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

Abstract

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 103 cfu g -1 faeces. These methods can be used to prepare template for PCR screening of bovine facets using any appropriate PCR primers.