肠球菌种属鉴定及万古霉素耐药基因型检测的多重聚合酶链反应方法的建立及应用

Establishment and application of multiplex polymerase chain reaction assay to identify Enterococcus and vancomycin-resistant genotypes

目的建立快速准确的肠球菌种属鉴定及万古霉素耐药基因检测的多重聚合酶链反应(multiplex polymerase chain reaction,MPCR)方法,并在人及动物源菌株中应用。方法针对肠球菌属、粪肠球菌、屎肠球菌、van A、van B、van C1、van C2/C3保守基因设计引物,建立肠球菌种属鉴定及万古霉素耐药基因型检测的MPCR方法;应用建立的MPCR方法检测98株人及动物源菌株菌种及万古霉素耐药基因,并比较MPCR方法检测结果与检测菌株的VITEK Compact 60自动微生物鉴定仪菌种鉴定结果及琼脂稀释法的万古霉素及替考拉宁药敏检测结果,分析MPCR方法检测结果与其他方法的一致性。结果成功建立了用于检测肠球菌属和常见肠球菌种及万古霉素耐药基因型的MPCR方法。MPCR方法和VITEK Compact 60自动微生物鉴定仪对98株检测菌株菌种鉴定结果一致;MPCR方法检测98株菌万古霉素耐药基因型与药敏检测其耐药表型相对应。结论建立的MPCR方法成功用于检测人及动物源肠球菌种属及万古霉素耐药基因型,为快速准确鉴别肠球菌种属及万古霉素耐药肠球菌耐药基因型提供方法,对于有效监测万古霉素耐药肠球菌,控制其传播、流行具有重要意义。

Objective To establish a multiplex polymerase chain reaction（ multiplex MPCR） to identify Enterococcus and its vancomycin-resistant genotypes simultaneously. Methods Eight pairs of primers targeting Enterococcus, E. faecalis and E. faecium specific genes vanA, vanB, vanC1, vanC2/C3, and 16S rDNA were used in one reaction tube. Ninety eight Enterococcus strains isolated from human and animal were identified and their vancomycin-resistant genotypes were detected by using the established multiplex PCR. Results The detection results of the 98 isolates by multiplex PCR were consistent with those by VITEK and antimicrobial susceptibility testing. Conclusion The established multiples PCR assay is simple and reliable for the rapid identification of vancomycin-resistant Enterococcu.