The Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei, China.
School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
Parasit Vectors. 2023 Jul 30;16(1):256. doi: 10.1186/s13071-023-05868-0.
Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.
This study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.
The results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi).
This study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.
刚地弓形虫是一种机会性的原生动物，广泛存在于人类和动物中。它可以侵犯任何人体器官，导致严重疾病，包括弓形虫眼病、脑膜脑炎和肝坏死。猪弓形虫病在中国流行。CRISPR（成簇的规律间隔的短回文重复序列）和 Cas（CRISPR 相关蛋白）系统广泛用于基因编辑和病原体检测。基于 CRISPR 的诊断是开发的分子检测方法，具有高灵敏度和特异性，可以检测寄生虫。
本研究旨在通过靶向 B1 基因和 529bp 重复元件（529RE），建立一种针对刚地弓形虫的 CRISPR/Cas12a 和 RPA 快速检测方法。检测结果可通过荧光或侧向流条（LFS）可视化。评估了该方法的灵敏度和特异性，并用于检测感染弓形虫的小鼠血液。
结果表明，刚地弓形虫检测方法的建立是令人满意的，两个靶位的检测限分别为 1.5cp/μl。此外，B1 基因可检测到每个反应中的 1 个速殖子，529RE 可检测到每个反应中的 0.1 个速殖子，与高度敏感的巢式聚合酶链反应（PCR）结果一致。该方法适用于 RH 等株系，与其他具有相似习性的原生动物 DNA 无交叉反应。感染弓形虫的小鼠血液样本在感染后 1、3 和 5 天均为弓形虫阳性。
本研究建立了一种快速、敏感、省时的刚地弓形虫 DNA 检测方法，有望成为现场检测刚地弓形虫的替代工具。
