羊源肠球菌属与屎肠球菌双重PCR检测方法的建立

摘要

Objective]To establish a duplex PCR assay for simultaneous and quick detection of sheep-originated clinical isolates of Enterococcus and Enterococcus faecium. [Methods]The genus specific and species specific primers for Enterococcus and Enterococcus faecium targeting at 16S rDNA and ddl gene were designed respectively, and the reaction system of duplex PCR assay was determined. The specificity and sensitivity of the established duplex PCR assay were evaluated. The simulated bacterial infection blood samples were prepared by using healthy sheep blood and mixed bacterial solution and were used to verify the specificity and sensitivity of the established duplex PCR assay in detection of clinical samples. [Results]The results showed that the duplex PCR assay was able to specifically amplify the DNA fragments of 294 bp from Enterococcus and 557 bp from Enterococcus faecium,but was negative for other common animal-originated pathogenic bacteria. The detection limits of the assay for Enterococcus and Enterococcus faecium were 5.71×10-5 ng/μL and 5.71×10-4 ng/μL, respectively. The duplex PCR assay was able to specifically detect the Enterococcus and Enterococcus faecium in simulated bacterial infection blood samples, and the detection limit for Enterococcus faecium was 1×101 CFU/mL. [Conclusion]It was indicated that the established duplex PCR assay is specific and sensitive for detection of clinical Enterococcus and Enterococcus faecium isolates.%[目的]建立羊源肠球菌属与屎肠球菌的双重PCR检测方法，对临床分离的菌株进行快速检测。[方法]设计肠球菌属特异性和屎肠球菌种特异性引物，建立双重PCR反应体系；评价双重PCR反应的特异性和敏感性；利用混合菌液制备模拟感染样品，确定双重PCR反应对模拟感染样品检测的特异性和敏感性。[结果]建立的双重PCR反应能够特异性的扩增肠球菌属（294 bp）和屎肠球菌（557 bp） DNA片段；该方法对常见的动物源细菌无交叉反应，对肠球菌属的最低检测量为5.71×10-5 ng/μL，对屎肠球菌的最低检测量为5.71×10-4 ng/μL；双重PCR反应能够特异性的检测模拟感染样品中的肠球菌属和屎肠球菌，对于模拟感染样品中的屎肠球菌检测量为1×101 CFU/mL。[结论]建立的双重PCR检测方法具有特异性强、敏感性高的特点，为快速鉴定屎肠球菌提供了快速、准确的方法。