Oral Abstract Submission
Malaria control and eradication have been hampered by asymptomatic carriage which serves as a parasite reservoir. Low density infections ( < 100 parasites/microliter) frequently fall below the limit of detection (LOD) of microscopy and rapid diagnostic tests (RDT) which are antigen-based tests. Molecular methods such as polymerase chain reaction are capable of higher sensitivity yet remain impractical for resource-limited settings. We describe development of an isothermal assay using the nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing), which may also be increasingly important as there has been rising detection of histidine-rich protein 2 (HRP2) gene deletions in Plasmodium spp. HRP2 is the most commonly used antigen in RDTs and deletion of this gene would render many RDTs obsolete.
SHERLOCK leverages the endonucleases of CRISPR-associated microbial adaptive immunity. Cas12a is a RNA-guided, DNA-cleaving enzyme, which can be programmed with guide RNAs to cleave non-target reporter ssDNA. We exploit the non-specific degradation of labeled ssDNA to detect the presence of the dsDNA target that activated Cas12a (Figure 1). Recombinase polymerase amplification (RPA) coupled with Cas12a detection enables a lower LOD. Plasmodium falciparum whole genomic DNA was compared to parasites cultured in red blood cells (RBCs) with known parasitemia and boiled at 95 ̊C for 5 minutes for lysis of RBCs/parasites then diluted 1:2.5 to prevent solidification.
This SHERLOCK assay detected simulated Plasmodium falciparum infection at attomolar LODs when applied to whole genomic DNA and simulated samples of infected RBCs spiked into whole blood. The genomic assay detected down to 0.2 parasites/microliter and the simulated sample detected to 10 parasites/microliter in the final reaction volume. In comparison, LODs from the initial input volume was 5aM and 250aM respectively (Figure 2).
We demonstrate an isothermal nucleic acid detection platform capable of diagnosis in 60 minutes in a one-pot assay requiring minimal sample preparation and reaching a LOD recommended by the WHO for malaria eradication. In summary, we illustrate the utility of the SHERLOCK platform in application to malaria and global health.