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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #180947
Title: COMPARISON OF PRIMERS FOR THE DETECTION OF PATHOGENIC E. COLI USING REAL-TIME PCR

Author
item Barak Cunningham, Jeri
item SANANIKONE, KATHY - UNIV. OF CALIFORNIA,DAVIS
item DELWICHE, MICHAEL - UNIV. OF CALIFORNIA,DAVIS

Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2005
Publication Date: 10/1/2005
Citation: Barak Cunningham, J.D., Sananikone, K., Delwiche, M. 2005 Comparison of primers for the detection of pathogenic e. coli using real-time pcr. Letters in Applied Microbiology. 41:112-118

Interpretive Summary: Aims: To evaluate polymerase chain reaction (PCR) primers for the detection of pathogenic E. coli in a real-time PCR assay and determine their utility in produce irrigation water testing. Methods and Results: Three previously published PCR primer sets and one set designed for this study were tested for their ability to produce products for several pathogenic E. coli from whole cells as template. Two of the previously published primer sets were chosen for real-time PCR detection limit determination. The coneaeA and PEH detection limit of E. coli O157:H7 was 1 and 10 CFU/rxn in sterile water, respectively. To detect E. coli O157:H7 in sprout irrigation water, the water required dilution due to PCR inhibitors. The detection limit of the coneaeA and PEH was 10 and between 100-1000 CFU/rxn in diluted sprout irrigation water, respectively. Conclusions: The primer set coneaeA was able to produce an amplification product from each E. coli, except O128:H7 and most sensitive for real-time PCR detection of pathogenic E. coli in diluted sprout irrigation water.

Technical Abstract: Aims: To evaluate PCR primers for the detection of pathogenic E. coli in a real-time PCR assay and determine their utility in produce irrigation water testing. Methods and Results: Three previously published PCR primer sets and one set designed for this study were tested for their ability to produce amplification products for several pathogenic E. coli serotypes from whole cells as template. Two of the previously published primer sets were chosen for real-time PCR detection limit determination. The coneaeA and PEH detection limit of E. coli O157:H7 was 100 and 101 CFU rxn-1 in sterile water, respectively. To detect E. coli O157:H7 in sprout irrigation water, the water required dilution due to PCR inhibitors. The detection limit of the coneaeA and PEH was 101 and between 102-3 CFU rxn-1 in diluted sprout irrigation water, respectively. Conclusions: The primer set coneaeA was able to produce an amplification product from each E. coli serotype, except O128:H7 and most sensitive for real-time PCR detection of pathogenic E. coli in diluted sprout irrigation water.