弓形虫,导致弓形虫病,在温血动物中普遍存在,比如猫,狗,和人类。弓形虫会给畜牧业造成经济损失,并对公众健康构成潜在风险。狗和猫是弓形虫病流行病学中的常见宿主。目前弓形虫感染的分子诊断工具需要很高的技术技能,实验室环境,复杂的工具。在这里,我们开发了一种重组酶聚合酶扩增(RPA)成簇的规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白12a(Cas12a)检测弓形虫。对于弓形虫B1基因,测定的最低检测限为31拷贝/μL。此外,我们建立了可视化RPA-CRISPR/Cas12a侧流带测定(RPA-CRISPR/Cas12a-LFA)结合数字可视化仪器,最大限度地减少了弱阳性样本的假阴性结果问题,避免了肉眼对结果的误解,使LFA测定结果更准确。本研究建立的方法可以在55分钟内鉴定弓形虫,具有很高的准确性和敏感性。没有交叉反应与其他测试的寄生虫。通过建立弓形虫病小鼠模型验证了所开发的方法。最后,该方法用于浙江省流浪猫和狗中弓形虫的患病率调查,中国东部。流浪猫、狗弓形虫感染阳性率分别为8.0%和4.0%,分别。总之,RPA-CRISPR/Cas12a-LFA是快速的,敏感,准确的早期诊断弓形虫,显示了现场监控的希望。
目的:弓形虫是一种毒性病原体,使数百万感染者面临慢性疾病再激活的风险。弓形虫的寄主分布在世界各地,猫和狗是弓形虫的常见宿主。因此,快速诊断早期弓形虫感染并调查其在流浪狗和猫中的患病率至关重要。这里,我们建立了可视化重组酶聚合酶扩增成簇的规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白12a测定,并结合了侧向流带测定和数字可视化仪器.详细分析发现,该方法可用于弓形虫的早期诊断,无假阴性结果。此外,我们在浙江省流浪猫和狗中检测到弓形虫的患病率,中国。我们开发的检测方法为弓形虫的早期诊断提供了技术支持,可用于流浪狗和猫的弓形虫患病率调查。
Toxoplasma gondii, which causes
toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of
toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/μL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of
toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern
China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance.
OBJECTIVE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province,
China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.