PMID: 11909738Mar 23, 2002Paper

A PCR-ELISA for detecting Shiga toxin-producing Escherichia coli

Microbes and Infection
Beilei GeJianghong Meng

Abstract

A sensitive and specific PCR-ELISA was developed to detect Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primer specific for Shiga toxin genes during PCR amplification. The labeled PCR products were bound to streptavidin-coated wells of a microtiter plate and detected by an ELISA. The specificity of the PCR was determined using 39 bacterial strains, including STEC, enteropathogenic E. coli, E. coli K12, and Salmonella. All of the STEC strains were positive, and non-STEC organisms were negative. The ELISA detecting system was able to increase the sensitivity of the PCR assay by up to 100-fold, compared with a conventional gel electrophoresis. The detection limit of the PCR-ELISA was 0.1-10 CFU dependent upon STEC serotypes, and genotypes of Shiga toxins. With the aid of a simple DNA extraction system, PrepMan, the PCR-ELISA was able to detect ca. 10(5) CFU of STEC per gram of ground beef without any culture enrichment. The entire procedure took about 6 h. Because of its microtiter plate format, PCR-ELISA is particularly suitable for large-scale screening and compatible with future automation.

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Citations

Jul 19, 2005·Journal of Clinical Laboratory Analysis·Jong-Wan KimSue-Nie Park
Jul 24, 2004·Molecular and Cellular Probes·Maria Isabel Queipo-OrtuñoPilar Morata
Oct 7, 2003·International Journal for Parasitology·J E HodgkinsonJ B Matthews
Jun 13, 2013·Foodborne Pathogens and Disease·Fei WangBeilei Ge
Jun 21, 2015·Journal of Microbiological Methods·Loreen R StrombergHarshini Mukundan
Feb 17, 2006·Clinica Chimica Acta; International Journal of Clinical Chemistry·Guangfu YuLiang Chen
Dec 1, 2005·Meat Science·H S Hussein, L M Bollinger
Apr 14, 2004·Journal of Applied Microbiology·K A O'HanlonD A McDowell
Apr 26, 2015·The Brazilian Journal of Infectious Diseases : an Official Publication of the Brazilian Society of Infectious Diseases·Jafar AmaniShahram Nazarian
Oct 26, 2005·Journal of Food Protection·Hussein S Hussein, Laurie M Bollinger
Jun 25, 2015·Microbiology Spectrum·Lothar Beutin, Patrick Fach

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