RPAcan3990: an Ultrasensitive Recombinase Polymerase Assay To Detect Angiostrongylus cantonensis DNA

Angiostrongylus cantonensis 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 A. cantonensis genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using A. cantonensis genomic DNA, the limit of detection was found to be 1 fg/μl by both fluorometer measurement and visual reading. All clinical samples known to be positive for A. cantonensis from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e., Toxocara sp. and Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35°C and 40°C. The assay successfully detected 1 fg/μl of A. cantonensis 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 A. cantonensis by producing visually interpretable results with minimal instrumentation.