通用性引物RT-nPCR扩增不同基因型戊型肝炎病毒基因组的探讨  

Genomic amplification of different genotypes of hepatitis E virus by RT-nPCR with universal primers

作　　者：周镇先[1] 戴星[2] 孟继鸿[3] 

机构地区：[1]东南大学医学院附属南京第二医院,江苏南京210003 [2]东南大学附属中大医院,江苏南京210009 [3]东南大学基础医学院病原生物学与免疫学系,江苏南京210009

出　　处：《东南大学学报（医学版）》2004年第3期141-144,共4页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目 (30 2 71 2 31 )

摘　　要：目的 :筛选戊型肝炎病毒 (HEV)通用性引物 ,用于不同基因型HEV基因组的逆转录 巢式聚合酶链扩增 (RT nPCR)。方法 :在HEV序列保守区设计和合成A、B、C、D 4组引物 ,用于扩增已知基因型的HEV参考株和HEVIgM阳性的临床标本 ,探讨扩增区域最小自由能与RT nPCR扩增效率的关系。结果 :4组引物均可扩增出不同基因型HEV基因片段 ,但得到的PCR滴度有差异 ,D组引物所得滴度最高 ,B组最低 ;5 4份临床标本的PCR阳性率也以D组最高、B组最低。D组引物扩增区段的RNA模板最小自由能的绝对值最低 ,B组的最高 ,与PCR扩增效率相关。结论 :在HEV基因组第 62 96～ 660 3核苷酸之间设计的D组通用性引物可扩增不同基因型HEV以及临床标本 ,扩增效率高 。Objective To screen out a set of universal primers of hepatitis E virus (HEV) for genomic amplification of different HEV genotypes by reverse transcription-nested polymerase chain reaction (RT?nPCR).Methods Four sets of primers numbered A,B,C and D were designed and synthesized based on several conserved regions of HEV nucleotide sequences.The primers were used to amplify HEV reference strains with known HEV genotypes and to amplify clinical serum samples with positive HEV?IgM.Finally,a relationship between the efficiency of RT?nPCR amplification and the minimum free energy of the amplified regions was analyzed.Results HEV gene fragments of different HEV genotypes were successfully amplified with the 4 sets of primers.However,different PCR titers for the known HEV reference strains and different PCR positive rates for 54 clinical samples were obtained with the different primer sets.The lower an absolute value of the minimum free energy of the amplified region was,the higher the efficiency of PCR amplification was.Conclusion The primer set D located at 6296?6603 nucleotide of HEV genome is a both universal and efficient primer set for amplification of different HEV genotypes.

关 键 词：通用性引物 RT-nPCR扩增 基因型 戊型肝炎病毒 基因组 逆转录-巢式聚合酶链反应 

分 类 号：R373.21[医药卫生—病原生物学] R394[医药卫生—基础医学]