Yao Liang, Shi-Chen Xie, Yi-Han Lv, Yuan-Hui He, Xiao-Nan Zheng, Wei Cong, Hany M Elsheikha, Xing-Quan Zhu
{"title":"一种新的单管LAMP-CRISPR/Cas12b方法用于环境中人畜共患病刚地弓形虫的快速和视觉检测。","authors":"Yao Liang, Shi-Chen Xie, Yi-Han Lv, Yuan-Hui He, Xiao-Nan Zheng, Wei Cong, Hany M Elsheikha, Xing-Quan Zhu","doi":"10.1186/s40249-024-01266-5","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":48820,"journal":{"name":"Infectious Diseases of Poverty","volume":"13 1","pages":"94"},"PeriodicalIF":8.1000,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11629535/pdf/","citationCount":"0","resultStr":"{\"title\":\"A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment.\",\"authors\":\"Yao Liang, Shi-Chen Xie, Yi-Han Lv, Yuan-Hui He, Xiao-Nan Zheng, Wei Cong, Hany M Elsheikha, Xing-Quan Zhu\",\"doi\":\"10.1186/s40249-024-01266-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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. 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A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment.
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.
期刊介绍：
Infectious Diseases of Poverty is an open access, peer-reviewed journal that focuses on addressing essential public health questions related to infectious diseases of poverty. The journal covers a wide range of topics including the biology of pathogens and vectors, diagnosis and detection, treatment and case management, epidemiology and modeling, zoonotic hosts and animal reservoirs, control strategies and implementation, new technologies and application. It also considers the transdisciplinary or multisectoral effects on health systems, ecohealth, environmental management, and innovative technology. The journal aims to identify and assess research and information gaps that hinder progress towards new interventions for public health problems in the developing world. Additionally, it provides a platform for discussing these issues to advance research and evidence building for improved public health interventions in poor settings.
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