Development of Recombinase Polymerase Amplification (RPA) Assays to Diagnose Infectious Diseases
Richards-Kortum, Rebecca Rae
Doctor of Philosophy
This thesis describes the development of Recombinase Polymerase Amplification (RPA) assays that can be used to improve access to diarrheal diagnostics and thereby reduce the number of preventable deaths that occur each year due to persistent diarrhea. In low-resource settings (LRS), where the majority of the almost 1.5 million annual diarrheal deaths occur, a major obstacle to receiving life-saving treatment is the inability to identify the specific cause of diarrhea. Diagnosis in LRS is usually done via stool smear microscopy, which fails to identify the cause of diarrhea up to half of the time. The widely considered gold standard diagnostic method is Polymerase Chain Reaction (PCR), which detects trace amounts of pathogen DNA from stool samples. While highly sensitive, PCR requires highly trained technicians and access to expensive thermal cycling equipment, restricting its use to centralized reference laboratories. The RPA diagnostics presented here amplifies trace amounts of pathogen DNA (much like PCR), but unlike PCR do not require the use of expensive thermal cycling equipment and can function at low temperatures, alleviating the need for any external heating equipment. RPA-based diagnostics and sample preparation protocols that are appropriate for low resource settings were developed to detect Cryptosporidium, Giardia, and Entamoeba, three of the leading causes of diarrhea. The three diagnostic assays were individually characterized on the benchtop where they demonstrated limits-of-detection and specificities comparable to the gold standard of PCR. The assays were further characterized in field studies using clinical samples where they demonstrated sensitivity and specificity nearly equivalent to that of the gold standard PCR. The three individual assays were then integrated into a multiplexed test designed to simultaneously amplify and detect DNA from Cryptosporidium, Giardia, and Entamoeba. This test was also characterized on the benchtop and in pre-clinical studies. All of the assays presented here are read using lateral flow strips that can easily be used in the field. This work demonstrates for the first time that multiplex RPA results can be read with lateral flow strips. By modifying the DNA primers, this diagnostic platform could be adapted to diagnose a broad variety of infectious diseases.
Recombinase Polymerase Amplification; diagnostic