EVALUATION OF DNA PREPARATION TECHNIQUES FOR DETECTION OF THE SLT-1 GENE OF ESCHERICHIA-COLI O157-H7 IN BOVINE FECES USING THE POLYMERASE-CHAIN-REACTION

The polymerase chain reaction (PCR) has the potential to detect low le vels of the human pathogen Escherichia coli O157:H7 in bovine faeces. To improve the utility of PCR for this application, several methods fo r preparing template DNA from bovine faeces, both directly and after n on-selective enrichment, were tested. These were boiling, enzyme treat ment, enzyme treatment plus phenol-chloroform extraction, and enzyme t reatment plus phenol-chloroform extraction plus Geneclean(R) purificat ion. Of these, the boiling method was the most and had a sensitivity o f approximately 3 cfu g(-1) faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separatio n (IMS) to detect E. coli O157:H7 in less than 8 h, but with a sensiti vity of approximately 10(3) cfu g(-1) faeces. These methods can be use d to prepare template for PCR screening of bovine faeces using any app ropriate PCR primers.