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Optimization of 6-carboxy-X-rhodamine concentration for real-time polymerase chain reaction using molecular beacon chemistry

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Abstract:

The optimal 6-carboxy-X-rhodamine (ROX) concentration, which is used as a passive reference dye for real-time quantitative polymerase chain reaction (PCR) with molecular beacon chemistry, was determined with the Mx4000™ Multiplex Quantitative PCR System. Additionally, the effects of changing ROX concentrations on PCR reproducibility, Ct values, and efficiency were investigated with this system by using the PCR data obtained from amplification of the Escherichia coli shiga toxin 2 (stx2) gene and the Campylobacter jejuni luxS gene. This study indicated that different ROX concentrations influence many aspects of the real-time PCR reaction. ROX concentration variation could have consequences in the analysis of quantitative data and may lead to erroneous results. This study further indicated that the optimal ROX concentration is 60 nmol/L for real-time PCR, using molecular beacon chemistry for PCR assay of luxS and stx2 genes.

Keywords: PCR efficiency; PCR en temps réel; ROX concentration; balise moléculaire; concentration de ROX; efficacité de la PCR; fluorescence intensity; intensité de fluorescence; molecular beacon; real-time PCR

Document Type: Research Article

DOI: https://doi.org/10.1139/W06-145

Publication date: 2007-03-01

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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