PCR指纹图技术筛选鼠疫耶尔森菌种特异性探针的研究

Screening for Yersinia pestis species-specific probes by PCR fingerprinting

目的 利用PCR指纹图技术筛选细菌种特异性探针 ,探索利用PCR指纹图技术实现病原菌通用检测的可能性。方法 以鼠疫耶尔森菌为实验对象 ,利用REP 1和REP 2引物对在非严谨的条件下进行PCR指纹图扩增 ;将所有扩增片段纯化后 ,克隆至pGEM T载体 ,转化大肠杆菌JM10 9;用生物素化的载体引物扩增克隆化片段 ,进行末端标记 ;在硝酸纤维素膜上固定鼠疫杆菌的染色体DNA以及相应对照菌株的染色体DNA ,以末端标记的扩增产物进行杂交 ,通过生物素 链霉亲和素 辣根过氧化物酶系统显色 ,判断扩增片段的特异性。结果 经杂交筛选 ,发现 1个长度约为 90 0bp的扩增具有较好的杂交特异性片段 ,将序列与鼠疫耶尔森菌已完成的基因组序列比较 ,仅发现 4个核苷酸的差异 ;根据这段序列设计的引物对可以特异性地扩增鼠疫耶尔森菌的模板。结论 利用PCR指纹图技术成功筛选到一段鼠疫耶尔森菌的种特异性探针 ,为细菌通用检测技术的研究开辟了新的思路。

Objective To screen a species-specific probe of individual bacteria species by PCR fingerprinting, which will facilitate a universal detection system for pathogenic bacteria. Methods Yersinia pestis was set as a model species. A pair of primers named REP-1 and REP-2 were employed to perform a PCR fingerprinting amplification. Every single amplified band of Yersinia pestis was purified, cloned into pGEM-T vector, which was transformed into E.coli JM109. The cloned fragments were then terminal-labeled by biotin with biotinylated pGEM-T vector primers. Meanwhile, the genomic DNAs of Yersinia pestis and of other references species were immobilized onto nitro-cellulose membranes. And then the biotinylated amplicons were hybridize with the membranes, and the specificity of every fragment was determined by the hybridization results. Results One fragment around 900 bp can specifically hybridize with Yersinia pestis genomic DNA but not with any other reference strains. Comparing the sequence of this fragment to the genome sequences of Yersinia pestis, only four base-pairs have been found to be different at 5′-end. A pair of primers derived from this fragment can specifically amplify Yersinia pestis templates. Conclusion One species-specific probe of Yersinia pestis is successfully screened by PCR fingerprinting, and the method presented in this paper proves to be a novel way to get specific probes of pathogenic bacteria.