Ortiz-Rodríguez Luis A, Cabanzo Rafael, Jaimes-Dueñez Jeiczon, Mendez-Sanchez Stelia C, Duque Jonny E
Centro de Investigaciones en Enfermedades Tropicales(Cintrop). Escuela de Medicina, Departamento de Ciencias Básicas, Universidad Industrial de Santander, Parque Tecnológico y de Investigaciones Guatiguará Km 2 El Refugio, Piedecuesta, Santander, Colombia.
Laboratorio de Espectroscopía Atómica y Molecular (LEAM), COL0012909, Escuela de Física, Facultad de Ciencias, Universidad Industrial de Santander, Bucaramanga, Colombia.
Sci Rep. 2025 May 31;15(1):19107. doi: 10.1038/s41598-025-04017-0.
Chagas disease, also known as American Trypanosomiasis, is a zoonosis with global distribution caused by the parasite Trypanosoma cruzi, primarily transmitted through the feces of infected triatomines. The emergence of new cases highlights the importance of early pathogen detection in vectors and reservoirs to generate effective control strategies and establish preventive policies. The objective of this study was to design and validate a detection system of T. cruzi based on specific DNA cleavage, activation of Cas12a and trans-cleavage, targeting the genes Cytochrome B (Cytb), 18 S ribosomal subunit (SR18 s), and histone (H2 A). This system was validated for their uses in both vectors and reservoirs of the parasite. The initial step involved performing a bioinformatic analysis of the target genes, followed by the design of RNA guides specific to each cleavage site, along with primers for amplifying the target region through PCR and RPA. Subsequently, we sequenced the amplified DNA target and validated the detection system using T. cruzi DNA extracted from naturally infected Rhodnius pallescens in the metropolitan area of Bucaramanga, Colombia. After standardizing the method, we tested the CRISPR/Cas system with Silvio X10 laboratory strain of T. cruzi and scaled up to blood samples of naturally infected Didelphis marsupialis. As a result, we observed DNA cleavage using the CRISPR/Cas system with the Cytb guide, achieving a detection sensitivity of 118 parasite equivalents/mL in PCR and 116 parasite equivalents/mL with RPA amplification. Sequencing of the Cytb gene showed no mutations in the cleavage site. However, point mutations and indels were found in SR18S and H2 A, avoiding the formation of the CRISPR/LbCas12 complex. Furthermore, we introduce the design of a fluorescent detection prototype with CRISPR/LbCas12a called "Tropical Diseases Detector" (TropD-Detector). This device operates with an excitation wavelength of 480 nm emitted by an LED and a high-pass light filter with a cutoff wavelength of 500 nm. We detected positive samples using any photographic camera system. The TropD-Detector provides a visual, viable, and sensitive method for detecting T. cruzi in both vectors and reservoirs from endemic areas.
恰加斯病，又称美洲锥虫病，是一种由克氏锥虫寄生虫引起的全球性人畜共患病，主要通过受感染锥蝽的粪便传播。新病例的出现凸显了在病媒和宿主中早期检测病原体对于制定有效控制策略和建立预防政策的重要性。本研究的目的是设计并验证一种基于特定DNA切割、Cas12a激活和反式切割的克氏锥虫检测系统，该系统靶向细胞色素B（Cytb）、18S核糖体亚基（SR18s）和组蛋白（H2A）基因。该系统在寄生虫的病媒和宿主中的应用均得到了验证。第一步是对目标基因进行生物信息学分析，随后设计针对每个切割位点的RNA引导序列，以及用于通过PCR和RPA扩增目标区域的引物。随后，我们对扩增的DNA靶点进行测序，并使用从哥伦比亚布卡拉曼加市大都会区自然感染的苍白红猎蝽中提取的克氏锥虫DNA验证了检测系统。在对方法进行标准化后，我们用克氏锥虫的西尔维奥X10实验室菌株测试了CRISPR/Cas系统，并扩大到对自然感染的大负鼠血液样本的检测。结果，我们观察到使用带有Cytb引导序列的CRISPR/Cas系统进行DNA切割，在PCR中检测灵敏度达到118个寄生虫当量/毫升，在RPA扩增中为116个寄生虫当量/毫升。Cytb基因的测序显示切割位点没有突变。然而，在SR18S和H2A中发现了点突变和插入缺失，这避免了CRISPR/LbCas12复合物的形成。此外，我们介绍了一种名为“热带疾病检测仪”（TropD-Detector）的带有CRISPR/LbCas12a的荧光检测原型的设计。该设备通过LED发出的480nm激发波长和截止波长为500nm的高通滤光片运行。我们使用任何摄影摄像系统检测阳性样本。TropD-Detector为在流行地区的病媒和宿主中检测克氏锥虫提供了一种可视化、可行且灵敏的方法。
