Fluorescence PCR detection reagent capable of discriminating source components of ruminant animal, preparation method and application thereof



The invention relates to a biological detection reagent capable of discriminating three types of ruminant animal varieties simultaneously, a preparation method and an application thereof. The invention selects the specificity of the cow, goat and sheep, and the conservative sequence segment of conservative mitochondrial gene as target, applies Primer Eexpress 3.0 software and Primer Select software in DNAStar, and designs and synthesizes a plurality of groups of primers and probes. After being synthesized and marked, the plurality of pairs of designed primers and the probes are carried out best pairing screening experiment so as to obtain the fittest combination of primers and probes. The reagent contains two pairs of specificity primers, and three Taq Man probes, wherein one pair of theprimer aims at cow source components, the other pair of primer aims at common primers of goats and sheep source component detection; the three probes are respective source components to the cows, thegoats and the sheep; and the amplified target fragment length to the source component detection of the cows, the goats and the sheep is respectively 92bp, 136bp and 136bp. The triple Taq Man fluorescence PCR detection method can simultaneously carry out accurate quantitative detection to the source components of the cows, the goats and the sheep, has simple and convenient operation, intuitive result, strong specificity, high sensitivity and good repeatability and can realize high throughput detection of various source components.
本发明涉及一种能同时鉴别三种反刍动物品种的生物检测试剂,以及这种试剂的制备方法和应用。本发明是选择牛、山羊和绵羊特异和保守的线粒体基因保守序列片段为靶目标,应用Primer Express 3.0软件和DNAStar中的Primer Select软件,设计合成多组引物和探针。将设计的多对引物和探针合成和标记后进行最佳配对筛选实验,得到最适合的引物和探针组合。本发明所述的试剂包含两对特异性引物和三条Taq Man探针,其中,一对引物针对牛源性成分,另一对是针对山羊和绵羊源性成分检测的通用引物,三条探针分别针对牛、山羊和绵羊源性成分,对牛、山羊和绵羊源性成分检测的扩增目标片段长度分别为92bp、136bp和136bp。本发明中的三重Taq Man荧光PCR检测方法可同时对牛、山羊和绵羊源性成分进行准确的定性检测,操作简便,结果直观,特异性强,灵敏度高,重复性好,可实现多种源性成分的高通量检测。




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