乙肝病毒基因组序列扩增的条件优化和结果分析
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摘要
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( Mg2 +),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 m M Mg2 +,200 n M 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.
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( Mg2 +),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 m M Mg2 +,200 n M 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.
引文
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[4]许军,王齐欣,范春蕾,等.中国南北两城市乙型肝炎病毒基因型与血清型的构成差异.中华实验和临床病毒学杂志,2003,17(4):327-329.
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[8]肖志壮,曲音波,汪天虹,等.过量DMSO显著降低低模板浓度PCR扩增的特异性.遗传,2001,23(4):341-343.
[9]姜鸾,邵蕾,胡旸,等.应用改良PCR法构建高GC含量启动子多位点定点突变载体.首都医科大学学报,2010,31(1):88-92.
[10]Ha SH,Park YM,Hong SP,et al.De Novo superinfection of hepatitis B virus in an anti-HBs positive patient with recurrent hepatitis C following liver transplantation.Gut Liver,2011,5(2):248-252.
[11]叶珺,熊自忠,尹华发,等.安徽部分医院HBV基因型的分布及其临床意义.安徽医科大学学报,2006,41(6):676-679.
[12]汪娟,董永海,汪松,等.安徽省某市级医院HBV感染住院患者HBV基因型检测分析.安徽医科大学学报,2011,46(10):1079-1081.
[13]郑欣,王文敬,单桂秋,等.广东阳江地区隐匿性乙型肝炎病毒检出率与分子生物学特性分析.中国输血杂志,2010(04):259-264.
[14]欧山海,林永财,倪宏英,等.闽南地区无偿献血者隐匿性乙型肝炎病毒感染研究.中国输血杂志,2010(12):1033-1037.
[15]叶贤林,郑欣,杜鹏.深圳献血人群隐匿性乙型肝炎病毒全基因序列系统进化树分析.中国输血杂志,2013,26(4):324-327.
[2]Okamoto H,Tsuda F,Sakugawa H,et al.Typing hepatitis B virus by homology in nucleotide sequence:comparison of surface antigen subtypes.J Gen Virol,1988,69(Pt 10):2575-2583.
[3]高俊薇,李雅娟,庄辉,等.中国11城市乙型肝炎病毒慢性感染者中乙型肝炎病毒基因型分布.中华流行病学杂志,2007,28(4):315-318.
[4]许军,王齐欣,范春蕾,等.中国南北两城市乙型肝炎病毒基因型与血清型的构成差异.中华实验和临床病毒学杂志,2003,17(4):327-329.
[5]Allain JP,Hsu J,Pranmeth M,et al.Quantification of viral inactivation by photochemical treatment with amotosalen and UV A light,using a novel polymerase chain reaction inhibition method with preamplification.J Infect Dis,2006,194(12):1737-1744.
[6]Yuan Q,Ou SH,Chen CR,et al.Molecular characteristics of occult hepatitis B virus from blood donors in southeast China.J Clin Microbiol,2010,48(2):357-362.
[7]吴光华,丁惠国,曾长青.公共核酸数据库乙肝病毒全基因组序列概况和中国HBV参照序列的建立.自然科学进展,2008,18(2):121-129.
[8]肖志壮,曲音波,汪天虹,等.过量DMSO显著降低低模板浓度PCR扩增的特异性.遗传,2001,23(4):341-343.
[9]姜鸾,邵蕾,胡旸,等.应用改良PCR法构建高GC含量启动子多位点定点突变载体.首都医科大学学报,2010,31(1):88-92.
[10]Ha SH,Park YM,Hong SP,et al.De Novo superinfection of hepatitis B virus in an anti-HBs positive patient with recurrent hepatitis C following liver transplantation.Gut Liver,2011,5(2):248-252.
[11]叶珺,熊自忠,尹华发,等.安徽部分医院HBV基因型的分布及其临床意义.安徽医科大学学报,2006,41(6):676-679.
[12]汪娟,董永海,汪松,等.安徽省某市级医院HBV感染住院患者HBV基因型检测分析.安徽医科大学学报,2011,46(10):1079-1081.
[13]郑欣,王文敬,单桂秋,等.广东阳江地区隐匿性乙型肝炎病毒检出率与分子生物学特性分析.中国输血杂志,2010(04):259-264.
[14]欧山海,林永财,倪宏英,等.闽南地区无偿献血者隐匿性乙型肝炎病毒感染研究.中国输血杂志,2010(12):1033-1037.
[15]叶贤林,郑欣,杜鹏.深圳献血人群隐匿性乙型肝炎病毒全基因序列系统进化树分析.中国输血杂志,2013,26(4):324-327.