Optimization of 6-carboxy-X-rhodamine concentration for real-time polymerase chain reaction using molecular beacon chemistry
Optimization of 6-carboxy-X-rhodamine concentration for real-time polymerase chain reaction using molecular beacon chemistry
复制标题
DOI：
10.1139/w06-145
复制
发表时间：
2007-03-01
影响因子：
2.8
通讯作者：
Mesa, Christine
中科院分区：
文献类型：
--
作者：
Wang, Gehua;Becker, Erin;Mesa, Christine
6-羧基- x -罗丹明（ROX）作为被动参比染料用于分子信标化学实时定量聚合酶链反应（PCR），用Mx4000（TM）多重定量PCR系统测定其最佳浓度。此外，利用大肠杆菌志贺毒素2 （stx2）基因和空肠弯曲杆菌luxS基因扩增的PCR数据，研究不同ROX浓度对该系统PCR重现性、Ct值和效率的影响。本研究表明，不同的ROX浓度会影响实时PUR反应的许多方面。ROX浓度的变化可能对定量数据的分析产生影响，并可能导致错误的结果。本研究进一步表明，实时PCR的最佳ROX浓度为60 nmol/L，采用分子信标化学对luxS和stx2基因进行PCR检测。
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(TM) 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 PUR 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.