The Use of Factorial Design, Image Analysis, and an Efficiency Calculation for Multiplex PCR Optimization

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...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Journal of clinical laboratory analysis 2013-05, Vol.27 (3), p.249-252
Hauptverfasser: Camacho, José Luis Villarreal, Torres, Evelyn Mendoza, Cadena, Christian, Prieto, Julieth, Prieto, Lourdes Luz Varela, Torregroza, Daniel Antonio Villanueva
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung: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 LabTM 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. Conclusion We propose the application of factorial design and image analysis to determine the most suitable conditions for multiplex PCR optimization.
ISSN:0887-8013
1098-2825
DOI:10.1002/jcla.21595