A Low-Cost Isothermal Amplification Microdevice for Rapid Colorimetric Detection Applied to Body Fluid Identification and Y-screening

As submitted by the proposer: There is no question that PCR has dominated the DNA amplification landscape as a result of the specificity inherent to thermocycling and the ability for rapid generation of billions of copies of an amplicon of a specific size. Isothermal amplification (LAMP) has lurked in the background for two decades and, despite the allure of dodging thermocycling and heat denaturation, it has not been widely-adopted. This is likely due to the need for more primers, and that the amplicons generated cover a broad size range. However, LAMP is ideal for qualitative assays because it is highly-specific, generates more amplicon DNA than PCR, and can be read-out colorimetrically; hence, the researchers propose application to body fluid identification (bfID) and Y-screening. With bfID, accurate presumptive/confirmatory tests are essential for gaining contextual information for crime scene investigators yet reliable assays are scarce. False positive results are not uncommon with biochemical-based tests that lack specificity, and many methods are known to be destructive to the sample and/or inhibit downstream processes. This has prompted a paradigm shift to nucleic acid testing for screening body-fluids and for Y-screening. The latter is equally important because a positive screen for the presence of a body fluid, is not always informative for the downstream DNA analysis. Y-screening is currently performed with real time PCR assays, requiring costly instrumentation; and rt-PCR requires the use of known DNA to create a standard curve for each plate. For the purposes of Y-screening, there is value in detecting a minimum threshold of male DNA – it is here that LAMP is ideal. We propose LAMP for rapid Y-screen and bfID including: venous and menstrual blood, semen, saliva, and vaginal fluid using colorimetric response and smartphone detection. The method could offer an inexpensive alternative to screening samples, and easily implementable into forensic casework. We show evidence that LAMP is superior to current presumptive/confirmatory testing in because: 1) mRNA is targeted with high tissue specificity because of multiple primer-sets; 2) proof-of-principle testing successfully identified blood, semen, and saliva, thus setting the stage for expanding the range of body fluids; 3) the method is simple, and will minimally disrupt forensic labs; and 4) the nature of LAMP reduces the instrument complexity we will build. Add to this, a 96-well format, and colorimetric detection that provides a yes/no read-out for six body fluids using a smartphone. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). ca/ncf