A multiplex PCR kit that detects three major virulence genes, gelE, hyl and asaI, in Enterococcus faecalis was developed. Analyses of the available sequences of the three major virulence genes and the designed primers allowed us to develop the three-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting three amplicon bands for gelE, hyl and asaI were even and distinct with product sizes of 213, 273 and 713 bp, respectively. The multiplex PCR procedure was validated with a total of 243 E. faecalis strains that included 02 ATCC strains, 109 isolates from marine samples (sediment, water and sea foods), 22 isolates from cattle fodder, 79 isolates fresh water samples and 31isolates from nosocomial samples. Specificity of the kit was indicated by amplification of only three major virulence genes gelE, hyl and asaI, and without any nonspecific bands. Tests for the limit of detection revealed that amplified genes from the sample with a minimum of 10⁴ CFU/g or CFU/mL (10 cells/reaction) of E. faecalis and lower cell load samples, after a 3 h enrichment in NIOT-E. faecalis enrichment medium at 37 °C, a sensitivity level of 10 CFU/g or CFU/mL was achieved.

检测三个主要毒力基因，多重PCR试剂盒凝胶E，HYL和ASA我，在粪肠球菌的开发。对三种主要毒力基因的可用序列和设计的引物进行分析，使我们能够开发出三基因多重PCR方案，该方案可保持每个引物对的特异性。所得到的三个凝胶E，hyl和asa I的扩增子条带是均匀且不同的，产物大小分别为213、273和713 bp。总共243条粪肠球菌对多重PCR程序进行了验证。这些菌株包括02种ATCC菌株，109种海洋样品（沉积物，水和海鲜）分离物，22种牛饲料分离物，79种淡水样品分离物和31种医院样品分离物。该试剂盒的特异性通过仅扩增三个主要毒力基因gel E，hyl和asa I来表明，并且没有任何非特异性条带。检测限的测试表明，在NIOT-中富集3 h后，从样品中扩增出的基因的粪肠球菌含量至少为10 ⁴  CFU / g或CFU / mL（10个细胞/反应），且细胞负荷较低。在37°C的粪肠球菌富集培养基中，灵敏度达到10 CFU / g或CFU / mL。