López-Andreo María, Garrido-Pertierra Amando, Puyet Antonio
Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
J Agric Food Chem. 2006 Oct 18;54(21):7973-8. doi: 10.1021/jf0615045.
Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine, horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle (C(t)) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactions were 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificity of the PCR products was tested by the identification of peaks in DNA melting curves, measured as the decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could be distinguished above the background even at the lowest amount of template DNA detected by the C(t) method. The system was also tested in duplex reactions, by use of either single-species DNA or DNA admixtures containing different shares of two species. The minimum proportions of each DNA species allowing the resolution of T(m) peaks in the duplex reactions were 5% (cattle or wallaroo) in cattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1% wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A loss in the sensitivity of the method was observed for some DNA combinations in the duplex assay. In contrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-species DNA were largely comparable to those obtained previously with species-specific TaqMan probes, showing the suitability of that simpler experimental approach for large-scale analytical applications.
对采用SYBR Green I PCR后熔解曲线分析的实时单重和双重聚合酶链反应（PCR）检测方法进行了评估，以鉴定和定量食品中的牛、猪、马和大袋鼠DNA。定量值来自从纯化DNA系列稀释液获得的阈值循环（C(t)）数据。单重反应中的检测限为猪和大袋鼠DNA 0.04 pg，牛和马DNA 0.4 pg。通过鉴定DNA熔解曲线中的峰来测试PCR产物的物种特异性，熔解曲线通过测量解离温度下SYBR Green I荧光的降低来确定。即使在C(t)方法检测到的最低模板DNA量下，也能在背景之上区分峰。该系统还通过使用单物种DNA或含有两种物种不同比例的DNA混合物进行双重反应测试。在牛/大袋鼠混合物中，允许分辨双重反应中T(m)峰的每种DNA物种的最小比例为5%（牛或大袋鼠），在猪/马混合物中为5%猪和1%马，在猪/大袋鼠混合物中为60%猪和1%大袋鼠，在牛/马混合物中为1%牛和5%马。在双重检测中，观察到某些DNA组合的方法灵敏度有所损失。相比之下，使用单物种DNA的SYBR Green I单重和双重反应获得的结果与先前使用物种特异性TaqMan探针获得的结果在很大程度上具有可比性，表明这种更简单的实验方法适用于大规模分析应用。
