Comparison of primers for the detection of pathogenic Escherichia coli using real-time PCR.
OBJECTIVE
To evaluate PCR primers for the detection of pathogenic Escherichia coli in a real-time PCR assay and determine their utility in produce irrigation water testing.


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 10(0) and 10(1) 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 10(1) and between 10(2) and 10(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.


CONCLUSIONS
The necessity of a dissociation analysis to distinguish positive samples from those with fluorescence of random dsDNA generation for real-time PCR in a complex background was established.
Barak
J D
JD
Produce Safety and Microbiology Research Unit, USDA Agricultural Research Service, Albany, CA 94710, USA. jbarak@pwusda.gov
Sananikone
K
K
Delwiche
M J
MJ
D018829:Adhesins, Bacterial / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics; D005516:Food Microbiology; D000455:Medicago sativa / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D014867:Water / Q000737:chemistry; D014871:Water Microbiology
D018829:Adhesins, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D014867:Water; C073819:eaeA protein, E coli