title: The use of factorial design, image analysis, and an efficiency calculation for multiplex PCR optimization.
abstract: BACKGROUND
  The quality of multiplex polymerase chain reaction (PCR) assays depends on several factors. Therefore, it is important to establish the optimal conditions to achieve efficient amplification. The objective of this study was to implement a 5 × 4 factorial design combined with image analysis using agarose gels and an efficiency calculation to optimize a multiplex PCR assays for the detection of Salmonella enterica serovar typhimurium.
  
  
  METHODS
  We used 12 ng of Salmonella DNA obtained from pure cultures and applied different annealing temperatures (65°C, 64.5°C, 63.3°C, 61.4°C, or 59°C) and different MgCl2 concentrations (1 mM, 1.5 mM, 2 mM, or 2.5 mM) to amplify regions of the fliC, rfbJ, and fljB genes. The 5 × 4 factorial design was performed using Statgraphics Plus software version 5.1, and the images were analyzed using Image Lab(TM) software.
  
  
  RESULTS
  Superior amplification was obtained using an annealing temperature of 65°C and 2 mM MgCl2 . This finding was confirmed by calculating the efficiency of multiplex PCR assays (6.1%) at these conditions.
  
  
  CONCLUSIONS
  We propose the application of factorial design and image analysis to determine the most suitable conditions for multiplex PCR optimization.
authors.affiliation: Medicine Program, Universidad Libre Seccional Barranquilla, Colombia. jvillarreal@unilibrebaq.edu.co
authors.forename: José Luis
authors.initials: JL
authors.lastname: Villarreal Camacho
authors.forename: Evelyn
authors.initials: E
authors.lastname: Mendoza Torres
authors.forename: Christian
authors.initials: C
authors.lastname: Cadena
authors.forename: Julieth
authors.initials: J
authors.lastname: Prieto
authors.forename: Lourdes Luz
authors.initials: LL
authors.lastname: Varela Prieto
authors.forename: Daniel Antonio
authors.initials: DA
authors.lastname: Villanueva Torregroza
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012107:Research Design; D012485:Salmonella typhi / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
chemical_list: D004269:DNA, Bacterial