title: Intra- and inter-laboratory evaluation of an improved multiplex-PCR method for detection and typing of Salmonella.
abstract: BACKGROUND
  We developed and evaluated a multiplex-PCR method for rapid detection of the most common Salmonella serovars in both developed and developing countries. Additionally, the stability of the premixed reagents at high room temperature was studied.
  
  
  METHODS
  Fifty-two Salmonella strains belonging to the collections of the University of Sassari, Italy, and to the University of the Basque Country, Spain, and a collection of a hundred blinded strains, were used to evaluate the multiplex-PCR. Primers targeting genes STY1599 and fliC were selected, and the method was evaluated both intra and inter-laboratories.
  
  
  RESULTS
  The inter-laboratory reproducibility was 95.92%, with a kappa index of 0.757 that indicates a substantial agreement and a high accuracy (80.81%). The sensitivity, specificity, accuracy and precision indexes for the Salmonella genus and S. Typhi targets were maximum, although the targets for Paratyphi A, Typhimurium and Enteritidis showed less accuracy. During a seven-week period, hot-start multiplex-PCR runs were performed with reagents mixed with wax to test their stability at 30ºC, and no significant variation in the patterns of amplification was observed.
  
  
  CONCLUSIONS
  An improved multiplex-PCR for rapid detection of the most common serovars of Salmonella operable in both developed and developing countries has been designed and tested intra and inter-laboratories. Following a careful optimization protocol will not only allow the confirmation of any suspicious colony by the amplification of the Salmonella genus target, but also the preliminary adscription to the prevalent serovars. Premixed reagents with wax facilitate the throughput and stability of reagents at high room temperatures.
authors.affiliation: Department of Immunology, Microbiology and Parasitology, University of the Basque Country, Vitoria-Gasteiz, Spain.
authors.forename: Ilargi
authors.initials: I
authors.lastname: Martinez-Ballesteros
authors.forename: Bianca
authors.initials: B
authors.lastname: Paglietti
authors.forename: Aitor
authors.initials: A
authors.lastname: Rementeria
authors.forename: Lorena
authors.initials: L
authors.lastname: Laorden
authors.forename: Maria
authors.initials: M
authors.lastname: Garcia-Ricobaraza
authors.forename: Joseba
authors.initials: J
authors.lastname: Bikandi
authors.forename: Salvatore
authors.initials: S
authors.lastname: Rubino
authors.forename: Javier
authors.initials: J
authors.lastname: Garaizar
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007558:Italy; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012475:Salmonella / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012480:Salmonella Infections / Q000175:diagnosis* / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013030:Spain
chemical_list: D017931:DNA Primers; D011933:Reagent Kits, Diagnostic