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

Abstract

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.

Keywords: Angiostrongylus; DNA; detection; meningitis; recombinase polymerase assay.

FIG 1

The visual- and fluorometer-derived limits of detection of RPAcan3990 are equivalent. The limit of detection of the RPAcan3990 was estimated using a one-tenth dilution series of Angiostrongylus cantonensis genomic DNA by fluorescence detection in a fluorometer. The same dilution series was then examined photographically using blue light illumination through an orange transparent filter (inset). The visual and fluorometer readouts of the RPA showed an equivalent limit of detection, 1 fg/μl.

FIG 2

RPAcan3990 performance on CSF samples from patients with eosinophilic meningitis of known parasitic causes and from uninfected control CSF. (A) RPAcan3990 was performed on archived CSF samples from cases of encephalitis caused by Angiostrongylus cantonensis, Toxocara, Gnathostoma, or by nonparasitic causes (negative controls). Products were visualized through digital photography using a blue light at a 470-nm wavelength (electrophoresis gel reader) and a transparent orange filter. (B) Fluorimetry of the corresponding reaction mixtures was performed and compared with the visualized fluorescence; the fluorimetry data are numbered according to the corresponding reaction in panel A (total time is equal to cycle time × 40 s). (C) RPAcan3990 did not produce visualizable fluorescence when applied to DNA extracted from a CSF sample from a patient with known gnathostomiasis (G. sp.). Also included are a positive control (A. cantonensis genomic DNA; Ac+) and a negative control (Neg).

FIG 3

Effects of temperature and incubation time on the limits of detection using RPAcan3990. (A) Schematic of the kinetics of RPAcan3990 performed on 100 pg/μl, 1 fg/μl, and negative control samples at 30°C (blue), 35°C (purple), and 40°C (red) with visualization of fluorescence at 10-min intervals for 60 min total. (B) Visualization at 30 min is shown in this representative set of products described in panel A. (C) Results obtained using RPAcan3990 performed in duplicate on 100 pg/μl genomic DNA, 1 pg/μl genomic DNA, 1 fg/μl genomic DNA, and negative control (NC) samples following a 30-min incubation in a shirt pocket.