Novel Real Time Isothermal DNA Amplification/Detection

描述（由申请人提供）：实时PCR仪器和试剂的出现允许快速和高度特异性的基因和信使rna定量（1-3）。实时PCR基因扩增极大地改变了微生物病原体检测、鉴定和定量的方式。存在几种实时PCR检测仪器，但是这些仪器的每个样品成本昂贵。这笔费用是由于荧光标记引物的成本，许可PCR专利的要求，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，目标特异性等温扩增和DNA定量，不依赖于聚合酶链反应，也不需要昂贵的荧光标记寡核苷酸引物。该方法使用“发夹环串联DNA扩增”（HLCA）过程扩增目标DNA序列，该过程在初始复制阶段将自引发夹寡核苷酸引物纳入线性DNA（5'8）。随后的几轮复制从这些发夹结构中引物，以获得等温、引物独立的串联DNA结构扩增。HCLA过程中扩增DNA的积累可以用SYBR绿色染料使用廉价的微量滴度板实时监测。HCLA过程可以用未标记的引物进行。I期数据显示，HLCA方法可以在不到1小时的时间内扩增微量目标基因组DNA。该方法和仪器如能完全实现，将大大节省成本，广泛适用于病原体和基因的快速定量。使用HLCA的每次检测成本将低于0.50美元，仪器成本低于7500美元。与目前的技术相比，每个样品的成本为5-15美元，每个仪器的成本为20,000至150,000美元。在二期试验中，将对该方法进行优化，并根据研究和临床病原体样本确定其灵敏度限。标准微量滴度板阅读器将被修改为检测方法处理和读取多达96个样品在一次分析。一种简化的DNA提取方法将纳入该程序，使其能够从原始病原体样本开始。第二阶段亦会发展研究应用。该产品具有7.5亿美元的市场潜力，可用于临床、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.