WHO-SPONSORED INTERNATIONAL COLLABORATIVE STUDY TO EVALUATE METHODS FOR SUBTYPING LISTERIA-MONOCYTOGENES - RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM (RFLP) ANALYSIS USING RIBOTYPING AND SOUTHERN HYBRIDIZATION WITH 2 PROBES DERIVED FROM L-MONOCYTOGENES CHROMOSOME

Seven laboratories participated in a WHO-sponsored international colla borative study, to evaluate methods for subtyping Listeria monocytogen es, by performing restriction fragment length polymorphism (RFLP) anal ysis-based subtyping of an international study set of 80 strains of L. monocytogenes that included 12 epidemiologically related groups. The RFLP analysis was done by Southern hybridization with one of two types of probes found in multiple copies on the chromosome of L. monocytoge nes. Six laboratories performed ribotyping. These laboratories used Ec oRI enzyme to restrict the L. monocytogenes DNA and ribosomal RNA or D NA as the probe for Southern hybridizations. The seventh laboratory us ed NciI to restrict the DNA, and two probes, one randomly cloned and t he other containing repeat sequences cloned from L. monocytogenes DNA. The overall discriminating power of ribotyping, as estimated by calcu lation of Simpson's index of diversity, ranged from 0.83 to 0.88 for t he six laboratories. The discriminating power of the combination of tw o probes used by Laboratory 7 was 0.91. Ribotyping and the cloned prob es used by Laboratory 7 discriminated poorly between serotype 4b strai ns. Neither method identified three atypical strains (identified by ot her subtyping methods) included in three apparently epidemiologically related groups. Ribotyping did not discriminate between strains of ser otypes 1b and 4b(X) in one epidemiologically related group of strains; one cloned probe used by Laboratory 7 discriminated between these str ains. Intra-laboratory reproducibilities for the seven laboratories ra nged from 80.0 to 100%, as determined by their abilities to correctly identify 11 pairs of duplicate strains included in the study set. Inte r-laboratory reproducibilities were generally very good considering th at no attempt was made to standardize protocols used by the participan ts.