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 Wolffs, P.
Right arrow Articles by Rådström, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wolffs, P.
Right arrow Articles by Rådström, P.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, March 2004, p. 1042-1047, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1042-1047.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid Quantification of Yersinia enterocolitica in Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR

Petra Wolffs,1 Rickard Knutsson,2 Börje Norling,3 and Peter Rådström1*

Applied Microbiology, Lund Institute of Technology, Lund University, SE-221 00 Lund,1 Medprobe, IDEON Science Park, Scheelevägen 17, SE-223 70 Lund,2 Quintessence Research AB, Målarvägen 9, SE-746 93 Bålsta, Sweden3

Received 25 August 2003/ Returned for modification 2 November 2003/ Accepted 14 November 2003

The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 ± 0.021 and 1.007 ± 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 ± 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y. enterocolitica was possible down to a level of at least 4.2 x 103 CFU per ml of meat juice, even in the presence of 106 CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.

* Corresponding author. Mailing address: Applied Microbiology, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: 46 46-222 3412. Fax: 46 46-222 4203. E-mail: Peter.Radstrom{at}tmb.lth.se.

Journal of Clinical Microbiology, March 2004, p. 1042-1047, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1042-1047.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Malorny, B., Lofstrom, C., Wagner, M., Kramer, N., Hoorfar, J. (2008). Enumeration of Salmonella Bacteria in Food and Feed Samples by Real-Time PCR for Quantitative Microbial Risk Assessment. Appl. Environ. Microbiol. 74: 1299-1304 [Full Text]  
  • Fukushima, H., Katsube, K., Hata, Y., Kishi, R., Fujiwara, S. (2007). Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR. Appl. Environ. Microbiol. 73: 92-100 [Abstract] [Full Text]  
  • Wolffs, P. F. G, Glencross, K., Thibaudeau, R., Griffiths, M. W. (2006). Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR.. Appl. Environ. Microbiol. 72: 3896-3900 [Abstract] [Full Text]  
  • Lim, D. V., Simpson, J. M., Kearns, E. A., Kramer, M. F. (2005). Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare. Clin. Microbiol. Rev. 18: 583-607 [Abstract] [Full Text]  
  • Wolffs, P., Norling, B., Hoorfar, J., Griffiths, M., Radstrom, P. (2005). Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR. Appl. Environ. Microbiol. 71: 5759-5764 [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»