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Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species

Tim Stakenborg, Jo Vicca, Patrick Butaye UGent, Dominiek Maes UGent, Thierry De Baere UGent, Rita Verhelst UGent, Johan Peeters, Aart de Kruif UGent, Freddy Haesebrouck UGent and Mario Vaneechoutte UGent (2005) BMC INFECTIOUS DISEASES. 5.
abstract
Background: Mycoplasmas are present worldwide in a large number of animal hosts. Due to their small genome and parasitic lifestyle, Mycoplasma spp. require complex isolation media. Nevertheless, already over 100 different species have been identified and characterized and their number increases as more hosts are sampled. We studied the applicability of amplified rDNA restriction analysis ( ARDRA) for the identification of all 116 acknowledged Mycoplasma species and subspecies. Methods: Based upon available 16S rDNA sequences, we calculated and compared theoretical ARDRA profiles. To check the validity of these theoretically calculated profiles, we performed ARDRA on 60 strains of 27 different species and subspecies of the genus Mycoplasma. Results: In silico digestion with the restriction endonuclease AluI (AG(boolean AND)CT) was found to be most discriminative and generated from 3 to 13 fragments depending on the Mycoplasma species. Although 73 Mycoplasma species could be differentiated using AluI, other species gave undistinguishable patterns. For these, an additional restriction digestion, typically with BfaI (C(boolean AND)TAG) or HpyF10VI (GCNNNNN(boolean AND)NNGC), was needed for a final identification. All in vitro obtained restriction profiles were in accordance with the calculated fragments based on only one 16S rDNA sequence, except for two isolates of M. columbinum and two isolates of the M. mycoides cluster, for which correct ARDRA profiles were only obtained if the sequences of both rrn operons were taken into account. Conclusion: Theoretically, restriction digestion of the amplified rDNA was found to enable differentiation of all described Mycoplasma species and this could be confirmed by application of ARDRA on a total of 27 species and subspecies.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
RIBOSOMAL-RNA GENES, POLYMERASE-CHAIN-REACTION, FRAGMENT-LENGTH-POLYMORPHISM, SUBSP MYCOIDES SC, CELL-CULTURES, AVIAN MYCOPLASMAS, PCR, DIFFERENTIATION, PHYLOGENY, DIAGNOSIS
journal title
BMC INFECTIOUS DISEASES
BMC Infect. Dis.
volume
5
article_number
46
pages
10 pages
Web of Science type
Article
Web of Science id
000230742600001
JCR category
INFECTIOUS DISEASES
JCR impact factor
1.956 (2005)
JCR rank
29/43 (2005)
JCR quartile
3 (2005)
ISSN
1471-2334
DOI
10.1186/1471-2334-5-46
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
338395
handle
http://hdl.handle.net/1854/LU-338395
date created
2006-06-08 08:59:00
date last changed
2010-08-12 10:20:06
@article{338395,
  abstract     = {Background: Mycoplasmas are present worldwide in a large number of animal hosts. Due to their small genome and parasitic lifestyle, Mycoplasma spp. require complex isolation media. Nevertheless, already over 100 different species have been identified and characterized and their number increases as more hosts are sampled. We studied the applicability of amplified rDNA restriction analysis ( ARDRA) for the identification of all 116 acknowledged Mycoplasma species and subspecies.
Methods: Based upon available 16S rDNA sequences, we calculated and compared theoretical ARDRA profiles. To check the validity of these theoretically calculated profiles, we performed ARDRA on 60 strains of 27 different species and subspecies of the genus Mycoplasma.
Results: In silico digestion with the restriction endonuclease AluI (AG(boolean AND)CT) was found to be most discriminative and generated from 3 to 13 fragments depending on the Mycoplasma species. Although 73 Mycoplasma species could be differentiated using AluI, other species gave undistinguishable patterns. For these, an additional restriction digestion, typically with BfaI (C(boolean AND)TAG) or HpyF10VI (GCNNNNN(boolean AND)NNGC), was needed for a final identification. All in vitro obtained restriction profiles were in accordance with the calculated fragments based on only one 16S rDNA sequence, except for two isolates of M. columbinum and two isolates of the M. mycoides cluster, for which correct ARDRA profiles were only obtained if the sequences of both rrn operons were taken into account.
Conclusion: Theoretically, restriction digestion of the amplified rDNA was found to enable differentiation of all described Mycoplasma species and this could be confirmed by application of ARDRA on a total of 27 species and subspecies.},
  articleno    = {46},
  author       = {Stakenborg, Tim and Vicca, Jo and Butaye, Patrick and Maes, Dominiek and De Baere, Thierry and Verhelst, Rita and Peeters, Johan and de Kruif, Aart and Haesebrouck, Freddy and Vaneechoutte, Mario},
  issn         = {1471-2334},
  journal      = {BMC INFECTIOUS DISEASES},
  keyword      = {RIBOSOMAL-RNA GENES,POLYMERASE-CHAIN-REACTION,FRAGMENT-LENGTH-POLYMORPHISM,SUBSP MYCOIDES SC,CELL-CULTURES,AVIAN MYCOPLASMAS,PCR,DIFFERENTIATION,PHYLOGENY,DIAGNOSIS},
  language     = {eng},
  pages        = {10},
  title        = {Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species},
  url          = {http://dx.doi.org/10.1186/1471-2334-5-46},
  volume       = {5},
  year         = {2005},
}

Chicago
Stakenborg, Tim, Jo Vicca, Patrick Butaye, Dominiek Maes, Thierry De Baere, Rita Verhelst, Johan Peeters, Aart de Kruif, Freddy Haesebrouck, and Mario Vaneechoutte. 2005. “Evaluation of Amplified rDNA Restriction Analysis (ARDRA) for the Identification of Mycoplasma Species.” Bmc Infectious Diseases 5.
APA
Stakenborg, T., Vicca, J., Butaye, P., Maes, D., De Baere, T., Verhelst, R., Peeters, J., et al. (2005). Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species. BMC INFECTIOUS DISEASES, 5.
Vancouver
1.
Stakenborg T, Vicca J, Butaye P, Maes D, De Baere T, Verhelst R, et al. Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species. BMC INFECTIOUS DISEASES. 2005;5.
MLA
Stakenborg, Tim, Jo Vicca, Patrick Butaye, et al. “Evaluation of Amplified rDNA Restriction Analysis (ARDRA) for the Identification of Mycoplasma Species.” BMC INFECTIOUS DISEASES 5 (2005): n. pag. Print.