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Comparison of primers for the detection of pathogenic Escherichia
coli using real-time PCR.
Lett Appl Microbiol. 2005;41(2):112-8.
Barak JD,
Sananikone K,
Delwiche MJ.
Produce Safety and Microbiology Research Unit, USDA Agricultural Research
Service, Albany, CA, USA.
Aims: 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. 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 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. Significance and Impact of the Study: 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.
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