刚果红荧光法测定ctDNA的研究

Spectrofluorometric Detection of ctDNA with Congo Red

为建立一种利用荧光测定ctDNA含量的新方法,利用荧光分析法的特点,对体系的pH、荧光试剂的浓度、试剂的添加顺序和干扰离子等实验条件进行优化,建立测定ctDNA含量的新方法。结果表明:在pH=7.4的Tris-HCl缓冲溶液中,十六烷基三甲基溴化铵(CTMAB)与DNA一起加入能大大增强刚果红的荧光强度。随着DNA浓度的不断增加,体系的荧光强度不断增强,在一定范围内,体系的△F与ctDNA的浓度呈良好的线性关系。在优化实验条件下,当ctDNA的浓度为0.005～3.600μg.mL-1时,回归方程为:△F=131.22c+3.854 3(c:mg.L-1),r=0.9988,检测限为0.01μg.mL-1。本方法首次成功地应用于羊驼血液中DNA的测定,回收率为96.25%～102.50%,结果令人满意。

The aim was to establish a new method for detecting ctDNA content by fluorescence.According to the characteristies of fluorescence analysis,each factor influencing ctDNA content detection such as pH value,the density of Congo red（CR）,the density of CTMAB,and the interference of the concomitant matter etc.were optimized and a new method for detecting ctDNA content was established.This paper discovers in the Tris-HCl buffer solution of pH=7.4,both ctDNA and CTMAB can strengthen significantly the fluorescence intensity of CR.With increasing of density of ctDNA,fluorescence intensity of the system of CR-CTMAB-ctDNA gets stronger and stronger,and what＇s more,within certain density range,△F of the system is directly proportional to the density of ctDNA.When ctDNA concentration is 0.005～3.600 μg·mL-1,the line square is △F=131.22c＋3.8543（c：mg·L-1）,the related coefficient is r =0.998 8,the detection limit is 0.01μg·mL-1.The proposed method has been successfully applied to the determination of DNA withdrawn from the alpaca blood and the recoveries were in the range of 96.25% to 102.50%with satisfactory results.