JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Evans, G. E.
Right arrow Articles by Chambers, S. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Evans, G. E.
Right arrow Articles by Chambers, S. T.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, July 2003, p. 3452-3453, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3452-3453.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Contamination of Qiagen DNA Extraction Kits with Legionella DNA


   LETTER
Top
 Letter
References
 
van der Zee and Crielaard recently reported the contamination of Qiagen DNA extraction columns with Legionella species (6). We have independently made this discovery and have attempted to further characterize the contaminating DNA.

We used the Qiagen columns (QIAamp DNA minikit; Qiagen, Hilden, Germany) for the extraction of DNA from a variety of clinical samples to be tested for Legionella DNA by PCR. When PCR assays targeting the Legionella 16S rRNA gene (1) or 5S rRNA gene (2) were used, positive signals were frequently recorded for the negative controls. The 5S rRNA amplicon contained a single TaqI restriction site, a characteristic of Legionella spp. that we have used to confirm the specificity of the PCR assay (2). Investigation of all potential sources of contamination indicated that the columns were the source and that contamination varied with different batches of columns. The 16S rRNA amplified product was sequenced by using primers for the Legionella 16S rRNA gene (1) and the Sanger dideoxy chain terminator method (5), and the sequence obtained was compared with those submitted to the GenBank database by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The closest matches for this sequence (with 96% identity) were Legionella-like amoebal pathogen (LLAP) strains 2, 3, 6, 7, 9, and 14, Legionella lytica strain L2, and Legionella birminghamensis. The alignment of the sequences from the contaminating product and these organisms can be seen in Table 1.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Partial sequence alignment of 16S rRNA region indicating alignment of legionella contaminant with nearest matches in a BLAST search

 
A PCR product was also obtained from a negative Qiagen extraction control, sequenced in the 23S-5S rRNA region (4), and submitted for identification in the same manner. Of the 312 bp of sequence submitted, only 246 bp shared any similarity with any reference species that had been submitted to the National Center for Biotechnology Information BLAST database. It was noted that no LLAP sequences for the 23S-5S rRNA region had been submitted; this may explain the poor degree of identification obtained. Alternatively, the product amplified in this region may not have been from the same source (organism) as that obtained in the 16S rRNA region. Limiting the sequence identification to the 5S rRNA region gave a 96% match with Legionella steigerwaltii (Z30463).

Sequencing using eubacterial primers (3) was also attempted, but this PCR was not adequately sensitive to detect a product in the water extract.

We agree with van der Zee and Crielaard (6) that the Qiagen columns are likely contaminated during production through the process of flushing with water containing Legionella spp. or LLAPs. Through this process the columns may also become contaminated with other environmental microorganisms. When using Qiagen QIAamp DNA minikit for extracting DNA for an Aspergillus PCR assay, we found that the columns and reagents were also frequently contaminated with fungal DNA (unpublished observations). Consequently, Qiagen columns at present may be unsuitable for the laboratory diagnosis of environmental microorganisms (especially those with a water habitat) and not just Legionella spp.


   REFERENCES
Top
 Letter
 References
 

  1. Jonas, D., A. Rosenbaum, S. Weyrich, and S. Bhakdi. 1995. Enzyme-linked immunoassay for detection of PCR-amplified DNA of Legionellae in bronchoalveolar fluid. J. Clin. Microbiol. 35:1247-1252.
  2. Murdoch, D. R., E. J. Walford, L. C. Jennings, G. J. Light, M. I. Schousboe, A. Y. Cheresky, S. T. Chambers, and G. I. Town. 1996. Use of the polymerase chain reaction to detect Legionella DNA in urine and serum from patients with pneumonia. Clin. Infect. Dis. 23:475-480.[Medline]
  3. Radstrom, P., A. Backman, N. Qian, P. Kragsbjerg, C. Pahlson, and P. Olcen. 1994. Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy. J. Clin. Microbiol. 32:2738-2744.[Abstract/Free Full Text]
  4. Robinson, P. N., B. Heidrich, F. Tiecke, F. J. Fehrenbach, and A. Rolfs. 1996. Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting. FEMS Microbiol. Lett. 140:111-119.[CrossRef][Medline]
  5. Sanger, F., S. Nicklen, and A. R. Coulsen. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.[Abstract/Free Full Text]
  6. van der Zee, A., and J. W. Crielaard. 2002. Qiagen DNA extraction kits for sample preparation for legionella PCR are not suitable for diagnostic purposes. J. Clin. Microbiol. 40:1126.[Free Full Text]
Gloria E. Evans
David R. Murdoch*
Trevor P. Anderson
Microbiology Unit
Canterbury Health Laboratories

Howard C. Potter
Peter M. George
Molecular Pathology
Canterbury Health Laboratories
P.O. Box 151
Christchurch
New Zealand

Stephen T. Chambers
Department of Pathology
Christchurch School of Medicine and Health Sciences
Christchurch
New Zealand

* Phone: 64 3 3641 530
Fax: 64 3 3640 238
E-mail: david.murdoch{at}cdhb.govt.nz

Journal of Clinical Microbiology, July 2003, p. 3452-3453, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3452-3453.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Evans, G. E.
Right arrow Articles by Chambers, S. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Evans, G. E.
Right arrow Articles by Chambers, S. T.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS