Development of Recombinase Polymerase Amplification and CRISPR-Cas12a-Enhanced Isothermal Amplification Assays for...

Soil-transmitted helminth (STH) infections are prevalent worldwide, but the true burden of strongyloidiasis is unclear due to lack of sensitive and field-friendly diagnostic tools. Diagnosis is often based on serological assays that are typically not point-of-care (POC). Although polymerase chain reaction (PCR) tests are sensitive and specific, the need for expensive equipment and highly skilled personnel limits their use in resource limited areas. Isothermal amplification assays are largely instrument-free, making them simpler to implement without loss of either sensitivity or specificity. We developed two recombinase polymerase amplification (RPA) assays to detect Strongyloides stercoralis (Ss) in human stool samples and a complementary CRISPR-Cas12a detection system with visual readouts. Primers, probes, and guide RNAs (crRNAs) for these assays were designed targeting the Ss-NIE sequence and Ss dispersed repetitive sequence (Ss-DRS). The assay's specificities and limits of detection (LOD) were assessed using gDNA from Ss L3 larvae or from other STH and filariae. The NIE RPA showed a LOD of 1 fg/µL, whereas the LOD for the Ss-DRS RPA was 1 pg/µL. The LOD was 500 fg/µL for the NIE RPA CRISPR-Cas12a assay. No cross-reactivity with any filarial parasite or other STH was observed. Because the NIE assays were more sensitive than the Ss-DRS assay, six patient samples positive for Ss by real-time PCR (qPCR) were tested using the NIE assays, of which four were positive. Though assay refinement and clinical validation are needed, this study establishes fast, highly sensitive and field-applicable POC diagnostic tools for Ss detection that are ideal for use in endemic areas with limited resources.