乙肝病毒基因组序列扩增的条件优化和结果分析

Evaluation of PCR conditions for the analyses of specific regions of Hepatitis B Virus

目的优化乙型肝炎病毒(HBV)不同长度基因片段PCR扩增条件,为后续机制研究提供实验基础。方法运用PCR或巢式PCR(Nested PCR)方法扩增6例临床乙肝患者HBV DNA,针对特异性目的片段,通过优化Mg2+浓度、引物浓度、Taq酶用量、DMSO浓度等,获得特异性扩增条带,PCR产物直接测序并使用CHROMAS、MEGA等生物学软件进行结果分析。结果通过综合分析各种实验条件对PCR扩增结果的影响,最终确认在50μL扩增体系中,2.5 mmol/L Mg2+、200 nmol/L特异性引物、1.5 U Taq酶、56℃退火在本实验室可获得良好的扩增效果,适量二甲基亚砜(DMSO)可减少非特异性干扰,同时建议对短片段可酌量减少Taq酶使用量。生物信息学分析示6例HBV感染标本中1例为B基因型,5例为C基因型,共发现S基因21个核酸变异及9个氨基酸可改变。结论建立了适合本实验室的HBV DNA扩增体系以及后续生物信息学分析方法,明确了不同的扩增体系条件对PCR扩增效果具有重要影响。

Objective To evaluate the PCR conditions and to analyze the sequences in specific regions of Hepatitis B Virus（ HBV）. Methods Samples from six hepatitis B patients were carried out by PCR or Nest PCR methods. The volume of reagents in PCR amplification,such as the concentration of magnesium（ Mg^2 ＋）,primers,Taq DNA polymerase and dimethyl sulfoxide（ DMSO）,were adjusted to achieve the specific amplicons of HBV. The specific fragments were directly sequenced and analyzed by CHROMAS and MEGA applications. Results In the 50 μL PCR reaction mixture,2. 5 mM Mg^2 ＋,200 nM specific primers and 1. 5 U Taq DNA polymerase at 56 ℃ annealing temperature could achieve the best results in HBV amplification. DMSO should be properly used in large fragment and DNA polymerase can be appropriately reduced when getting shorter fragments. Among the 6 patients,1 sample was identified as genotype B and 5 as genotype C.Twenty-one nucleotide variations and 9 amino acid mutations were observed in HBV S gene of these samples. Conclusion PCR amplification of specific HBV gene was established and the sequences of S gene were analyzed by bioinformatic applications. The concentrations of reagents in PCR mixtures are very important in obtaining specific results.