AP—PCR结合进化树分析在幽门螺杆菌地域起源特征研究中的价值

目的探讨随机引物PCR（Arbitrary primerPCR，AP—PCR）结合克隆测序及生物信息学分析等方法在研究幽门螺杆菌（Helicobacter pylori，Hpylori）菌株地域起源特征中的价值和意义。方法针对临床分离培养的Hpylori菌株的基因组DNA，采用一组10nt的寡核苷酸引物进行随机PCR扩增，选取相对保守的片段进行回收、克隆及测序，测序的基因序列提交GenBank数据库进行序列相似性的BLAST比对，收集BLAST比对得到的同源性较高不同地域来源螺杆菌的对应序列，用ClustalX软件进行排序，采用Mega4．0软件中的邻位相连法（Neighbor-joining）和最大简约法（Maximum—parsimony）进行进化树分析。结果随机引物扩增及筛选克隆测序得到的基因产物为NADH脱氢酶G和H亚单位的部分编码序列，与27株不同地域来源H．pylori及1株猫科动物来源螺杆菌菌株的同源性均高达90％以上，表明Hpylori中NADH脱氢酶基因序列为保守结构，进化树分析显示：采用AP．PCR方法得到的Hpylori临床菌株的基因序列，显示出东亚菌株来源的遗传特征，与具有东亚菌株特征的美洲秘鲁Sat464和Shi470菌株、韩国的52、51菌株、日本的1757菌株遗传距离较近，与南亚、欧洲菌株距离较远，与非洲的SouthAffica7菌株和猫科动物来源的Sheeba菌株的遗传距离最远。结论不仅某些特殊基因可以反映地域差异，随机定位相对保守的基因片段同样可以反映Hpylori的地域起源特征。AP—PCR、测序等技术方法与进化树分析相结合是探讨Hpylori地域起源特征的一种更为便捷有效的新方法。

The value of AP-PCR combined with phylogenetic tree in characterizing the geographical origin of Helicobacter pylori

Objective To explore the value and significance of the method which combined Arbitrary primer PCR with cloning,sequencing and bioinformatics analysis for characterizing geographical origin of Helicobacter pylori(H.pylori).Method H.pylori strains were isolated from patients and cultivated,and then genomic DNA was extracted.PCR was performed by using a pair of 10 nt random oligonucleotide primers.Befitting PCR products were selected for cloning and sequencing.By submitting the nucleotide sequence to GenBank database and Blasting,we obtained 28 corresponding similar sequences with different geographical origins.All the sequences were aligned using ClustalX 1.8 software,the phylogenetic relationship was inferred using the neighbor-joining and maximum-parsimony methods with 1000 bootstrap replicates implemented in the MEGA4 software.Result The sequencing result showed that the product of AP-PCR which we selected was a part of coding sequence of NADH dehydrogenase′s G and H subunits.The sequence of our clinical isolates showed query coverage of more than 95% with the other 27 H.pylori sequences with different origin and 1 Helicobacter acinonychis sequence obtained from public database,indicating that NADH dehydrogenase of H.pylori was fairly conservative.The phylogenetic analysis showed the sequence obtained by AP-PCR was genetically characterized with East Asia type strains.The isolates from Chinese patient were close related to South America isolates characterized with East Asia type and isolates from South Korea and Japan,but separating from the strains from southern Asian,Europe and Africa.Conclusion The different origin was not only reflected by some especial gene,but also by the gene fragment of H.pylori obtained from AP-PCR.The method combining the AP-PCR,sequencing and phylogenetic tree was fast,efficient and convenient to characterize the geographical origin of H.pylori.