描述了一种利用特殊的双链引物突触引物，用于核酸扩增的特异定量检测，在传统的引物的5，端标记荧光物质，而在引物的互补序列的3，端标记荧光淬灭剂以封闭其延伸，二者杂交即成双链突触引物，在制备PCR反应混合物阶段以及加热的初期，突触引物保持稳定的双链结构而不能引导扩增，在退火阶段，突触引物部分解链导致引物延伸，荧光物质与淬灭剂分开而产生荧光，利用人β珠蛋白基因对此方法进行了验证，这种可自行退火的荧光引物不仅简单地实现了实时扩增，同时比常规的热启动PCR更有效地提高了扩增的特异性。

Simple and reliable genotyping technology is a key to success for high-throughput genetic screening in the post-genome era. Here we have developed a new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes. The speci

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.

A rapid and simple homogeneous fluorescence PCR assay was developed for the clinical diagnosis of infectious diseases based on a molecular beacon. The established method could reproducibly detect Mycobacterium tuberculosis at the 10 bacteria/mL level. The analytical specificity was tested with 14 strains of mycobacterium, four unrelated bacteria and 220 negative samples; no false positive results were obtained. A blind test was also performed to evaluate its performance in Mycobacterium tuberculosis diagnosis. The results showed that both the clinical sensitivity and the specificity were 100%, and that the detection limit was in the range of 1-10 bacteria/ml. A clinical study with 466 patient samples demonstrated that fluorescence PCR assay correlated well with smear (93.6%) and culture (98.4%) methods for positive samples. However, fluorescence PCR could detect positive samples (62.9%) more than smear (30.3%) and culture (31.4%), indicating a higher sensitivity of the present method than the traditional ones. The feasibility of this method was further approved by successful detection of Neisseria gonorrhoeae and Chlamydia trachomatis.

将对虾白斑杆状病毒的一段特异性DNA设计成分子信标探针，用于该病毒的PCR检测，温度与荧光强度之间的关系表明，所设计探针的发夹既可以形成也可以打开，符合PCR对分子信标探针的要求，结果表明，在PCR同时加入分子信标探针不影响PCR扩增，分子信标探针只能与目的DNA杂交，具有较高的特异性，随着PCR循环数的增加以及含目的DNA的质粒拷贝数的增加，荧光强度都随之增强。

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。∀

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。

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。

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。

目的建立改良分子信标2双重实时PCR同时检测霍乱弧菌和副溶血弧菌的快速方法，应用于霍乱监测、副溶血弧菌食物中毒的快速诊断和海产品检验。方法根据GenBank公布的霍乱弧菌肠毒素基因A亚单位（ctxA）和副溶血弧菌的耐热直接溶血毒素基因（TDH）的保守序列，分别设计引物和改良分子信标探针，以10种细菌作对照，建立双重实时PCR2改良分子信标检测体系，应用于副溶血弧菌食物中毒快速诊断和霍乱监测。结果改良分子信标2双重实时PCR 反应体系DNA灵敏度为102.4～166.6fgPμl，菌液灵敏度为32～64CFUPml或3～6CFUPPCR反应体系，无交叉反应。此反应体系同时检测40株副溶血弧菌和50株霍乱弧菌，均出现特异的荧光信号，两种细菌检测互不干扰。对3起细菌性食物中毒共48份样品和100份海产品进行检测，9份副溶血弧菌实时PCR阳性，其中7份副溶血弧菌细菌培养阳性，其余样品都为阴性。从样品处理到检测结果仅需1天时间。结论改良分子信标2双重实时PCR 检测体系快速、灵敏度高、特异性强，可用于霍乱和副溶血弧菌食物中毒的快速诊断，为食源性疾病的分子流行病学调查提供新的检测手段。