Establishment And Application Of A Improved Genotyping Method For Hepatitis B Virus By PCR Using Type-Specific Primers

Hepatitis B poses a great threat to life and health. In China Hepatitis B virus (HBV) prevails at a high infection rate and causes considerable morbidity. HBV divides into eight different genotypes from A to H according to the homology in complete gene sequence(≥92%) or S gene sequence(≥96%). The division of HBV genotypes results from virus mutation and long-term filtration and accumulation by the host. The present research suggests that there have the differences between HBV genotypes in some clinical phenomenon, such as epidemiology characters,virus mutation,consequent disease and treatment response and so on, therefore the study of HBV genotyping have the great significance to further clarify the pathogenesis of hepatitis B, improve the efficiency of detection, Search treatment strategy and achieve epidemiological control. At present,there are several major methods to determine genotypes, such as the nucleotide sequence mensuration method,the genotype-specific primers PCR method, the PCR-RFLP method and the ELISA with monoclonal antibodies method and so on. The genotype-specific primers PCR method is a simple, rapid typing one that has good application value. Because of the significant geographic differences of the prevalence of HBV and HBV genotype, and remarkable differences may exist in the distribution of genotype-specific nucleotides, which are the guarantee of specificity of this typing method,on HBV genome in different places , simultaneously the specificity of the same genotype-specific nucleotides in same genotype HBV is likely to present great differences in different geographical locations too, So it is very important for the rationality of type-specific primers design and the geographical applicability of typing system. At present, due to the lack of effective genotype-specific primer design standards and assessment system, for domestic HBV genotypes related research it has the great significance to improve actual genotype-specific primers design standards and assessment system to establish a sensitive, accurate and fast, suitable for domestic application HBV genotype-specific primers PCR typing system.Objective: To improve actual genotype-specific primers design standards and assessment system and establish a sensitive, accurate and fast, suitable for domestic application HBV genotype-specific primers PCR typing method.Methods: 1.The establishment of the improved genotype-specific primers PCR method to determine genotypes B and C of HBV: First, We collect standard complete genome sequence of all genotypes HBV from GeneBank database, and set related definitions and criteria about genotype-specific nucleotide. After this we use DNAman software to do multiple sequence aligment to the standard sequences for geting all genotype-specific nucleotides in HBV S gene, and design genotype- specific primers in the area that genotype-specific nucleotides gather together. At last, we establish the improved genotype-specific primers PCR method to determine genotypes B and C of HBV by using the standard B and C genotypes sreum. Second, we evaluate the method capability by doing PCR reaction condition optimization experiments, sensitivity experiments, reproducibility and specificity experiments. Third, comparing this method with the nucleotide sequence mensuration method. 2. Comparison of two HBV genotype-specific primers PCR typing systems: To establish the comparison genotype-specific primers PCR typing system according to the reference papers, we compare two typing systems's performance. 3. Preliminary application of the improved HBV genotype-specific primers PCR typing method: We use this typing system to detect 187 patients with chronic hepatitis B in Guangdong province.Results: 1. Using genotype-specific nucleotides(nt321 A, nt324 T,nt330 G)in S gene of B genotype HBV and genotype-specific nucleotides(nt293 A, nt294 C, nt300 C)in S gene of C genotype HBV we successfully establish the improved genotype-specific primers PCR method to determine genotypes B and C of HBV. Compared with the improved genotype-specific primers PCR method and the nucleotide sequence mensuration method, the results coincidence rate is 100%. The distribution and specificity of genotype-specific nucleotides in S gene of genotype B HBV exist significant difference between theoretic value which be got according to the analysis of standard HBV sequences and practical value which come from the analysis of domestic patients with chronic hepatitis B. 2. To compare of two HBV genotype-specific primers PCR typing systems, the results coincidence rate is 86%. 3. In 187 patients with chronic hepatitis B in Guangdong province, we successfully typed 185 samples, 2 samples failed detected. In the 185 samples, genotype B are 107, genotype C are 70, 8 are genotype B plus genotype C.Conclusion: The study has successfully established a sensitive, accurate and fast, suitable for domestic application HBV genotype-specific primers PCR typing method. This method was applied to HBV genotype prevalence surveys in Guangdong province.