抑制消减杂交技术筛选肺炎链球菌多耐药相关DNA片段

目的筛选肺炎链球菌多耐药相关DNA片段,探索肺炎链球菌多耐药的分子机制。方法提取多耐药菌株09062407与标准菌株ATCC49619基因组DNA,应用抑制性消减杂交技术得到富含差异DNA片段的消减混合物,与pEASY-T3克隆载体连接并转化到TP10感受态细胞中,构建多耐药肺炎链球菌差异DNA消减文库。结果耐药株与敏感株间差异片段大量富集,得到大小200～1 500 bp弥散状DNA条带;将这些差异DNA片段富集后构建成消减文库,随机挑取192个白色克隆,经PCR扩增,155个克隆插入有200～800 bp大小片段。结论抑制消减杂交技术能有效筛选肺炎链球菌多耐药株与敏感株间差异DNA片段,可进一步通过克隆鉴定等生物信息学方法寻找与肺炎链球菌多耐药相关基因。

Screening of multi-drug resistance associated DNA fragments of streptococcus pneumonia using suppression subtractive hybridization

Objective To screen multi-drug resistance（MDR）associated DNA fragments for providing insight into the molecular mechanism of multi-drug resistant streptococcus pneumonia.Methods The genomic DNAs were purified from MDR 09062407 and ATCC 49619.The mixture of subtracted DNA fragments were obtained using suppression subtractive hybridization（SSH） and were ligated with pEASY-T3 vector and transformed to competent cells TP10.Then the differential subtraction library was constructed.Results The fragments from 200 to 1500 bp were enriched in streptococcus pneumonia MDR,155 positive clones included 192 randomly selected clones were screened out inserted DNA sequence by PCR.The length of the inserted fragments was from 200 to 800 bp.Conclusions SSH technique can efficiently identify differential DNA fragments between MDR and susceptible strain in streptococcus pneumonia.Many bioinformatics methods will be used to analyze and find genes associated with MDR.