The use of recombinase polymerase amplification for the development of a point of care vertical flow immunoassay to detect radiation dosimetry genes
In a mass-casualty radiological emergency, it is important to quickly differentiate between exposed and non-exposed individuals in order to effectively organize treatment. To accomplish this discrimination, there is a need to develop a point-of-care (POC) device to assess radiation exposure. Previously, a proof of concept was established for the detection of radiation dosimetry genes in a powered vertical flow immunoassay (VFI). However, to develop a true POC platform, new methods of amplifying genes and running the VFI that do not require power supply need to be explored. Therefore, this study aimed to implement (1) an unpowered VFI run entirely by capillary forces and gravity and (2) an isothermal recombinase polymerase amplification (RPA) using a power-free heat source to detect biodosimetry genes CDKN1A, DDB2, and housekeeping gene MRPS5. First, the thermal kinetics of a hot compress were tested to determine its suitability for RPA. Results showed that on average, a hot compress can reach a maximum temperature of 42°C and maintain temperatures above 37°C for the 20 minutes required for RPA. Using the hot compress, samples irradiated at 0 and 2 Gy from 3 different donors were then amplified, and data showed that the unpowered VFI could detect their gene expression level and discriminate between irradiated and non-irradiated samples. Interestingly, neither RPA with a hot compress nor the standard thermal cycler approach demonstrated a superior discrimination between 0 and 2 Gy samples. These results indicate that using RPA on an unpowered VFI platform is a viable option to detect radiation dosimetry gene expression.
