title: A multiplex real-time PCR assay for the identification and differentiation of Salmonella enterica serovar Typhimurium and monophasic serovar 4,[5],12:i:-.
abstract: Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is considered to be a monophasic variant of Salmonella Typhimurium and is increasingly associated with human infections. The use of PCR for the unequivocal identification of strains identified by conventional serotyping as 4,[5],12:i:- has been recommended by the European Food Safety Authority (EFSA), in particular the conventional multiplex PCR developed by Tennant et al. (2010). An alternative protocol for the identification and differentiation of S. Typhimurium and S. Typhimurium-like strains, including its monophasic variants, based on a multiplex real-time PCR assay was developed in our laboratory. A panel of 206 Salmonella strains was used to validate our multiplex real-time PCR against the conventional multiplex PCR recommended by EFSA, i.e. 43 Salmonella strains of serovars other than Typhimurium and 163 routine isolates determined by slide agglutination serotyping to have an incomplete antigenic formula compatible with the S. Typhimurium formula 4,[5],12:i:1,2. Both methods correctly identified the 43 Salmonella strains as non S. Typhimurium. Among the 163 isolates of undetermined serovar by conventional serotyping, both PCR protocols identified 54 isolates as S. Typhimurium, 101 as monophasic S. Typhimurium and 8 as non-S. Typhimurium. Twenty isolates phenotypically lacking the phase-2 H antigen were positive for the fljB.1,2 gene. These strains have been recently described in the literature by other workers and have been referred to as "inconsistent" variants of S. Typhimurium. Antimicrobial resistance and phage typing were also performed on the S. Typhimurium isolates, including monophasic variants, and approximately half of the isolates identified as monophasic S. Typhimurium by our multiplex real-time PCR protocol were DT193 with the resistance pattern ASSuT. There was 100% concordance between the conventional PCR and the multiplex real-time PCR method developed in this study which proved that our protocol is equivalent to the one recommended by EFSA. In comparison to the conventional PCR, this new protocol is faster and is currently being applied routinely in our laboratory to all isolates that could potentially be S. Typhimurium.
authors.affiliation: National Reference Laboratory for Salmonella (Food, Feed and Animal Health), Department of Agriculture, Food and the Marine, Backweston Complex, Celbridge, Co. Kildare, Ireland, Republic of Ireland. deirdre.prendergast@agriculture.gov.ie
authors.forename: Deirdre M
authors.initials: DM
authors.lastname: Prendergast
authors.forename: Darren
authors.initials: D
authors.lastname: Hand
authors.forename: Eadaoin
authors.initials: E
authors.lastname: Nί Ghallchóir
authors.forename: Evonne
authors.initials: E
authors.lastname: McCabe
authors.forename: Seamus
authors.initials: S
authors.lastname: Fanning
authors.forename: Margaret
authors.initials: M
authors.lastname: Griffin
authors.forename: John
authors.initials: J
authors.lastname: Egan
authors.forename: Montserrat
authors.initials: M
authors.lastname: Gutierrez
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D015373:Bacterial Typing Techniques / Q000379:methods*; D001434:Bacteriophage Typing; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction / Q000592:standards*; D012486:Salmonella typhimurium / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012703:Serotyping; D013045:Species Specificity
chemical_list: D000890:Anti-Infective Agents
keywords: 4,[5],12:i:–; Monophasic; Multiplex real-time PCR; Salmonella; Typhimurium