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Title: USE OF A GENUS- AND SPECIES-SPECIFIC MULTIPLEX PCR FOR IDENTIFICATION OF ENTEROCOCCI

Author
item Jackson, Charlene
item Cray, Paula
item Barrett, John

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2004
Publication Date: 8/20/2004
Citation: Jackson, C.R., Cray, P.J., Barrett, J.B. 2004. Use of a genus- and species-specific multiplex PCR for identification of enterococci. Journal of Clinical Microbiology. 42(8):3558-3565.

Interpretive Summary: Presently, no procedure exists that will allow genus and species identification of Enterococcus in a single reaction in a single day. The current classification and identification scheme for Enterococcus is both tedious and laborious and is based upon phenotypic analysis. To solve this problem, a multiplex PCR for genus and species identification of Enterococcus was developed. For the multiplex PCR, primers for detecting the genus Enterococcus were added to primers that will simultaneously detect the species. Because over 25 species of Enterococcus exist, 7 different multiplex reactions were designed for detection of the species. This invention will provide a cost-effective, rapid, and accurate procedure for identification of enterococci and will be useful for scientists in clinical laboratories and in basic research.

Technical Abstract: The 16S rRNA gene has previously been used to develop genus specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (sodA) has been identified as a potential target for species differentiation of enterococci. In this study, Enterococcus genus specific primers developed by Deasy et al (E1/E2) were incorporated with species specific primers based upon the superoxide dismutase (sodA) gene for development of a multiplex PCR, This assay provides simultaneous genus and species identification of 25 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources and retail food were evaluated and, overall, 90% of the isolates tested by PCR agreed with results obtained using VITEK. Eighty-five percent and 82% of PCR results agreed with results from API Rapid ID 32 Strep and BBL Crystal, respectively. With the exception of concurrence between identification using BBL Crystal/VITEK (83%), percent agreement for PCR was higher than any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.