基因多态性分析在凝固酶阴性葡萄球菌菌种鉴定中的应用

Application of PCR for identification of coagulase-negative Staphylococcus

目的建立快速凝固酶阴性葡萄球(CNS)菌种的基因鉴定方法。方法对培养获得的、经AutoScan4微生物分析仪和APIStaph鉴定系统鉴定为CNS的100株临床分离菌株和质控菌株应用tRNA基因间长度多态性PCR分析(tDNAPCR)和肠杆菌基因间相同序列(ERIC)两种分子生物学方法进行常见的CNS菌种鉴定,同时改良实验条件,并将tDNAPCR及ERICPCR两种分子生物学方法的鉴定结果与传统的AutoScan4微生物分析仪和APIStaph鉴定系统进行比较。结果tDNAPCR及ERICPCR两种分子生物学方法对质控菌株及经AutoScan4微生物分析仪和APIStaph系统鉴定的12种临床分离CNS菌株均可以得到各自不同的电泳图谱,它们可以快速、准确地对常见12种CNS,包括表皮葡萄球菌、溶血葡萄球菌、人葡萄球菌、华纳葡萄球菌、模仿葡萄球菌、孔氏葡萄球菌、心耳葡萄球菌、松鼠葡萄球菌、头状葡萄球菌、施氏葡萄球菌、木糖葡萄球菌和里昂葡萄球菌进行菌种鉴定。从图谱的肉眼观察看,12种CNS均可以很好地鉴别开。tDNAPCR及ERICPCR与AutoScan4和APIStaph鉴定系统的一致率为95%。经Taq酶聚合后进行电泳,tDNAPCR在100～600bp产生6～10条DNA片段;ERICPCR在100～1200bp仅产生1～8条DNA片段。结论tDNAPCR及ERICPCR是快速、敏感的CNS菌种鉴定的方法,具有高度的特异性。

Objective To set up a rapid identification of Coagulase-negative Staphylococcus. Methods One hundred strains of Clinical isolates identified as members of the CNS by Auto Scan-4 and API Staph system, were subjected to be identified to the species level with molecular methods of tRNA gene intergenic spacer length polymorphism （tDNA-ILP） and enterobactefial repetitive intergenic consensus PCR （ERIC- PCR）. The comparison was conducted among the results of tDNA-ILP,ERIC-PCR and Auto Scan-4 and API Staph system. Results tDNA -PCR and ERIC -PCR exhibited accurately for identification of 11 kinds of clinical CNS isolates, such as S. epidermidis, S. haemolyticus, S. hominis S. wamefi, S. simulans, S. cohhni, S. auricularis, S. sciuri, S. capitis, S. schleifefi, S. xylosus, and S. lugdunensis. The coincidency of the methods between Auto Scan-4, API Staph and tDNA-PCR, ERIC -PCR was 95%. Taq tDNA-PCR yielded profiles of 6 - 10 bands ranging in size from 100 -600 bp, and ERIC-PCR yielded profiles of 1 -8 bands ranging in size from 100 -600 bp. Conclusion Both tDNA-PCR and ERIC-PCR were a methods for accurate, rapid, and sensitive identify ication of 11 kinds of clinical CNS isolates.