Preparation of PCR samples from food by a rapid and simple centrifugation technique evaluated by detection of Escherichia coli O157:H7.
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 BacXtractorTM) 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 x 10(4) cfu g-1), respectively. The enrichment of spiked samples in buffered peptone water at 37 degrees 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.
Lindqvist
R
R
National Food Administration, Uppsala, Sweden. roli@slv.se
D002499:Centrifugation, Density Gradient; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005516:Food Microbiology; D018189:Immunomagnetic Separation; D016133:Polymerase Chain Reaction