Establishment of a Duplex PCR Assay for Simultaneous Detection of Sheep-originated Enterococcus and Enterococcus faecium

[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 16 S r DNA 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 5.71×10-5ng/μL and 5.71×10-4ng/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 1×101CFU/m L. [Conclusion]It was indicated that the established duplex PCR assay is specific and sensitive for detection of clinical Enterococcus and Enterococcus faecium isolates.