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

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.

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

“…To compensate for such limitations, the standard strategy is priorly using nucleic-acid amplification to increase the quantity of the target dsDNA substrate in the CRISPR/Cas12a system. The amplification methods include polymerase chain reaction (PCR) and isothermal amplification such as recombinase polymerase amplification ,− and loop-mediated amplification (LAMP). , Preamplification significantly improved the sensitivity of CRISPR Cas12a by 10 7 -fold or even 10 10 -fold . However, in contrast to Cas12a protein, which can be activated at room temperature, preamplification methods require sophisticated control over temperature.…”

Acute kidney injury in the tropics

“…37 Recently, the RPA-CRISPR/Cas12 targeting the LipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. 38 Antibiotics is the mainstay therapy, which provide benefits not only in a significant reduction in leptospira antigen but also have a dramatic effect on infected patients, especially those with multiple organ failure. 39 Based on WHO recommendations, severe leptospirosis should be treated with intravenous penicillin, ceftriaxone or cefotaxime for the duration of 7 days.…”

“…Rapid diagnostic test kit of serum IgM antibody which was most widely used and available, produced low sensitivity especially during the first week of illness 37 . Recently, the RPA‐CRISPR/Cas12 targeting the LipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires 38 …”

Ocular leptospirosis: lack of awareness among ophthalmologists and challenges in diagnosis

“…Diagnostic performance of lipL32-LAMP presented 84.2% sensitivity and 93.2% specificity [70 ▪ ]. The recombinase polymerase amplification-CRISPR/Cas12 (clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a) targeting the lipL32 gene demonstrated good sensitivity and excellent specificity for detection of leptospirosis [71]. Carbo-Lip probe was recently demonstrated.…”