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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
Author(s) -
Diana Stewart,
Tortorello,
Gendel
Publication year - 1998
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1046/j.1472-765x.1998.00279.x
Subject(s) - immunomagnetic separation , polymerase chain reaction , escherichia coli , feces , chromatography , biology , dna extraction , chloroform , microbiology and biotechnology , chemistry , biochemistry , gene
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.