Novel Real Time Isothermal DNA Amplification/Detection

描述(由申请人提供)：实时聚合酶链式反应仪器和试剂的出现使基因和信使RNA的快速和高度特异的量化成为可能(1-3)。实时聚合酶链式反应基因扩增显著改变了微生物病原体的检测、鉴定和量化的方式。有几种仪器可用于实时聚合酶链式反应检测，但这些仪器的每份样本成本较高。这笔费用是由于荧光标记引物的成本、对聚合酶链式反应专利的许可要求以及扩增/检测仪器和软件的复杂性(14-17)。在第二阶段，我们将进一步开发一种新的方法和仪器，用于实时、靶点特异的等温扩增和DNA定量，该方法不依赖于聚合酶链式反应，也不需要昂贵的荧光标记寡核苷酸引物。这种方法使用“发夹环链接体DNA扩增”(HLCA)过程扩增目标DNA序列，该过程在复制的最初几轮(5‘8)中将自启动的发夹寡聚DNA引物掺入到线性DNA中。随后的几轮复制是从这些发夹结构中启动的，以提供等温、不依赖于引物的串联DNA结构的扩增。从HCLA过程中扩增的DNA的积累可以用SYBR绿染料使用廉价的微滴定板进行实时监测。HCLA方法可以用未标记的引物进行。第一阶段获得的数据表明，HLCA方法可以在不到一小时的时间内扩增出微量的目标基因组DNA。如果完全实现，该方法和仪器将极大地节省成本，并在病原体和基因的快速定量方面具有广泛的适用性。使用HLCA的每份化验成本将不到0.50美元，而一台仪器的成本不到7500美元。这与目前的技术相比是有利的，每个样本的成本为5-15美元，每个仪器的成本为20,000-150,000美元。在第二阶段，将对该方法进行优化，并用研究和临床病原体样本确定其灵敏度限度。标准的微滴定板读取器将针对检测方法进行改进，以便在一次检测中处理和读取多达96个样本。一种简化的DNA提取方法将被纳入程序，允许从粗略的病原体样本开始。研究应用也将在第二阶段开发。该产品具有7.5亿美元的市场潜力，适用于临床、研究和国防应用。

DESCRIPTION (provided by applicant): The advent of real-time PCR instrumentation and reagents allows rapid and highly specific quantification of genes and messenger RNAs (1-3). Real-time PCR gene amplification has significantly changed the way microbial pathogens are detected, identified and quantified. Several instruments exist for real-time PCR detection, however these instruments have expensive per sample costs. This expense is due to the cost of fluorescent labeled primers, the requirement to license PCR patents, and the complexity of the amplification/detection instruments and software (14-17). In Phase II, we will further develop a.new method and instrument for real-time, target-specific isothermal amplification and DNA quantitation that does not rely on the polymerase chain reaction and does not require expensive fluorescent labeled oligo primers. This method amplifies the target DNA sequence using the "hairpin loop concatamer DNA amplification" (HLCA) process which incorporates self-priming hairpin oligo DNA primers into linear DNA during the initial rounds of replication (5'8). Subsequent rounds of replication are primed from these hairpin structures to give isothermal, primer-independent amplification of concatameric DNA structures. The accumulation of amplified DNA from HCLA process can be monitored in real-time with SYBR green dye using an inexpensive microtiter plate. The HCLA process can be performed with unlabeled primers. Data obtained in Phase I showed that the HLCA method could amplify trace amounts of target genomic DNA in less than one hour. The method and instrument, if fully realized, have tremendous cost saving and generalized applicability for rapid pathogen and gene quantitation. The cost per assay using HLCA will be less than $0.50, with an instrument costing less than $7500. This compares favorably to current technology costing $5-15 per sample and $20,000 to $150,000 per instrument. In Phase II, the method will be optimized and its sensitivity limits determined with research and clinical pathogen samples. A standard microtiter plate reader will be modified for the detection method to process and read up to 96 samples in one assay. A simplified DNA extraction method will be incorporated into the procedure, allowing it to begin with crude pathogen samples. Research applications will also be developed in Phase II. The product has a $750 million market potential addressing clinical, research and defense applications.