Department of Immunity and Biochemistry, Institute of Parasitic Disease, Zhejiang Academy of Medical Sciences 310013 Hangzhou PR China - Hangzhou Medical College 310053 Hangzhou PR China.
Parasite. 2021;28:41. doi: 10.1051/parasite/2021039. Epub 2021 May 3.
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.
刚地弓形虫是一种专性细胞内寄生的原虫，可引起弓形体病，威胁着全球温血动物和人类的健康。迫切需要简单适用的诊断方法来指导开发有效的弓形虫病防治方法。大多数用于弓形虫感染的分子诊断工具都需要较高的技术技能、复杂的设备和受控的实验室环境。在这项研究中，我们开发了一种环介导等温扩增-侧流层析（LAMP-LFD）检测方法，该方法专门针对 529bp 区域，用于检测弓形虫感染。这种新型便携式设备通用、快速、用户友好，保证了实验的灵敏度和低气溶胶污染风险。我们的 LAMP-LFD 检测方法的检测限为 1fg 的弓形虫 DNA，与其他寄生虫病原体（包括微小隐孢子虫、杜氏利什曼原虫和间日疟原虫）无交叉反应。我们通过检测从浙江省德清、温州、义乌、丽水和舟山五个城市采集的 318 只流浪猫和狗的血液样本中的 DNA 来验证该检测方法的有效性。LAMP-LFD 设备在流浪猫和狗中分别检测到 4.76%和 4.69%的弓形虫 DNA。总之，开发的 LAMP-LFD 检测方法效率高，最大限度地减少了气溶胶污染，因此适合在基本医疗机构和现场环境中检测弓形虫。
