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J Appl Microbiol. 2001 Feb;90(2):180-9.

Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms.

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1
Boston Probes, Bedford, MA, USA.

Abstract

AIMS:

A method for rapid and simultaneous detection, identification and enumeration of specific micro-organisms using Peptide Nucleic Acid (PNA) probes is presented.

METHODS AND RESULTS:

The method is based on a membrane filtration technique. The membrane filter was incubated for a short period of time. The microcolonies were analysed by in situ hybridization, using peroxidase-labelled PNA probes targeting a species-specific rRNA sequence, and visualized by a chemiluminescent reaction. Microcolonies were observed as small spots of light on film, thereby providing simultaneous detection, identification and enumeration. The method showed 95-100% correlation to standard plate counts along with definitive identification due to the specificity of the probe.

CONCLUSION:

Using the same protocol, results were generated approximately three times faster than culture methods for Gram-positive and -negative bacterial species and yeast species.

SIGNIFICANCE AND IMPACT OF THE STUDY:

The method is an improvement on the current membrane filtration technique, providing rapid determination of the level of specific pathogens, spoilage or indicator micro-organisms.

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