Detection of leptospires by RPA-NALFIA and CRISPR-Cas12a

Abstract

The key barrier in leptospirosis diagnosis is a lack of available sensitive point-of-care testing. Therefore, we aimed to develop and validate nucleic acid lateral flow immunoassay (NALFIA) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patients' DNA samples. A recombinase polymerase amplification (RPA)-NALFIA and RPA-CRISPR/Cas12a assay was designed to detect the LipL32, SecY and lfb1 genes of pathogenic Leptospira spp. The RPA-NALFIA targeting LipL32 observed the LOD at 105 copies/reaction. In comparison, the RPA-CRISPR/Cas12a targeting LipL32 and SecY demonstrated a limit of detection (LOD) of 100 copies/reaction, with no cross-reactivity against other acute febrile illnesses. However, RPA-CRISPR/Cas12a targeting lfb1 failed to detect the leptospira spp. The clinical performance of the RPA-CRISPR/Cas12a assay targeting LipL32 was validated with DNA extracted from 110 clinical specimens and then compared with qPCR detection of Leptospira spp. Relative to the qPCR detection, the RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make this test more accessible for use and easier to read. The combined LFDA showed a similar LOD of 100 copies/reaction could correctly distinguish between known positive and negative clinical samples in a pilot study. The RPA-NALFIA targeting LipL32 demonstrated acceptable sensitivity and excellent specificity for leptospires detection. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.