A Rapid and Simple Integrated extraction amplificaion and detection device for Y. pestir

This proposal is directed toward the development of a rapid, simple and efficient method for extraction, purification, amplification and detection of nucleic acids found in biological or environmental samples. This proposal will demonstrate the feasibility of using a novel self contained isothermal particle (SCIP) assay to detect Yersinia pestis. SCIP integrates extraction, purification, amplification and detection into a single testing device. The assay is expected to take less than an hour, yet provide the sensitivity and specificity afforded by nucleic acid based assays. The self contained nature of the device elminates possibility of contamination from previously ran samples. The extraction method is extremely efficient, with expected recoveries greater than 65%, and does not require the use of centrifugation or vacuum source. The nucleic acid is purified and free from inhibitors of amplification enzymes. The amplification strategy proposed is isothermal, eliminating the use of thermocycling equipment and produces high copy in less than 30 minutes. The amplified product is labeled for detection during the amplification reaction. The detection mechanism is based on microparticle and lateral flow chromatography technology. The assay will be easy to perform and can be completed in the field or at the first level of medical care. BENEFITS: The successful development of a SCIP assay for Y. pestis detection will make testing for plague in biological or environmental samples easy and rapid. Early detection will provide an opportunity for treatment before the disease progresses to fatality. SCIP testing increases the sensitivity, specificity and precision for detection of Y pestis. SCIP can be used in the field, eliminating the use of skilled laboratory personnel and sophisticated equipment. Based on preliminary cost analysis, SCIP, will provide substantial cost savings in terms of medical care. The SCIP concept is adaptable for detection of almost any organism with unique and determined genetic sequences.