结合荧光双链置换探针的特异性和实时PCR的准确定量能力，建立了一种新颖、准确、价廉且高通量的测定DNA池等位基因频率的方法，该方法采用不同荧光标记的双链置换探针1次PCR反应即可测定出等位基因频率，实验以β-地中海贫血CDs41-42(-TCTT)突变为对象 分别用荧光染料FAM和ROX标记野生型和突变型探针，由实时CR检测建立的等位基因浓度与循环阈值的响应曲线计算DNA池的等位基因频率，结果表明，建立的系列等位基因浓度与循环阈值呈线性关系，线性相关系数分别为0.9977(野生型等位基因)和0.9938（突变型等位基因），可检测的最低等位基因频率达1%等位基因频率在1%～90%范围内测定误差小于4%该方法可广泛用于流行病学调查、遗传连锁分析以及全基因组连锁不平衡扫描等领域。

以荧光染料Eam和Joe分别标记野生型和突变型双链探针作为均相检测探针，以构建的DNA模板作为研究模型 采用固定波长差同步荧光分析法对PCR反应产物进行终点检测，通过对HFE基因C282Y点突变的检测，并以限制性内切核酸酶Rsa1证实，该方法是一种廉价、快速、可靠的筛查遗传性血色病基因C282Y突变的方法，该法可扩展到各种基因的突变检测。

基于分子信标的原理，设计了一种发夹型荧光探针作为限制性内切酶作用的专一底物，来研究限制性内切酶的剪切作用。检测Bg/II和NcoI表明，这种探针不仅可以灵敏、实时地指示内切酶的活性，采用不同荧光标记的探针还可以同时特异地显示双酶切反应中两个酶各自的活性。并且以Rotor2Gene 2000实时荧光PCR仪作仪器检测，实现了高通量的操作。利用其特有的作变温曲线的功能，能很好的区分限制性内切酶与发夹型荧光探针的特异剪切与非特异的结合。

目的建立改良分子信标-实时PCR检测霍乱弧菌的快速方法，应用于霍乱监测。方法根据GenBank公布的霍乱弧菌肠毒素基因A亚单位(ctxA)的保守序列，设计一对引物和改良分子信标探针，并进行特异性和灵敏度分析； 同时以11种细菌作对照，建立改良分子信标检测霍乱弧菌的实时PCR反应体系，应用于霍乱监测。结果霍乱弧菌改良分子信标检测体系检测12种细菌，只有霍乱弧菌有荧光信号，与其他细菌无交叉反应，DNA灵敏度为102.4fg/ul，菌液灵敏度为32cfu/ml或3cfu反应体系，对100份海产品和30份腹泻病人大便标本进行检测，结果都为阴性。检测时间仅需2h结论改良分子信标-实时PCR检测体系快速、灵敏度高、特异性强，可用于霍乱的快速诊断，为霍乱的分子流行病学调查提供新的检测手段。∀

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant.