Evaluation of DNA preparation techniques for detection of the SLT-1 gene of Escherichia coli O157: H7 in bovine faeces using the polymerase chain reaction.
The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157: H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g-1 faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157: H7 in less than 8 h, but with a sensitivity of approximately 10(3) cfu g-1 faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers.
Stewart
D S
DS
U.S. Food and Drug Administration, National Center for Food Safety and Technology, Summit-Argo, IL 60501, USA. dss@vm.cfsan.fda.gov
Tortorello
M L
ML
Gendel
S M
SM
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis*; D019453:Escherichia coli O157 / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D022622:Shiga Toxin 1
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D022622:Shiga Toxin 1