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United States Department of Agriculture

Agricultural Research Service

Title: Simultaneous Identification and Enumeration of Microorganisms by Filter Based in Situ Hybridization Using Enzyme-Labeled Peptide Nucleic Acid Probes

Authors
item Stender, H - BOSTON PROBES, INC.
item Perry-O'keefe, H - BOSTON PROBES, INC.
item Hyldig-Nielsen, J - BOSTON PROBES, INC.
item Broomer, A - BOSTON PROBES, INC.
item Oliveira, K - BOSTON PROBES, INC.
item Kurtzman, Cletus
item Coull, J - BOSTON PROBES, INC.

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 25, 2000
Publication Date: N/A

Technical Abstract: Peptide nucleic acid (PNA) probes open new possibilities for molecular diagnostic assays. PNA probes are able to hybridize to complementary nucleic acid targets obeying Watson-Crick base pairing rules. Due to their uncharged, neutral backbone, PNA probes have improved hybridization characteristics such as higher specificity and faster binding. In this study, we present a new filter-based in situ hybridization method using PNA probes for simultaneous identification and enumeration of microorganisms in liquid samples. The sample is filtered to isolate and separate individual microorganisms onto a membrane filter, which is then placed on a culture medium prior to testing. Micro-colonies are detected by in situ hybridization using peroxidase-labeled PNA probes targeting species-specific rRNA sequences. Excess probe is removed by washing, and hybridized probe is detected by a chemiluminescent reaction via exposure to a film. Each micro-colony is observed as a small dot on the film providing simultaneous identification and enumeration. This method has been applied successfully to bacteria and yeasts. Compared to traditional plating methods, this method is faster as micro-colonies are detected before they are visible by eye and definitive as no further identification of the microorganism is required. An assay for identification and enumeration of Dekkera bruxellensis, a spoilage yeast in wine, has already been developed. In the future, this method seems promising for a variety of different applications within food and clinical microbiology where both identification and enumeration are essential.

Last Modified: 4/17/2014
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