Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR.
OBJECTIVE
To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater.


RESULTS
Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater.


CONCLUSIONS
The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples.


CONCLUSIONS
The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.
Ibekwe
A M
AM
USDA-ARS-George E. Brown Jr Salinity Laboratory, 450 W. Big Springs Road, Riverside, CA 92507, USA. aibekwe@ussl.ars.usda.gov
Grieve
C M
CM
D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D003612:Dairying / Q000379:methods; D004926:Escherichia coli / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D008372:Manure / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D014871:Water Microbiology
D003470:Culture Media; D004269:DNA, Bacterial; D008372:Manure