title: Rapid determination of multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis.
abstract: This report presents a new method for identifying multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis (mu-TGGE). Genomic comparison of L. monocytogenes serovar 1/2a strains EGD-e and F6854 allowed selection of novel polymorphic sequences lmo0386 and lmo0428 as optimum regions for mu-TGGE analysis, in addition to the previously identified lmo0297 gene. Sequence analysis of a total of 48 standard strains revealed that the strains could be grouped into 7 (lmo0386), 8 (lmo0428) and 12 (lmo0297) sequence types. The PCR products from 2, 4 and 4 sequence types of the lmo0386, lmo0428 and lmo0297 genes were selected as marker alleles, and mu-TGGE analysis of the mixture revealed adequate band separation on a single gel. Furthermore, the primer sets could be successfully mixed in a single tube for multiplex PCR, yielding a rapid and easy strategy for sequence type identification. For practical application, multiplex PCR was performed with Cy3-labeled primers against a sequence type-unknown sample isolated from meat. The resulting products were mixed with Cy5-labeled products of marker alleles whose sequence types were known, and mu-TGGE analysis was performed. Overlapping Cy3 and Cy5 patterns allowed identification of the sequence types at all 3 loci on a single gel. Moreover, the mu-TGGE analysis step took only 9 min. Thus, this novel method of multiplex PCR followed by mu-TGGE analysis could prove useful as a rapid and discriminative tool for tracing the strain types, contamination routes and sources of L. monocytogenes.
authors.affiliation: Saitama Industrial Technology Center North Institute, 2-133, Suehiro, Kumagaya, Saitama 360-0031, Japan. tominaga@saitama-itcn.jp
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tominaga
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D002645:Chickens; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005849:Gerbillinae; D006168:Guinea Pigs; D006801:Humans; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011200:Poultry; D011817:Rabbits; D012756:Sheep; D013696:Temperature