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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.

La concentration optimale de 6-carboxy-X-rhodamine (ROX), utilisée comme colorant passif de référence dans les réactions en chaîne de polymérase quantitatives en temps réel (PCR) avec balise moléculaire, a été déterminée avec le système de PCR quantitatif Mx4000™ Multiplex. De plus, les effets d’un changement des concentrations de ROX sur la reproductibilité, les valeurs de Ct et l’efficacité de la PCR ont été examinés dans ce système en utilisant des données obtenues suite à l’amplification du gène de la toxine shiga 2 de Escherichia coli (stx2) et du gène luxS de Campylobacter jejuni. Cette étude a indiqué que différentes concentrations de ROX influencent plusieurs aspects de la PCR en temps réel. La variation de la concentration de ROX pourrait avoir des conséquences dans l’analyse de données quantitatives et pourrait conduire à des résultats erronés. Cette étude indique de plus que la concentration optimale de ROX est de 60 nmol/L pour la PCR en temps réel avec balise moléculaire lors des essais PCR des gènes luxS et stx2.

Document Type: Research Article

Publication date: March 1, 2007

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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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