The combination of RPA-CRISPR/Cas12a and Leptospira IgM RDT enhances the early detection of leptospirosis

Jirawannaporn, Sirawit; Limothai, Umaporn; Tachaboon, Sasipha; Dinhuzen, Janejira; Kiatamornrak, Patcharakorn; Chaisuriyong, Watchadaporn; Srisawat, Nattachai

doi:10.1371/journal.pntd.0011596

Cited by 2 publications

(1 citation statement)

References 22 publications

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“…To address these issues, RPA integrated with CRISPR/Cas12 (RPA-CRISPR/Cas12) methods has been developed and utilized for detecting pathogens [ 35 , 36 ]. In the present study, we first introduced a method for S. Indiana detection via combining RPA with the CRISPR/Cas12b system.…”

Section: Discussionmentioning

confidence: 99%

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

Gong,

Zhang,

et al. 2024

Microorganisms

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Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5′-/6-FAM/CCCCCCCC/BHQ1/-3′) presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection.

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Section: Discussionmentioning

confidence: 99%

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

Gong,

Zhang,

et al. 2024

Microorganisms

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Accuracy of rapid lateral flow immunoassays for human leptospirosis diagnosis: A systematic review and meta-analysis

Nualnoi,

Lomlim,

Naorungroj

2024

PLoS Negl Trop Dis

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Background In the last two decades, several rapid lateral flow immunoassays (LFIs) for the diagnosis of human leptospirosis were developed and commercialized. However, the accuracy and reliability of these LFIs are not well understood. In this study, we aimed to evaluate the accuracy of leptospirosis LFIs as well as the factors affecting the test efficiency using systematic review and meta-analysis. Methods and results Original articles reporting the accuracy of human leptospirosis LFIs against microagglutination tests (MAT) or immunofluorescent assays (IFA) were searched from PubMed, Embase, and Scopus, and selected as per pre-set inclusion and exclusion criteria. A total of 49 data entries extracted from 24 eligible records published between 2003 and 2023 were included for meta-analysis. A meta-analysis was performed using STATA. The quality of the included studies was assessed according to the revised QUADAS-2. Only nine studies (32.1%) were considered to have a low risk of bias and no concern for applicability. Pooled sensitivity and specificity were calculated to be 68% (95% confidence interval, CI: 57–78) and 93% (95% CI: 90–95), respectively. However, the ranges of sensitivity (3.6 – 100%) and specificity (53.5 – 100%) of individual entries are dramatically broad, possibly due to the heterogeneity found in both study designs and LFIs themselves. Subgroup analysis demonstrated that IgM detection has better sensitivity than detection of IgG alone. Moreover, the test performance seems to be unaffected by samples from different phases of infection. Conclusions The pooled specificity of LFIs observed is somewhat acceptable, but the pooled sensitivity is low. These results, however, must be interpreted with caution because of substantial heterogeneity. Further evaluations of the LFIs with well-standardized design and referent test will be needed for a greater understanding of the test performance. Additionally, IgM detection type should be employed when leptospirosis LFIs are developed in the future.

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The combination of RPA-CRISPR/Cas12a and Leptospira IgM RDT enhances the early detection of leptospirosis

Cited by 2 publications

References 22 publications

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

Accuracy of rapid lateral flow immunoassays for human leptospirosis diagnosis: A systematic review and meta-analysis

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