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地定量基因和信使RNA（1-3）。实时PCR基因扩增显著改变了微生物病原体检测、鉴定和定量的方式。存在几种用于实时PCR检测的仪器，然而这些仪器具有昂贵的每份样品成本。该费用是由于荧光标记引物的成本、PCR专利许可的要求以及扩增/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a.new方法和仪器，用于实时、靶特异性等温扩增和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“发夹环多联体DNA扩增”（HLCA）过程扩增靶DNA序列，该过程在最初的几轮复制期间将自引发发夹寡聚DNA引物掺入线性DNA中（5 '8）。随后的几轮复制从这些发夹结构引发，以产生多联体DNA结构的等温、引物非依赖性扩增。可以使用廉价的微量滴定板，用SYBR绿色染料实时监测来自HCLA过程的扩增DNA的积累。HCLA过程可以用未标记的引物进行。在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痕量的靶基因组DNA。该方法和仪器，如果完全实现，具有巨大的成本节约和快速病原体和基因定量的普遍适用性。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。这与目前每个样品5 -15美元和每个仪器20，000 - 150，000美元的技术相比是有利的。在第二阶段，将对该方法进行优化，并使用研究和临床病原体样本确定其灵敏度限度。将对检测方法的标准微量滴定板读数器进行修改，以在一次试验中处理和读取多达96份样本。一种简化的DNA提取方法将被纳入程序，使其开始与粗病原体样品。第二阶段还将开发研究应用程序。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.