Preparation of PCR samples from food by a rapid and simple centrifugation technique evaluated by detection of Escherichia coli O157:H7

Abstract

A sample treatment method based on buoyant density centrifugation which separates bacteria from food, concentrates bacteria and removes PCR inhibitors is described. The method involves a one minute centrifugation of food homogenate layered over a gradient medium (Percoll® or BacXtractor ™) in Eppendorf tubes, followed by a single wash step. The small scale of this treatment makes it possible to process many samples in a short time. To evaluate the method beef and minced beef samples, spiked with strains of Escherichia coli O157;H7, were treated and then analysed by PCR aimed at verocytotoxin- ( VT1 and VT2) and eae-genes. The detection limits in 1:10 ( w v ) beef and minced beef homogenates were 125–250 cfu ml −1 (1250–2500 cfu g −1) and 1000 cfu ml −1 (1 × 10 4 cfu g −1), respectively. The enrichment of spiked samples in buffered peptone water at 37 °C for 6 hours before buoyant density centrifugation and PCR, allowed 0.5 cfu g −1 beef and 5 cfu g −1 minced beef to be detected. This combination of enrichment and buoyant density centrifugation was also used for analysis of 43 beef samples from a consignment in which E. coli O157:H7 had been detected, and detected VT-genes in all 43 samples. E. coli O157:H7 was also separated and detected in spiked samples of milk, lettuce, shrimps, and blue cheese at arbitrary concentrations of 3000 cfu ml −1. The present sample preparation method has the potential to be applicable to many other combinations of bacteria and food, and in connection with other detection methods than PCR as well.