LAUSR

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.