Advanced

Towards an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.

Lübeck, P S; Wolffs, Petra LU ; On, S L; Ahrens, P; Rådström, Peter LU and Hoorfar, J (2003) In Applied and Environmental Microbiology 69(9). p.5664-5669
Abstract
As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors... (More)
As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Applied and Environmental Microbiology
volume
69
issue
9
pages
5664 - 5669
publisher
American Society for Microbiology
external identifiers
  • wos:000185437000075
  • pmid:12957958
  • scopus:17344386538
ISSN
0099-2240
DOI
10.1128/AEM.69.9.5664-5669.2003
language
English
LU publication?
yes
id
609b5bfd-6f0a-475e-9a63-c648ed35ad26 (old id 132812)
date added to LUP
2007-06-28 16:16:50
date last changed
2018-01-07 05:51:06
@article{609b5bfd-6f0a-475e-9a63-c648ed35ad26,
  abstract     = {As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.},
  author       = {Lübeck, P S and Wolffs, Petra and On, S L and Ahrens, P and Rådström, Peter and Hoorfar, J},
  issn         = {0099-2240},
  language     = {eng},
  number       = {9},
  pages        = {5664--5669},
  publisher    = {American Society for Microbiology},
  series       = {Applied and Environmental Microbiology},
  title        = {Towards an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.},
  url          = {http://dx.doi.org/10.1128/AEM.69.9.5664-5669.2003},
  volume       = {69},
  year         = {2003},
}