Open AccessThe Use of Factorial Design, Image Analysis, and an Efficiency Calculation for Multiplex PCR Optimization
Author(s) -
Camacho José Luis Villarreal,
Torres Evelyn Mendoza,
Cadena Christian,
Prieto Julieth,
Prieto Lourdes Luz Varela,
Torregroza Daniel Antonio Villanueva
Publication year - 2013
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.21595
Subject(s) - multiplex polymerase chain reaction , factorial experiment , multiplex , salmonella enterica , agarose , simulated annealing , salmonella , polymerase chain reaction , mathematics , biology , microbiology and biotechnology , gene , bioinformatics , genetics , statistics , bacteria , algorithm
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 MgCl 2 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 MgCl 2 . This finding was confirmed by calculating the efficiency of multiplex PCR assays (6.1%) at these conditions. Conclusion We propose the application of factorial design and image analysis to determine the most suitable conditions for multiplex PCR optimization.