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Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation

Knutsson, Rickard LU ; Fontanesi, M; Grage, Halfdan LU and Rådström, Peter LU (2002) In International Journal of Food Microbiology 72(3). p.185-201
Abstract
A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation <5%. When a background flora was present at concentrations greater than or equal to10(6)... (More)
A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation <5%. When a background flora was present at concentrations greater than or equal to10(6) (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment, Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10(1) (CFU/ml) Y enterocolitica in the presence of at least an inoculation concentration of 10(3) (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR. (C) 2002 Elsevier Science B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
dynamic detection range, amplification efficiency, PCR inhibitors, real-time PCR, Yersinia enterocolitica, sample preparation, experimental design, medium, YPCE
in
International Journal of Food Microbiology
volume
72
issue
3
pages
185 - 201
publisher
Elsevier
external identifiers
  • pmid:11845818
  • wos:000173701300001
  • scopus:0037022311
ISSN
0168-1605
DOI
10.1016/S0168-1605(01)00636-5
language
English
LU publication?
yes
id
ad10e93f-7858-4aa0-824a-703f6ae9d9d1 (old id 343866)
date added to LUP
2007-11-02 11:17:08
date last changed
2017-01-01 04:53:00
@article{ad10e93f-7858-4aa0-824a-703f6ae9d9d1,
  abstract     = {A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation &lt;5%. When a background flora was present at concentrations greater than or equal to10(6) (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment, Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10(1) (CFU/ml) Y enterocolitica in the presence of at least an inoculation concentration of 10(3) (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR. (C) 2002 Elsevier Science B.V. All rights reserved.},
  author       = {Knutsson, Rickard and Fontanesi, M and Grage, Halfdan and Rådström, Peter},
  issn         = {0168-1605},
  keyword      = {dynamic detection range,amplification efficiency,PCR inhibitors,real-time PCR,Yersinia enterocolitica,sample preparation,experimental design,medium,YPCE},
  language     = {eng},
  number       = {3},
  pages        = {185--201},
  publisher    = {Elsevier},
  series       = {International Journal of Food Microbiology},
  title        = {Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation},
  url          = {http://dx.doi.org/10.1016/S0168-1605(01)00636-5},
  volume       = {72},
  year         = {2002},
}