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Development of a PCR assay for rapid detection of enterococci
D Ke1, F J Picard, F Martineau
1Centre de Recherche en Infectiologie de l'Université Laval, Sainte-Foy, Québec, Canada G1V 4G2.
Abstract:
Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.
Insights
A new PCR assay detects enterococci, common hospital pathogens, rapidly at the genus level. This method targets the tuf gene for faster identification than traditional culture methods.
Area of Science:
- Microbiology
- Molecular Biology
Background:
- Enterococci are significant nosocomial pathogens.
- Increasing vancomycin resistance in enterococci is a concern.
- Conventional identification methods are time-consuming (2-3 days).
Purpose of the Study:
- To develop a rapid, culture-independent PCR assay for genus-level enterococcal detection.
- To target the tuf gene for broad enterococcal identification.
- To overcome limitations of existing species-specific PCR assays.
Main Methods:
- Designed degenerate PCR primers targeting conserved regions of the tuf gene.
- Sequenced amplified 803 bp regions from four enterococcal species.
- Identified conserved regions for genus-specific primer design.
- Tested assay with 15 enterococcal species and 79 non-enterococcal species.
Main Results:
- The tuf gene PCR assay successfully identified 14 of 15 enterococcal species.
- No amplification was observed for most non-enterococcal species, with exceptions.
- The assay efficiently detected 159 clinical enterococcal isolates.
- Sequence variations within amplicons suggest potential for species-specific probes.
Conclusions:
- A rapid PCR assay targeting the tuf gene enables genus-level detection of enterococci.
- This assay offers a faster alternative to conventional methods in clinical microbiology.
- Potential exists for further development into species-specific identification tools.