The use of factorial design, image analysis, and an efficiency calculation for multiplex PCR optimization.
J Clin Lab Anal
; 27(3): 249-52, 2013 May.
Article
en En
| MEDLINE
| ID: mdl-23686781
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.CONCLUSION:
We propose the application of factorial design and image analysis to determine the most suitable conditions for multiplex PCR optimization.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proyectos de Investigación
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Procesamiento de Imagen Asistido por Computador
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Técnicas de Tipificación Bacteriana
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Reacción en Cadena de la Polimerasa Multiplex
Idioma:
En
Revista:
J Clin Lab Anal
Asunto de la revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Año:
2013
Tipo del documento:
Article
País de afiliación:
Colombia
Pais de publicación:
EEUU
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ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA