Chesneau O, Allignet J, el Solh N
National Reference Centre for Staphylococci, Institut Pasteur, Paris, France.
Mol Cell Probes. 1993 Aug;7(4):301-10. doi: 10.1006/mcpr.1993.1044.
DNA fragments, 450 bp in length, were amplified by polymerase chain reaction (PCR) from the thermonuclease gene (nuc) carried by seven epidemiologically independent Staphylococcus aureus isolates. Sequencing of the PCR products led us to characterize 210 bp strictly conserved. A 186 bp piece from within this conserved region was cloned into pUC18. The resulting recombinant plasmid, pIP1608, was used as a probe against the cellular DNA of 360 staphylococcal isolates belonging to 28 species. Only the 146 S. aureus isolates, including four which were not thermonuclease producers, had DNA that hybridized with pIP1608. Among the 214 non-S. aureus staphylococci, 55 exhibited a thermonuclease activity. For 32 of these, the enzymatic activity was inhibited by a commercially available polyclonal antiserum directed against the thermonuclease of an S. aureus strain. These results are in favour of the use of pIP1608 as a probe to specifically recognize S. aureus. Furthermore, we propose a method based on colony blot hybridization and potentially useful to enumerate S. aureus cells in biological samples.
从七株流行病学上独立的金黄色葡萄球菌分离株携带的热核酸酶基因（nuc）中，通过聚合酶链反应（PCR）扩增出长度为450 bp的DNA片段。对PCR产物进行测序后，我们鉴定出210 bp的序列严格保守。从该保守区域内选取一段186 bp的片段克隆到pUC18中。所得重组质粒pIP1608用作针对属于28个种的360株葡萄球菌分离株细胞DNA的探针。只有146株金黄色葡萄球菌分离株（包括4株不产生热核酸酶的菌株）的DNA与pIP1608杂交。在214株非金黄色葡萄球菌中，有55株表现出热核酸酶活性。其中32株的酶活性被一种针对金黄色葡萄球菌菌株热核酸酶的市售多克隆抗血清所抑制。这些结果支持将pIP1608用作特异性识别金黄色葡萄球菌的探针。此外，我们提出了一种基于菌落杂交印迹的方法，该方法可能有助于对生物样品中的金黄色葡萄球菌细胞进行计数。
