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Transferring PCR assays into isothermal platforms: How to make sure it works in real world settings.
Timothy Miles: California State University-Monterey Bay
<div>PCR technologies have been the backbone of molecular diagnostics in plant pathology for almost 30 years. Utilizing these tools researchers have been able to detect various species and genotypes in a variety of environmental samples (e.g. soil, air, and plant tissue). While PCR is powerful it is not without limitations including; 1) the requirement of very clean template DNA, 2) the amount of time required to extract this DNA from environmental samples, and 3) a requirement of a high level of skill to run the assays. Isothermal assays offer significant advantages over traditional PCR assays. Many approaches exist but the two main techniques include loop mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Like any molecular technology there are advantages and disadvantages to either technique but both approaches are tolerant of PCR inhibitors, can be completed within 30 minutes and can utilize more crude template DNA. Another plus is these assays require significantly less training to perform and have been successfully deployed in field settings to many different groups of plant pathogens. We will discuss examples used in transferring a mitochondrial based PCR marker system for <i>Phytophthora</i> species to LAMP and RPA as well as other examples and compare these approaches in terms of sensitivity, specificity and overall accuracy of the platforms.</div>

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