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. 2024 Dec 10;13(1):94.
doi: 10.1186/s40249-024-01266-5.

A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment

Yao Liang  1 , Shi-Chen Xie  1 , Yi-Han Lv  1 , Yuan-Hui He  1 , Xiao-Nan Zheng  1 , Wei Cong  2 , Hany M Elsheikha  3 , Xing-Quan Zhu  4
Affiliations

A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment

Yao Liang et al. Infect Dis Poverty. .

Abstract

Background: Toxoplasma gondii oocysts, excreted in cat feces, pose a significant health risk to humans through contaminated soil and water. Rapid and accurate detection of T. gondii in environmental samples is essential for public health protection.

Methods: We developed a novel, single-tube detection method that integrates loop-mediated isothermal amplification (LAMP), the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system, and lateral flow immunoassay strips for rapid, visual identification of T. gondii. This method targets the T. gondii B1 gene, initially amplifies it with LAMP, directed by a single-guide RNA (sgRNA). It then recognizes the amplified target gene and activates trans-cleavage, cutting nearby single-stranded DNA (ssDNA) reporters. Fluorescence detection was performed using a 6-Carboxyfluorescein (FAM)-12N-Black Hole Quencher-1 (BHQ1) reporter, while Fluorescein Isothiocyanate (FITC)-12N-Biotin enabled visual detection on lateral flow strips. The method was tested for its ability to detect various T. gondii genotypes and related parasites, assessing its specificity and broad-spectrum applicability. It was further applied to real-world environmental samples to evaluate its practicality.

Results: The LAMP-CRISPR/Cas12b method exhibited high specificity and broad-spectrum detection capability, successfully identifying nine T. gondii genotypes and distinguishing them from 11 other parasitic species. Sensitivity testing at both molecular (plasmid) and practical (oocyst) levels showed detection limits of 10 copies/μL and 0.1 oocyst, respectively. When applied to 112 environmental samples (soil, water, and cat feces), the method demonstrated 100% sensitivity, accurately reflecting known infection rates.

Conclusions: This LAMP-CRISPR/Cas12b single-tube method offers a robust, innovative approach for monitoring zoonotic T. gondii in environmental samples, with significant implications for public health surveillance.

Keywords: Toxoplasma gondii; CRISPR/Cas12b; Environmental detection; Lateral flow immunoassay; Loop-mediated isothermal amplification; Molecular diagnostics; Zoonotic parasites.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: The procedures involving animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Shanxi Agricultural University (Approval No. SXAU-EAW-2021XM121001). Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Detection mechanisms of the LAMP-CRISPR/Cas12b single-tube method. A Step-by-step operational workflow of the detection process. B Functional principles of each component in the LAMP-CRISPR/Cas12b system
Fig. 2
Fig. 2
Optimization results of the LAMP and CRISPR/Cas12b systems. A Screening results for LAMP primers. B Optimization of LAMP reaction temperature. C Optimization of conditions for the CRISPR/Cas12b detection system. D Screening results of sgRNAs
Fig. 3
Fig. 3
Evaluation of the LAMP-CRISPR/Cas12b single-tube method’s broad-spectrum capability, specificity, and sensitivity. A Positive and negative control detection results. B Evaluation of broad-spectrum detection with different T. gondii strains. C Specificity assessment of the method. D Sensitivity analysis based on plasmid concentration. E Sensitivity analysis based on oocyst counts. F Gel electrophoresis results from the semi-nPCR sensitivity test using oocyst counts. *F: First detection line; S: Second detection line
Fig. 4
Fig. 4
Environmental sample detection using the LAMP-CRISPR/Cas12b single-tube method. A Soil sample detection: 14 positives and 16 negatives. B Water sample detection: 30 positives and 20 negatives. C Cat fecal sample detection: 10 positives and 11 negatives. D Comparison of the LAMP-CRISPR/Cas12b and semi-nPCR in detecting T. gondii in 11 cat fecal samples collected 3 days post-infection. *F: First detection line; S: Second detection line
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