中文摘要：

结合双重PCR（Duplex PCR,dPCR）和扩增产物熔点的测定,建立了一种定量分析近缘物种混合样品中各物种相对含量的新方法。即针对混合样品中2种物种的DNA序列,各自设计高特异性引物,使2种物种的DNA同时进行PCR扩增而互不干扰,且扩增产物的熔点有足够的差别（〉1℃）;然后进行PCR扩增并测得不同温度下PCR产物同荧光染料结合后的荧光值;该荧光值对温度进行微分所得微分曲线上会产生2个峰（在两物种各自PCR产物熔点温度处）,根据这2个峰的峰高比值同其相对含量的关系,即可完成对各物种含量的定量分析。本研究以长鳍和黄鳍金枪鱼的混合样品为例,对此方法的可行性进行了验证。结果表明,2种成分的含量比与对应峰值比之间线性关系良好;当混合样品中含有10%以上长鳍金枪鱼或5%以上黄鳍金枪鱼时,能够进行较准确的定量检测,证明了本方法切实可行。这一新方法解决了在混合样品中难以准确定量物种的难题,在食品安全和基因诊断领域有广泛的应用前景。

英文摘要：

A novel quantitative analysis method for measuring the content of two close-related species in their mixture were developed based on the combination of duplex PCR（dPCR）and melting temperature（Tm）measurement of PCR products.At first,primers were designed to carry out dPCR to amplify simultaneously one DNA fragment from one species and another fragment from the other species.The two PCR products were designed to have enough difference in Tm（1℃）.Then dPCR was carried out,and the Tms of PCR products were measured by recoding the fluorescence change with temperature,which is caused by different intensity of fluorescence between dsDNA and ssDNA in the presence of a dsDNA-binding dye.There should be two peaks on the plot of first differential of fluorescence value to temperature at Tms of each PCR product,and the quantitative analysis of relative content can be carried out according to the ratio between the heights of two peaks.Here,mixtures of albacore and yellowfin tuna were taken as an example to assess the feasibility and efficiency of this method.The results showed that there is a good linearity between the content ratio and peak height ratio.The method can accurately quantify the content of each species,e.g.more than 10% of albacore or more than 5% of yellowfin tuna,indicating that this approach is practical.This novel method can solve the problem of quantitative analysis of mixed species and have a wide range of applications in food safety,genetic diagnosis and so on.