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Updated: Dec 8, 2025
A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters
Published on: March 9, 2016
A multiplex PCR kit for the detection of three major virulent genes in Enterococcus faecalis
Balakrishnan Meena1, Lawrance Anburajan1, Karthik Srinivasan Varma2
1Atal Centre for Ocean Science and Technology for Islands, National Institute of Ocean Technology, Port Blair 744103, Andaman and Nicobar Islands, India.
Abstract:
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 three major virulence genes and 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 31 isolates from nosocomial samples. Specificity of the kit was indicated by amplification of only three major virulent genes gelE, hyl and asaI 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.
Insights
A new multiplex PCR kit accurately detects three key virulence genes (gelE, hyl, asaI) in Enterococcus faecalis. This validated method offers high specificity and sensitivity for identifying this important bacterium.
Area of Science:
- Microbiology
- Molecular Biology
- Diagnostic Development
Background:
- Enterococcus faecalis is an opportunistic pathogen associated with various infections.
- Virulence factors like gelE, hyl, and asaI play crucial roles in E. faecalis pathogenicity.
- Rapid and accurate detection methods are essential for clinical and environmental monitoring.
Purpose of the Study:
- To develop and validate a multiplex PCR kit for the simultaneous detection of three major virulence genes (gelE, hyl, asaI) in E. faecalis.
- To assess the specificity and sensitivity of the developed multiplex PCR assay.
Main Methods:
- Design and optimization of primers for three target genes (gelE, hyl, asaI).
- Development of a multiplex PCR protocol for simultaneous amplification.
- Validation of the assay using a diverse collection of 243 E. faecalis strains from various sources.
- Determination of the limit of detection through serial dilutions and enrichment.
Main Results:
- The multiplex PCR assay successfully amplified specific products of 213 bp (gelE), 273 bp (hyl), and 713 bp (asaI) without non-specific amplification.
- The assay demonstrated high specificity, detecting only the targeted virulence genes.
- A sensitivity of 10 CFU/g or CFU/mL was achieved after a 3-hour enrichment step, indicating robust detection capabilities.
Conclusions:
- The developed multiplex PCR kit provides a specific, sensitive, and efficient tool for detecting key virulence genes in E. faecalis.
- This assay has potential applications in clinical diagnostics, food safety, and environmental surveillance of E. faecalis.
