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Rapid quantification of yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR

Wolffs, Petra LU ; Knutsson, Rickard LU ; Norling, B and Rådström, Peter LU (2004) In Journal of Clinical Microbiology 42(3). p.1042-1047
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Clinical Microbiology
volume
42
issue
3
pages
1042 - 1047
publisher
American Society for Microbiology
external identifiers
  • pmid:15004051
  • wos:000220376900016
  • scopus:1542513866
ISSN
1098-660X
DOI
10.1128/JCM.42.3.1042-1047.2004
language
English
LU publication?
yes
id
0198b852-c039-432b-b60a-b4ac3f0d3f11 (old id 140490)
date added to LUP
2007-06-28 14:41:51
date last changed
2017-05-28 04:24:35
@article{0198b852-c039-432b-b60a-b4ac3f0d3f11,
  abstract     = {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.},
  author       = {Wolffs, Petra and Knutsson, Rickard and Norling, B and Rådström, Peter},
  issn         = {1098-660X},
  language     = {eng},
  number       = {3},
  pages        = {1042--1047},
  publisher    = {American Society for Microbiology},
  series       = {Journal of Clinical Microbiology},
  title        = {Rapid quantification of yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR},
  url          = {http://dx.doi.org/10.1128/JCM.42.3.1042-1047.2004},
  volume       = {42},
  year         = {2004},
}