This Article
Right arrow Full Text
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Killgore, G. E.
Right arrow Articles by Tenover, F. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Killgore, G. E.
Right arrow Articles by Tenover, F. C.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, July 2000, p. 2516-2519, Vol. 38, No. 7
0095-1137/00/$04.00+0

A 5' Nuclease PCR (TaqMan) High-Throughput Assay for Detection of the mecA Gene in Staphylococci

George E. Killgore,1,* Brian Holloway,2 and Fred C. Tenover1

Hospital Infections Program1 and Scientific Resources Program,2 Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 27 January 2000/Returned for modification 24 March 2000/Accepted 15 April 2000

In an effort to find a rapid, efficient, and reliable method of screening large numbers of bacterial isolates for specific antimicrobial resistance genes, we compared conventional PCR results to the results generated using the TaqMan 5' nuclease PCR kit in conjunction with an ABI Prism 7700 Sequence Detector for detecting the mecA gene in various species of staphylococci. DNA was extracted using two techniques. The first used a high-salt extraction method suitable for conventional PCR but resulted in a 7.2% rate of PCR inhibition with the TaqMan technique. PCR inhibition could be overcome by diluting samples 1:5 prior to testing. The second method used the Qiagen QIAamp Tissue Kit; no instances of PCR inhibition were encountered with this method. A total of 197 (96%) of the 206 samples with no inhibition showed agreement between the two methods. Eight of the nine disagreements were likely the result of low-level DNA cross contamination caused by frequent specimen handling. Target DNA in all eight of these samples was first detected in the initial tests only after >30 PCR cycles, and all were negative upon repeat testing even after 40 PCR cycles using freshly extracted DNA. Among those positive samples in agreement, target DNA was invariably detected before 30 PCR cycles. The TaqMan assay eliminated the need to load, run, stain, and read agarose gels and provided the advantage of instant detection of PCR product by laser-activated fluorescence. Thus, final results were obtained 2 h after PCR was initiated, as opposed to a requirement of 2 days to examine 96 samples by agarose gel electrophoresis.

* Corresponding author. Mailing address: Hospital Infections Program (G08), Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-0192. Fax: (404) 639-1381. E-mail: gek1{at}cdc.gov.

Journal of Clinical Microbiology, July 2000, p. 2516-2519, Vol. 38, No. 7
0095-1137/00/$04.00+0


This article has been cited by other articles:

  • Tenover, F. C., McAllister, S., Fosheim, G., McDougal, L. K., Carey, R. B., Limbago, B., Lonsway, D., Patel, J. B., Kuehnert, M. J., Gorwitz, R. (2008). Characterization of Staphylococcus aureus Isolates from Nasal Cultures Collected from Individuals in the United States in 2001 to 2004. J. Clin. Microbiol. 46: 2837-2841 [Abstract] [Full Text]  
  • Ruimy, R., Dos-Santos, M., Raskine, L., Bert, F., Masson, R., Elbaz, S., Bonnal, C., Lucet, J.-C., Lefort, A., Fantin, B., Wolff, M., Hornstein, M., Andremont, A. (2008). Accuracy and Potential Usefulness of Triplex Real-Time PCR for Improving Antibiotic Treatment of Patients with Blood Cultures Showing Clustered Gram-Positive Cocci on Direct Smears. J. Clin. Microbiol. 46: 2045-2051 [Abstract] [Full Text]  
  • Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., Yao, J. D. C., Wengenack, N. L., Rosenblatt, J. E., Cockerill, F. R. III, Smith, T. F. (2006). Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 19: 165-256 [Abstract] [Full Text]  
  • Wisplinghoff, H., Ewertz, B., Wisplinghoff, S., Stefanik, D., Plum, G., Perdreau-Remington, F., Seifert, H. (2005). Molecular Evolution of Methicillin-Resistant Staphylococcus aureus in the Metropolitan Area of Cologne, Germany, from 1984 to 1998. J. Clin. Microbiol. 43: 5445-5451 [Abstract] [Full Text]  
  • Warren, D. K., Liao, R. S., Merz, L. R., Eveland, M., Dunne, W. M. Jr. (2004). Detection of Methicillin-Resistant Staphylococcus aureus Directly from Nasal Swab Specimens by a Real-Time PCR Assay. J. Clin. Microbiol. 42: 5578-5581 [Abstract] [Full Text]  
  • Carneiro, L. A.M., Queiroz, M. L.P., Merquior, V. L.C. (2004). Antimicrobial-resistance and enterotoxin-encoding genes among staphylococci isolated from expressed human breast milk. J Med Microbiol 53: 761-768 [Abstract] [Full Text]  
  • Mothershed, E. A., Cassiday, P. K., Pierson, K., Mayer, L. W., Popovic, T. (2002). Development of a Real-Time Fluorescence PCR Assay for Rapid Detection of the Diphtheria Toxin Gene. J. Clin. Microbiol. 40: 4713-4719 [Abstract] [Full Text]  
  • Knutsson, R., Lofstrom, C., Grage, H., Hoorfar, J., Radstrom, P. (2002). Modeling of 5' Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica. J. Clin. Microbiol. 40: 52-60 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»