通过抑制性消减杂交技术鉴定来自非溃疡性消化不良（NUD）和胃溃疡患者的两株临床分离株之间幽门螺杆菌菌株特异性DNA序列。

Identification of H. pylori strain specific DNA sequences between two clinical isolates from NUD and gastric ulcer by SSH.

作者信息

Han Feng-Chan, Gong Min, Ng Han-Chong, Ho Bow

机构信息

Department of Microbiology, Faculty of Medicine, National University of Singapore.

出版信息

World J Gastroenterol. 2003 Aug;9(8):1747-51. doi: 10.3748/wjg.v9.i8.1747.

DOI:10.3748/wjg.v9.i8.1747

PMID:12918113

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4611536/

Abstract

AIM

The genomes of Helicobacter pylori (H. pylori) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH).

METHODS

Two clinical H. pylori isolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to Alu I digestion. Then two different adaptors were ligated respectively to the 5'-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting.

RESULTS

Among the 240 colonies randomly chosen, 50 contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1 036 bp. Fifteen inserts belonged to the H.pylori plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H.pylori in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GC). But a 670 bp DNA fragment (GU198) that was a bit homologous to the 3'-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GU-DU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H.pylori isolates, 2 from GU and 2 from GU-DU.

CONCLUSION

Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.

摘要

目的

不同个体的幽门螺杆菌（H. pylori）基因组存在差异。本项目旨在通过抑制性消减杂交（SSH）鉴定两株临床幽门螺杆菌分离株之间的菌株特异性DNA序列。

方法

培养两株临床幽门螺杆菌分离株，一株来自胃溃疡（GU，测试组），另一株来自非溃疡性消化不良（NUD，驱动组），制备基因组DNA并进行Alu I酶切。然后将两种不同的接头分别连接到测试组DNA片段的两个等份的5'端，并在测试组和驱动组DNA之间进行SSH。未杂交的测试组DNA序列通过两轮连续PCR扩增，并克隆到pGEM-T-Easy载体中。通过斑点杂交筛选测试菌株特异性插入片段并鉴定疾病相关DNA序列。

结果

在随机挑选的240个菌落中，50个含有测试菌株特异性DNA序列。对23个插入片段进行测序，大小范围为261 bp至1 036 bp。15个插入片段属于约3.5 kb的幽门螺杆菌质粒pHPO100，其编码复制蛋白A。其他插入片段与GenBank中幽门螺杆菌的基因有同源片段。给出了各种斑点杂交模式，当使用4个插入片段作为探针筛选来自27株临床分离株（8株来自GU，12株来自十二指肠溃疡（DU），4株来自GU-DU，2株来自NUD，1株来自胃癌（GC））的基因组DNA时，未发现GU菌株独特的DNA序列。但是一个670 bp的DNA片段（GU198）与胸苷酸激酶基因的3'端有一定同源性，在7株GU菌株（7/8）、3株GU-DU菌株（3/4）和3株DU菌株（3/12）中呈阳性。复制基因A（repA）的一个384 bp片段（GU79）仅在4株幽门螺杆菌分离株中呈阳性，2株来自GU，2株来自GU-DU。