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.
