多种绿色DNA嵌入染料实时荧光PCR检测猪DNA效果比较

Comparison of Multiple Green Fluorescence DNA Intercalating Dyes for Identify Pork DNA Using Real-Time PCR

实时荧光定量PCR是食品科学领域一种高效、灵敏的研究技术,荧光染料的种类和浓度会对实时PCR反应产生一定影响。以8种绿色荧光DNA嵌入染料SYTO 11~14,SYTO 16,SYTO 21,SYTO 24~25为研究对象,体系选择荧光染料浓度分别为10,5,2,1,0.5μmol/L,以从猪肉中提取的基因组DNA为模板进行扩增,产物长度149 bp。通过分析Ct值和熔解曲线,评价染料浓度对扩增效果的影响,初步筛选出最适染料及相应浓度:SYTO 11(5μmol/L,2μmol/L),SYTO 13(10μmol/L,5μmol/L),SYTO 16(10μmol/L,5μmol/L),SYTO 21(1μmol/L)和SYTO 24(0.5μmol/L)。根据筛选结果自配反应预混液,以2种商业实时荧光PCR预混液为参照,通过模板10倍梯度稀释绘制标准曲线,计算扩增效率,R2均大于0.99,线性范围均在40 ng^0.04 pg之间,其中染料SYTO 13(10μmol/L)和SYTO 16(10μmol/L,5μmol/L)扩增效率分别为99.35%,100.50%和99.71%,接近商业试剂盒2(BYRT染色,扩增效率99.61%),高于商业试剂盒1(Evagreen染色,扩增效率97.80%),具有实际应用价值。

Real-Time Polymerase Chain Reaction（PCR） is a highly efficient and sensitive technique in the field of food science. The variety and concentration of dyes have certain effects on the PCR reaction. In this study, eight kinds of green fluorescence DNA intercalating dyes including SYTO 11-14, SYTO 16, SYTO 21, SYTO 24 and SYTO 25 are selected as the object of study, using the primer pair of DNA extracted from the pork as a template for real-time PCR amplification with a production length of 149 bp. By analyzing Ctvalue and dissociation curve, and evaluating the effects of concentration of dyes on the amplification result, the following DNA intercalating dyes with corresponding concentrations are selected： SYTO 11（5 μmol/L, 2 μmol/L）, SYTO 13（10 μmol/L, 5 μmol/L）, SYTO 16（10 μmol/L, 5 μmol/L）,SYTO 21（2 μmol/L, 1 μmol/L）, SYTO 24（0.5 μmol/L）. Ten-fold dilution method was used to generate standard curves of all these laboratory-prepared mix and the screening results were compared with two kinds of commercial real-time PCR master mix being set as a reference. As a result, all the curves indicated R2〉0.985 with linear range between 40 ng and 0.04 pg. Particularly, the amplification efficiency of SYTO 13（10 μmol/L）, SYTO 16（10 μmol/L, 5 μmol/L） are respectively 99.35%, 100.50% and 99.71%, showing a similar efficiency with the referenced commercial master mix 2（made with BYRT, amplification efficiency 99.61%） and a higher efficiency than the referenced commercial master mix1（made with Eva Green, amplification efficiency 97.80%）, and have high value of further study for practical application.