Liang Yao, Xie Shi-Chen, Lv Yi-Han, He Yuan-Hui, Zheng Xiao-Nan, Cong Wei, Elsheikha Hany M, Zhu Xing-Quan
Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, 030801, Shanxi, People's Republic of China.
Marine College, Shandong University, Weihai, 264209, Shandong, People's Republic of China.
Infect Dis Poverty. 2024 Dec 10;13(1):94. doi: 10.1186/s40249-024-01266-5.
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.
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.
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.
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.
猫粪便中排出的刚地弓形虫卵囊，通过污染土壤和水对人类健康构成重大风险。快速准确地检测环境样本中的刚地弓形虫对于公共卫生保护至关重要。
我们开发了一种新型单管检测方法，该方法整合了环介导等温扩增（LAMP）、成簇规律间隔短回文重复序列（CRISPR）/Cas12b系统和侧向流免疫分析试纸条，用于快速、可视化鉴定刚地弓形虫。该方法靶向刚地弓形虫B1基因，首先用LAMP由单向导RNA（sgRNA）引导进行扩增。然后识别扩增的靶基因并激活反式切割，切割附近的单链DNA（ssDNA）报告基因。使用6-羧基荧光素（FAM）-12N-黑洞猝灭剂-1（BHQ1）报告基因进行荧光检测，而异硫氰酸荧光素（FITC）-12N-生物素则可在侧向流试纸上进行可视化检测。测试了该方法检测各种刚地弓形虫基因型和相关寄生虫的能力，评估其特异性和广谱适用性。进一步将其应用于实际环境样本以评估其实用性。
LAMP-CRISPR/Cas12b方法表现出高特异性和广谱检测能力，成功鉴定出9种刚地弓形虫基因型，并将它们与11种其他寄生虫物种区分开来。在分子（质粒）和实际（卵囊）水平上的敏感性测试分别显示检测限为10拷贝/μL和0.1个卵囊。当应用于112个环境样本（土壤、水和猫粪便）时，该方法显示出100%的敏感性，准确反映了已知的感染率。
这种LAMP-CRISPR/Cas12b单管方法为监测环境样本中的人畜共患刚地弓形虫提供了一种强大的创新方法，对公共卫生监测具有重要意义。
