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Applied and Environmental Microbiology, March 2007, p. 2020-2023, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.01718-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry{triangledown}

Jeremy Lenaerts,1* Hilary M. Lappin-Scott,2 and Jonathan Porter3

Dako UK Ltd., Denmark House, Angel Drove, Ely, Cambridgeshire, United Kingdom,1 Department of Biological Sciences, University of Exeter, Prince of Wales Road, Exeter, Devon, United Kingdom,2 National Laboratory Service, Starcross Laboratory, Staplake Mount, Starcross, Devon, United Kingdom3

Received 21 July 2006/ Accepted 20 January 2007

Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.

* Corresponding author. Mailing address: Dako UK Ltd., Denmark House, Angel Drove, Ely, Cambridgeshire CB7 4ET, United Kingdom. Phone: 44-1353669911. Fax: 44-1353668989. E-mail: jeremy.lenaerts{at}dako.com.

{triangledown} Published ahead of print on 2 February 2007.

Applied and Environmental Microbiology, March 2007, p. 2020-2023, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.01718-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mahmoud, K. K., McNeely, D., Elwood, C., Koval, S. F. (2007). Design and Performance of a 16S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization. Appl. Environ. Microbiol. 73: 7488-7493 [Abstract] [Full Text]  


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