WHO-sponsored international collaborative study to evaluate methods for subtyping Listeria monocytogenes: Restriction fragment length polymorphism (RFLP) analysis using ribotyping and Southern hybridization with two probes derived from L-monocytogenes chromosome

Seven laboratories participated in a WHO-sponsored international collaborative study, to evaluate methods for subtyping Listeria monocytogenes, by performing restriction fragment length polymorphism (RFLP) analysis-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. monocytogenes. Six laboratories performed ribotyping. These laboratories used EcoRI enzyme to restrict the L. monocytogenes DNA and ribosomal RNA or DNA as the probe for Southern hybridizations. The seventh laboratory used NciI to restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences cloned from L. monocytogenes DNA. The overall discriminating power of ribotyping, as estimated by calculation of Simpson's index of diversity, ranged from 0.83 to 0.88 for the six laboratories. The discriminating power of the combination of two probes used by Laboratory 7 was 0.91. Ribotyping and the cloned probes used by Laboratory 7 discriminated poorly between serotype 4b strains. Neither method identified three atypical strains (identified by other subtyping methods) included in three apparently epidemiologically related groups. Ribotyping did not discriminate between strains of serotypes 1b and 4b(X) in one epidemiologically related group of strains; one cloned probe used by Laboratory 7 discriminated between these strains. Intra-laboratory reproducibilities for the seven laboratories ranged from 80.0 to 100%, as determined by their abilities to correctly identify 11 pairs of duplicate strains included in the study set. Inter-laboratory reproducibilities were generally very good considering that no attempt was made to standardize protocols used by the participants.