应用非变性毛细管电泳检测乙肝病毒基因型B和基因型C

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

目的建立非变性毛细管电泳分离乙肝病毒特异性巢式PCR产物方法，用于乙肝病毒B基因型和C基因型判断。方法建立线性聚丙烯．聚乙二胺一TBE非变性毛细管电泳方法，分离母婴阻断失败患儿血清中乙肝病毒基因型特异性巢式PCR的扩增产物，采用表观分子量鉴定B、C基因型特异性和非特异性扩增产物。结果毛细管电泳条件为30．2cm×50μm毛细管，分离缓冲液为2％（w／v）线性聚丙烯酰胺＋0．4％（w／v）聚乙二醇＋1×TBE缓冲液（pH8．3）。在9kV电压下12分钟内可分离DNA分子量梯度标准品（50bp至300bp）及PCR扩增产物，表观分子量与电泳迁移时间的曲线适合度大于0．9999。B型扩增产物表观分子量范围为282bp至285bp，非特异性扩增产物为276bp至280bp。C型为119bp至120bp，当前扩增条件下不产生非特异性扩增产物。

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 eleetrophoresis method with linear polyaerylamide-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 ×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 rain under 9 kV. The goodness-of-fit between molecular weights and migration times was over O. 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.