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Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells

Wolffs, Petra LU ; Norling, B and Rådström, Peter LU (2005) In Journal of Microbiological Methods 60(3). p.315-323
Abstract
Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred... (More)
Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized. (C) 2004 Published by Elsevier B.V. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Microbiological Methods
volume
60
issue
3
pages
315 - 323
publisher
Elsevier
external identifiers
  • wos:000226646300003
  • pmid:15649533
  • scopus:12144272204
ISSN
1872-8359
DOI
10.1016/j.mimet.2004.10.003
language
English
LU publication?
yes
id
412e843b-9b48-4e04-aad3-1c443ecf4e83 (old id 151273)
date added to LUP
2007-06-28 14:43:35
date last changed
2017-11-05 03:44:55
@article{412e843b-9b48-4e04-aad3-1c443ecf4e83,
  abstract     = {Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized. (C) 2004 Published by Elsevier B.V.},
  author       = {Wolffs, Petra and Norling, B and Rådström, Peter},
  issn         = {1872-8359},
  language     = {eng},
  number       = {3},
  pages        = {315--323},
  publisher    = {Elsevier},
  series       = {Journal of Microbiological Methods},
  title        = {Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells},
  url          = {http://dx.doi.org/10.1016/j.mimet.2004.10.003},
  volume       = {60},
  year         = {2005},
}