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Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria.

Blixt, Ylva; Knutsson, Rickard LU ; Borch, Elisabeth and Rådström, Peter LU (2003) In International Journal of Food Microbiology 83(1). p.15-26
Abstract
A random amplified polymorphic DNA (RAPD) protocol was developed for interlaboratory use to discriminate food-borne Yersinia enterocolitica O:3 from other serogroups of Y. enterocolitica and from Y. enterocolitica-like species. Factors that were studied regarding the RAPD performance were choice of primers and concentration of PCR reagents (template DNA, MgCl2, primer and Taq DNA polymerase). A factorial design experiment was performed to identify the optimal concentrations of the PCR reagents. The experiment showed that the concentration of the PCR reagents tested significantly affected the number of distinct RAPD products. The RAPD protocol developed was evaluated regarding its discrimination ability using 70 different Yersinia strains.... (More)
A random amplified polymorphic DNA (RAPD) protocol was developed for interlaboratory use to discriminate food-borne Yersinia enterocolitica O:3 from other serogroups of Y. enterocolitica and from Y. enterocolitica-like species. Factors that were studied regarding the RAPD performance were choice of primers and concentration of PCR reagents (template DNA, MgCl2, primer and Taq DNA polymerase). A factorial design experiment was performed to identify the optimal concentrations of the PCR reagents. The experiment showed that the concentration of the PCR reagents tested significantly affected the number of distinct RAPD products. The RAPD protocol developed was evaluated regarding its discrimination ability using 70 different Yersinia strains. Cluster analysis of the RAPD patterns obtained revealed three main groups representing (i) Y. pseudotuberculosis, (ii) Y. enterocolitica and (iii) Y. kristensenii, Y. frederiksenii, Y. intermedia and Y. ruckeri. Within the Y. enterocolitica group, the European serovar (O:3) and the North American serovar (O:8) could be clearly separated from each other. All Y. enterocolitica O:3 strains were found in one cluster which could be further divided into two subclusters, representing the geographical origin of the isolates. Thus, one of the subclusters contained Y. enterocolitica O:3 strains originating from Sweden, Finland and Norway, while Danish and English O:3 strains were found in another subcluster together with O:9 and O:5,27 strains. The repeatability (intralaboratory) and reproducibility (interlaboratory) of the RAPD protocol were tested using 15 Yersinia strains representing different RAPD patterns. The intralaboratory and the interlaboratory studies gave similarity coefficients of the same magnitude (generally >70%) for the individual strains. In the present study, it was shown that interreproducible RAPD results could be achieved by appropriate optimisation of the RAPD protocol. Furthermore, the study reflects the heterogeneous genetic diversity of the Y. enterocolitica species. (Less)
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Contribution to journal
publication status
published
subject
in
International Journal of Food Microbiology
volume
83
issue
1
pages
15 - 26
publisher
Elsevier
external identifiers
  • wos:000182692300002
  • pmid:12672589
  • scopus:0037466732
ISSN
0168-1605
DOI
10.1016/S0168-1605(02)00319-7
language
English
LU publication?
yes
id
05818fc6-2bc5-4bb9-b041-f82780dfcbe1 (old id 132771)
date added to LUP
2007-06-28 11:31:19
date last changed
2018-05-29 12:25:59
@article{05818fc6-2bc5-4bb9-b041-f82780dfcbe1,
  abstract     = {A random amplified polymorphic DNA (RAPD) protocol was developed for interlaboratory use to discriminate food-borne Yersinia enterocolitica O:3 from other serogroups of Y. enterocolitica and from Y. enterocolitica-like species. Factors that were studied regarding the RAPD performance were choice of primers and concentration of PCR reagents (template DNA, MgCl2, primer and Taq DNA polymerase). A factorial design experiment was performed to identify the optimal concentrations of the PCR reagents. The experiment showed that the concentration of the PCR reagents tested significantly affected the number of distinct RAPD products. The RAPD protocol developed was evaluated regarding its discrimination ability using 70 different Yersinia strains. Cluster analysis of the RAPD patterns obtained revealed three main groups representing (i) Y. pseudotuberculosis, (ii) Y. enterocolitica and (iii) Y. kristensenii, Y. frederiksenii, Y. intermedia and Y. ruckeri. Within the Y. enterocolitica group, the European serovar (O:3) and the North American serovar (O:8) could be clearly separated from each other. All Y. enterocolitica O:3 strains were found in one cluster which could be further divided into two subclusters, representing the geographical origin of the isolates. Thus, one of the subclusters contained Y. enterocolitica O:3 strains originating from Sweden, Finland and Norway, while Danish and English O:3 strains were found in another subcluster together with O:9 and O:5,27 strains. The repeatability (intralaboratory) and reproducibility (interlaboratory) of the RAPD protocol were tested using 15 Yersinia strains representing different RAPD patterns. The intralaboratory and the interlaboratory studies gave similarity coefficients of the same magnitude (generally >70%) for the individual strains. In the present study, it was shown that interreproducible RAPD results could be achieved by appropriate optimisation of the RAPD protocol. Furthermore, the study reflects the heterogeneous genetic diversity of the Y. enterocolitica species.},
  author       = {Blixt, Ylva and Knutsson, Rickard and Borch, Elisabeth and Rådström, Peter},
  issn         = {0168-1605},
  language     = {eng},
  number       = {1},
  pages        = {15--26},
  publisher    = {Elsevier},
  series       = {International Journal of Food Microbiology},
  title        = {Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria.},
  url          = {http://dx.doi.org/10.1016/S0168-1605(02)00319-7},
  volume       = {83},
  year         = {2003},
}