RPAcan3990: an ultrasensitive recombinase polymerase assay to detect Angiostrongylus cantonensis DNA.

Sears, William J; Qvarnstrom, Yvonne; Nutman, Thomas B

Angiostrongylus cantonensis (Ac) is one of the leading causes of eosinophilic meningitis worldwide. A field deployable molecular detection method could enhance both environmental surveillance and clinical diagnosis of this emerging pathogen. Accordingly, RPAcan3990, a recombinase polymerase assay (RPA) was developed to target a region predicted to be highly repeated in the Ac genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using Ac genomic DNA, the limit of detection was found to be 1fg/μl by both fluorometer measurement and visual reading. All clinical samples known to be positive for Ac from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e. Toxocara sp., Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35-40°C. The assay successfully detected 1fg/μl of Ac genomic DNA after incubation at human body temperature (in a shirt pocket). In conclusion, these data suggest RPAcan3990 is potentially a point of contact molecular assay capable of sensitively detecting Ac by producing visually interpretable results with minimal instrumentation.