Development of multilocus single strand conformation polymorphism (MLSSCP) analysis of virulence genes of Listeria monocytogenes and comparison with existing DNA typing methods.
Development of rapid and simple typing methods is required for analyzing the distribution and contamination routes of food-borne pathogens. We established a simple typing method for Listeria monocytogenes using MLSSCP (Multilocus Single Strand Conformation Polymorphism) analysis. Four virulence genes, hlyA, iap, actA and inlB were amplified by PCR, digested with endonucleases and applied to gels for SSCP. As banding patterns have been shown to reflect even a single nucleotide difference, this method has a potential discriminatory power comparable to that of sequencing analysis. The 64 strains isolated from five meat processing plants were divided into 18 groups by this MLSSCP. Additionally, clustering obtained with this method showed strong correspondence with phylogenetic lineages I and II, and was achieved with much less expenditure in time and cost than is required for other methods, such as MLST. The validity of the MLSSCP lineage classification was confirmed by PFGE, AFLP and ribotyping results. This newly developed MLSSCP method is suitable when obtaining accurate results quickly and simply is crucial.
Takahashi
Hajime
H
Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
Handa-Miya
Satoko
S
Kimura
Bon
B
Sato
Miki
M
Yokoi
Asami
A
Goto
Seitaro
S
Watanabe
Itaru
I
Koda
Takashi
T
Hisa
Kazuo
K
Fujii
Tateo
T
D016000:Cluster Analysis; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D005838:Genotype; D008089:Listeria monocytogenes / Q000145:classification / Q000472:pathogenicity*; D008461:Meat Products / Q000382:microbiology*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity; D013045:Species Specificity; D037521:Virulence Factors / Q000235:genetics*