Jansson Linda, Koliana Marianne, Sidstedt Maja, Hedman Johannes
Applied Microbiology, Department of Chemistry, Lund University, Lund, SE-221 00, Sweden.
Swedish National Forensic Centre, Linköping, SE-591 94, Sweden.
Biotechnol Rep (Amst). 2017 Apr 5;14:34-37. doi: 10.1016/j.btre.2017.02.002. eCollection 2017 Mar.
The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.
实时荧光定量聚合酶链反应（qPCR）分析的成功部分受到核酸样品中抑制性化合物的限制。例如，土壤和水沉积物中的腐殖酸（HA）会干扰扩增，还会淬灭双链（ds）DNA结合染料的荧光，从而阻碍扩增子检测。我们旨在通过混合互补的dsDNA结合染料来抵消HA的荧光淬灭效应，从而提高染料饱和水平并增加荧光信号。与单独使用的染料和双染料混合物相比，EvaGreen、ResoLight、SYBR Green和SYTO9这四种染料的混合物在有和没有HA的情况下都能产生明显更高的荧光强度。我们提出混合染料是提高qPCR荧光信号从而在存在淬灭物质的情况下实现检测的一种普遍适用的方法。
