Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32

PLoS Negl Trop Dis. 2022 Jan 6;16(1):e0010112. doi: 10.1371/journal.pntd.0010112. eCollection 2022 Jan.

Abstract

Background: One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples.

Methodology/principal findings: A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4-6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study.

Conclusions/significance: The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

MeSH terms

  • Bacterial Outer Membrane Proteins / genetics*
  • CRISPR-Cas Systems / genetics
  • Female
  • Humans
  • Leptospira / genetics*
  • Leptospira / isolation & purification
  • Leptospirosis / diagnosis*
  • Limit of Detection
  • Lipoproteins / genetics*
  • Male
  • Mass Screening / methods
  • Middle Aged
  • Molecular Diagnostic Techniques / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • Point-of-Care Testing
  • Real-Time Polymerase Chain Reaction
  • Sensitivity and Specificity

Substances

  • Bacterial Outer Membrane Proteins
  • LipL32 protein, Leptospira
  • Lipoproteins

Grants and funding

Sirawit Jirawannaporn received funding from the The 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, and the 90th Anniversary of Chulalongkorn University Scholarship (Ratchadaphiseksomphot Endowment Fund). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.