Application of non-denaturing capillary electrophoresis in detection of hepatitis B virus genotypes B and C

Objective To establish the non-denaturant capillary electrophoresis method to separate nested PCR products with HBV genotype specific primers,and confirm HBV genotypes B and C.Methods The non-denaturant capillary electrophoresis method with linear polyacrylamide-polyethylene-TBE buffer system was first established.The nested PCR method with HBV genotype specific primers was used to amply HBV DNA segments from serums of children who are failed in prevention of mother-to-children transmission.The apparent molecular weights indicate.specific and non-specific amplicons of genotype B and C.Results Capillary electrophoresis conditions applied a 30.2cm x 50μm i.d.fused silica capillary,running buffer containining 2％(w/v) linear polyacrylamide,0.4％ (w/v) polyethylene and 1 × TBE buffer (pH8.3).Separation of 50-bp DNA step ladder and PCR products were completed within 12 min under 9 kV.The goodness-of-fit between molecular weights and migration times was over 0.9999.Apparent molecular weights of specific amplification products for genotype B range from 282 bp to 285 bp,non-specific products range 276 bp to 280 bp.For genotype C,the products were 119 bp to 120 bp,and no non-specific amplification was observed under current conditions.Conclusion The non-denaturant capillary electrophoresis method could conveniently separate and identify non-specific amplification products of genotype B. Key words: Hepatitis B;  HBV genotype;  Capillary Electrophoresis;  prevention of mother-to-children transmission