三种荧光染料SYBR GreenⅠ、LCGreen PLUS、EvaGreen在实时定量PCR应用中的比较

Comparison of SYBR GreenⅠ,LCGreen PLUS,EvaGreen in quantitative real-time PCR

目的探讨定量PCR中三种荧光染料SYBR GreenⅠ、LC Green PLUS、EvaGreen定量效果的差异。方法以三个样品基因组DNA为模板,应用巢氏PCR法先扩增G6PDH大片段,纯化回收产物,定量,并分别以10倍、5倍和2倍倍比稀释,作为定量PCR制作标准曲线的模板。应用real-time PCR仪指定试剂盒检测模板质量,再分别以含有相同Taq酶并分别含有SYBR GreenⅠ、LC Green PLUS、EvaGreen染料的试剂进行小片段定量PCR扩增,每个浓度3复孔。标准曲线拟合,结果分析用仪器自带分析软件完成。结果应用三种荧光染料获得的标准曲线回归系数≥0.98（除1个样品为0.97）,10倍、5倍梯度稀释标准曲线PCR效率为0.8~1.0,2倍稀释标准曲线PCR效率应用SYBR GreenⅠ有2/3样品〉1.2,而后两种染料为0.9~1.2。应用SYBR GreenⅠ较另两种染料的标准曲线误差较大,且样品间分散度也较大。结论在定量PCR中饱和染料LC Green PLUS、EvaGreen的定量效果好于SYBR GreenⅠ。

Objective To compare with SYBR GreenⅠ,LCGreen PLUS,EvaGreen in quantitative real- time PCR（ qPCR）. Methods Large G6 PDH fragment（ 960 bp） was amplified from 3 g DNA samples by nest PCR. PCR products were purified and quantified,then diluted 7 times with 10-,5-,2- fold as the templates of qPCR. The quality of templates was tested with qPCR kit of Roche,which is the start kit of the qPCR instrument. Short G6 PDH fragment（ 76bp） was amplified by qPCR with the same Taq enzyme and different dye,SYBR GreenⅠ,LCGreen PLUS,EvaGreen respectively. Commercial software was used to analyze data. Results R2 of all standard curves from 3 dyes were equal or more than 0. 98（ 0. 97 in a sample）. PCR efficiency of 10- fold and 5- fold dilution standard curve were 0. 8- 1. 0 in all 3 dyes. PCR efficiency of 2- fold dilution was spacemore than 1. 2 in 2 /3 samples with SYBR GreenⅠ,while 0. 9-1. 2 in all others. The estimate error in standard curve with SYBR GreenⅠwas larger and more dispersed than in those with other two dyes. Conclusion Saturated dye,LCGreen PLUS and EvaGreen may be better in quantity of qPCR.