An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

• Published in: Journal of microbiological methods 2003-11, Vol.55 (2), p.425-432
• Main Authors: Moorhead, Sandra M., Dykes, Gary A., Cursons, Ray T.
• Format: Article
• Language: English
• Subjects: Alleles; Animals; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cluster Analysis; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Food Microbiology; Fundamental and applied biological sciences. Psychology; Humans; Lineage; Listeria innocua; Listeria monocytogenes; Listeria monocytogenes - classification; Listeria monocytogenes - genetics; Listeriosis - diagnosis; Microbiology; Phylogeny; Polymerase Chain Reaction - methods; Polymorphism, Single Nucleotide; Sequence Alignment; Sequence Analysis, DNA; sigB; sigB gene; Sigma Factor - chemistry; Sigma Factor - genetics; Single nucleotide polymorphisms (SNPs); Systematics
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(03)00188-X

The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions. This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping. The sigB sequence was resolved in four local L. monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined. The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages. Based on nucleotide sequences L. monocytogenes lineage I/serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L. innocua strains clustered together as an outgroup. SNPs differentiating the three lineages were identified. Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L. monocytogenes isolates into the three lineages.