Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p |
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Authors: | SE Pasteris & AM Strasser de Saad |
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Institution: | U.S. Food and Drug Administration, National Center for Food Safety and Technology, Summit-Argo, IL, USA |
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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 faeces using any appropriate PCR primers. |
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