PCR detection of Escherichia coli O157:H7 directly from stools: evaluation of commercial extraction methods for purifying fecal DNA.
Rapid identification of Escherichia coli O157:H7 is important for patient management and for prompt epidemiological investigations. We evaluated one in-house method and three commercially available kits for their ability to extract E. coli O157:H7 DNA directly from stool specimens for PCR. Of the 153 stool specimens tested, 107 were culture positive and 46 were culture negative. The sensitivities and specificities of the in-house enrichment method, IsoQuick kit, NucliSens kit, and QIAamp kit were comparable, as follows: 83 and 98%, 85 and 100%, 74 and 98%, and 86 and 100%, respectively. False-negative PCR results may be due to the presence of either inherent inhibitors or small numbers of organisms. The presence of large amounts of bacteria relative to the amount of the E. coli O157:H7 target may result in the lower sensitivities of the assays. All commercial kits were rapid and easy to use, although DNA extracted with the QIAamp kit did not require further dilution of the DNA template prior to PCR.
Holland
J L
JL
Department of Microbiology, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ontario, Canada.
Louie
L
L
Simor
A E
AE
Louie
M
M
D003470:Culture Media; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000382:microbiology; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
D003470:Culture Media; D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic