Identification of Listeria Monocytogenes by Accuprobe™(Accuprobe Rapid DNA Probe Test)

Laboratory Procedure MFLP-88
October 1996

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Health Protection Branch
Ottawa

Jeffrey M. Farber and Elaine Daley
Microbiology Research Division
Bureau of Microbial Hazards, Food Directorate
HPB, Postal Locator: 2204A2
Ottawa, ON KIA OL2

1. Application

This method can be used to identify presumptive colonies of Listeria monocytogenes isolated using methods MFLP-74 and MFHPB-30.

2. Principle

The Accuprobe™ Listeria Monocytogenes Identification Test offers a rapid objective method for the definitive identification of L. monocytogenes based on the detection of specific ribosomal RNA sequences that are unique to L. monocytogenes.

Nucleic acid hybridization tests are based on the ability of complementary nucleic acid strands to specifically align and associate to form stable double-stranded complexes. The Accuprobe™ system uses a chemiluminescent acridinium ester labelled, single-stranded DNA probe, that is complementary to the ribosomal RNA of the target organism. When the labelled DNA probe hybridizes with the target organism's ribosomal RNA to form a stable DNA: RNA hybrid, the acridinium is protected from chemical hydrolysis and can react with hydrogen peroxide under basic conditions to produce chemiluminescence. If the probe remains unbound, the ester bond undergoes hydrolysis and renders the acridinium permanently nonchemiluminescent. The labelled DNA:RNA hybrids are measured by a Gen-Probe luminometer. A positive result is a luminometer reading equal to or greater than a cut-off value. Total time of performing the test is less than one hour.

AccuProbe is a trademark of Gen-Probe Incorporated, San Diego, CA 92121.

3. Definition of Terms

See Appendix A of Volume 3.