Plasmid DNA positive control standards for molecular environmental assays

TECHNICAL ABSTRACT: We believe that employing current molecular biology techniques it is possible to develop plasmid DNA based positive control standard kits for a wide variety molecular assays for environmental organisms, and that this represents a significant commercial opportunity. In Phase I we demonstrated that plasmid DNA-based qPCR positive controls are stable, perform as well as genomic DNA positive controls and are technically feasible to produce at a commercially viable cost. In Phase II we intend to both improve the stability and ease of use of plasmid DNA based controls in laboratory PCR and qPCR assays as well as explore a wider variety of potential applications for such controls. Our objectives are to test methods to limit evaporation, determine minimum handling requirements and performance with & without background DNA of plasmid DNA controls. Additionally, we will test the utility of plasmids DNA controls to define the detection limits of multiple targets at different ratios in a multiplex qPCR, validate the detection sensitivity of new amplification technologies, and measure the stability and recovery efficiency of environmental DNA during collection, preservation and DNA extraction of environmental DNA samples. SUMMARY OF ANTICIPATED RESULTS: The anticipated results of this project will be readily available, accurately quantified positive control kits for molecular assays for environmental organisms that can provide more accurate and trustworthy results from testing that can have economic or public health impacts, better comparisons between results from different laboratories or procedures, or even different assays for the same organism.