Rapid quantification of Yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR.
The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 +/- 0.021 and 1.007 +/- 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 +/- 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y. enterocolitica was possible down to a level of at least 4.2 x 10(3) CFU per ml of meat juice, even in the presence of 10(6) CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.
Applied Microbiology, Lund Institute of Technology, Lund University, SE-221 00 Lund, Sweden.
Petra
P
Wolffs
Rickard
R
Knutsson
Börje
B
Norling
Peter
P
Rådström
D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D005243:Feces / Q000382:microbiology; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013552:Swine / Q000382:microbiology*; D015008:Yersinia enterocolitica / Q000235:genetics / Q000302:isolation & purification*