具發展成為沙門氏菌特異性探針潛力之基因體片段選殖

Cloning of the Genomic DNA Fragments Containing Salmonella-Specific Sequences

黃進發(Chin-Fa Hwang) ； 林建谷(Chien-Ku Lin)

《弘光學報》 47期 (2005/11) Pp. 61-67

https://doi.org/10.6615/HAR.200511.47.07

摘要

DNA探針與聚合酶連鎖反應(PCR)已成為快速檢測食品微生物的重要技術之一。這些技術所使用的寡核苷酸探針或PCR引子，均需具有清楚的DNA序列才能進行設計。由於沙門氏菌各血清型間並無共同之毒素基因或致病因子；因此，沙門氏菌基因體成為篩選特異性探針之重要來源。本研究參考前人相關報告下，由Salmonella typhimurium染色體DNA，經Bam HI切割完全後，回收可能帶有沙門氏菌特異性DNA片段4.1kb 及4.9 kb附近之DNA片段，繼以高轉殖效率的pUC13載體黏接；經轉型作用後，由101個轉型細胞中，篩選到64個Lac^-轉形細胞。這些轉形株所含重組殖體再以限制酶Bam HI和EcoRl分別進行切割，分析其DNA大小，其中有5株純系(clone)，可能插入S. typhimurium 4.1kb及4.9 kb特意DNA片段。初步以Southern blotting進行沙門氏菌及E. coli基因體DNA之雜交測試，結果顯示這些DNA片段可與沙門氏DNA反應，而不會與E. coli DNA反應。因此，這些純系所攜帶之DNA片段又應具有發展成為沙門氏菌特異性探針之潛力，未來將近一不與更多其他腸內菌進行DNA雜交測試，並進一步次選殖以利定序，及作為設計特意性較高且序列較短之寡核苷酸探針或PCR引子之用。

關鍵字

沙門氏菌 ； 基因體探針 ； 選殖

並列摘要

DNA probe and polymerase chain reaction (PCR) have been important techniques for the rapid detection of foodborne pathogens. The knowledge of DNA sequences is essential to the designing of oligonucleotide probes or PCR primers. There are no universal toxin genes or other virulence factors among all Salmonella serotypes. Therefore, genomic DNA of Salmonella becomes an important source for searching specific DNA probes for their detection. In this study, the genomic DNA isolated from S. typhimurium was digested with BamHI. The DNA fragments of approximate 4.1 and 4.9 Kb, which were possible candidates of DNA probes for Salmonella as Fitts et al. (6) reported, were collected and ligated with pUC13 vector. After transformation of ligated DNA, 64 Lac^- transformants were obtained. The recombinant plasmids from these strains were isolated and digested with BamHI and EcoRI. Five clones with inserts of 4.1 or 4.9 Kb were identified to be with the potential of Salmonella specific fragments after prelimary tests. These cloned fragments would further be evaluated the detection specificity of Salmonella through DNA hybridization with other enterobacteria, and subcloned for DNA sequencing and the designing of oligonucleotide probes or PCR primers.

並列關鍵字

Salmonella ； genomic DNA ； DNA cloning

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