Diagnosis | Parasitic Diseases
BACKGROUND: CRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection.
METHODS: We developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We further tested these assays using a diverse panel of clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. In addition, we developed a prototype SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine resistance.
FINDINGS: The suite of Plasmodium assays achieved analytical sensitivities ranging from 2*5-18*8 parasites per reaction when tested against laboratory strain genomic DNA. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity during testing of 123 clinical samples. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities.
INTERPRETATION: These novel SHERLOCK assays demonstrate the versatility of CRISPR-based diagnostics and their potential as a new generation of molecular tools for malaria diagnosis and surveillance.
FUNDING: National Institutes of Health (T32GM007092, R21AI148579, K24AI134990, R01AI121558, UL1TR002489, P30CA016086).
CRISPR, Cas13a, Diagnostic, Malaria, SHERLOCK
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© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
DOI of Published Version
Cunningham CH, Hennelly CM, Lin JT, Ubalee R, Boyce RM, Mulogo EM, Hathaway N, Thwai KL, Phanzu F, Kalonji A, Mwandagalirwa K, Tshefu A, Juliano JJ, Parr JB. A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, species differentiation, and drug-resistance genotyping. EBioMedicine. 2021 Jun;68:103415. doi: 10.1016/j.ebiom.2021.103415. Epub 2021 Jun 14. PMID: 34139428; PMCID: PMC8213918. Link to article on publisher's site
Cunningham CH, Hennelly CM, Lin JT, Ubalee R, Boyce RM, Mulogo EM, Hathaway NJ, Thwai KL, Phanzu F, Kalonji A, Mwandagalirwa K, Tshefu A, Juliano JJ, Parr JB. (2021). A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, species differentiation, and drug-resistance genotyping. Open Access Publications by UMass Chan Authors. https://doi.org/10.1016/j.ebiom.2021.103415. Retrieved from https://escholarship.umassmed.edu/oapubs/4773
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