cloned in pMD18-T plasmid vecter that constructs HBV genotypes A,D,E~H templates. Nested PCR was used to get fragments for hybridization.2. We designed a series of optimization experiments to optimize the probe carrier, the probe concentraion and modification, hybridization conditions, stringent elution congditions and antibody concentration, screened the best reaction conditions could be detect of HBV genotypes specifically and sensitively.3. Standard strain plasmids of HBV genotypes A~H were used to evaluate the specificity and sensitivity of this procedure. To assess the potential of the method detection of mixed infections, PCR products of genotypes B and C plasmids were mixed at various ratios. 70 specimens in Chongqing area were collected and detected by this method, and results were evaluated using direct sequencing. The consistency of the two methods were compared .Result: 1.HBV genotypes A~H recombinant plasmids were constructed. Primers and probes were designed at conserved regins.2. The optimum reaction conditions were: positively charged nylon membranes as a probe carrier, probes were tailed poly(dC) and the probe concentration was 10μmol/L, UV cross-linking plus high-temperature baking were used to fix probes, DIG antibody conjugate should be diluted 1:10000, hybridized at 38℃for 1h and washed stringently for 15 min at 38℃with 0.5×SSC/0.1% SDS, alkaline phosphatase catalyzed NBT / BCIP as a color manner.3. The specificity of this method was so well that there was no cross reactivity. The detectong sensitivety of the method was found to 103 or greater DNA copies /ml, detected of 5% of the mixed infection. Of 70 clinical specimens, 67 clinical samples were genotyped accurately. The results of our method agreed with the results of sequencing in 98.51%(66/67). Three cases were not genotyped by our method or by direct sequencing because the PCR failed to amplify positive band. The results showed that genotype B was the predominant genotype in Chongqing area.Conclusion: We successfully constructed a rapid, inexpensive and accurate method for detection of HBV genotypes. This procedure was simple and rapid enough for large-scale clinical usage. The method would reach to the detection of all eight genotyptes only need to design the other type-specific probes.It would be a wide range of applications. |
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