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

Sirawit Jirawannaporn,Patcharakorn Kiatamornrak,Watchadaporn Chaisuriyong,Nattachai Srisawat,Oraphan Mayuramart,Umaporn Limothai,Sasipha Tachaboon,Jom Bhumitrakul,Sunchai Payungporn,Janejira Dinhuzen,Kelly Hawley

doi:10.1371/journal.pntd.0010112

Abstract

BackgroundOne 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 findingsA 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/SignificanceThe 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.

Highlights

Leptospirosis is a zoonotic disease that affects global health with over one million cases and 58,900 deaths annually [1]
We developed an Recombinase Polymerase Amplification (RPA)-clustered regularly-interspaced short palindromic repeat (CRISPR)/Cas12a -based detection platform to identify the lipL32 gene of pathogenic Leptospira spp
The results showed that the limit of detection (LOD) was approximately 102 cells/mL without cross-reactivity against other infectious diseases

Summary

Introduction

Leptospirosis is a zoonotic disease that affects global health with over one million cases and 58,900 deaths annually [1]. The second is dark field microscope diagnosis from sample cultures collected from the patient’s blood at the early stage of Leptospira spp. infection. The third technique is the use of polymerase chain reaction (PCR), a nucleic acid detection method that is faster and more accurate. The drawback of qPCR is the high-cost of equipment and the lack of availability in every hospital, especially in rural areas [5,6] where most leptospirosis cases occur. The development of a novel diagnostic tool that can detect pathogenic Leptospira spp. early, providing rapid and accurate results and is not cost-prohibitive to resource-limited hospitals and clinics is essential for public health [7]. 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

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Conclusion