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


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.


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


CONCLUSIONS
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.
Perry-O'Keefe
H
H
Boston Probes, Bedford, MA, USA.
Stender
H
H
Broomer
A
A
Oliveira
K
K
Coull
J
J
Hyldig-Nielsen
J J
JJ
D001419:Bacteria / Q000145:classification / Q000254:growth & development / Q000302:isolation & purification; D015169:Colony Count, Microbial; D003470:Culture Media; D005374:Filtration / Q000295:instrumentation / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D008163:Luminescent Measurements; D008851:Micropore Filters / Q000382:microbiology*; D015335:Molecular Probes / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000235:genetics*; D009195:Peroxidase / Q000378:metabolism; D013045:Species Specificity; D014965:X-Rays; D015003:Yeasts / Q000145:classification / Q000254:growth & development / Q000302:isolation & purification
D003470:Culture Media; D015335:Molecular Probes; D020135:Peptide Nucleic Acids; D009195:Peroxidase