Prathyusha Sunkara, Radhika R, Lakshmanan Bindu, Syamala K, Thomas Marykutty, Justin Davis K, Devada K
Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, 680 651, India; Kerala Veterinary and Animal Sciences University, Pookode, Wayanad, Kerala, India.
Kerala Veterinary and Animal Sciences University, Pookode, Wayanad, Kerala, India; Department of Veterinary Animal Genetics and Breeding, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, 680 651, India.
Exp Parasitol. 2025 Sep;276:108983. doi: 10.1016/j.exppara.2025.108983. Epub 2025 Jul 30.
Theileria orientalis, the causative agent of oriental theileriosis is a globally distributed protozoan parasite affecting livestock. Rapid and accurate parasite detection is crucial for effective disease management and evaluating therapeutic interventions. The high prevalence of T. orientalis in Kerala, a south Indian state demanded the development of a sensitive field tool for specific detection. Loop-mediated isothermal amplification (LAMP) is a rapid and highly sensitive nucleic acid amplification technique conducted under isothermal conditions. In this study, a LAMP assay was developed targeting the major piroplasm surface protein (MPSP) gene of T. orientalis using colourimetric dyes (both hydroxy naphthol blue (HNB) and phenol red) for improved visual detection of amplification. This assay utilised a set of six specifically designed primers, recognizing eight distinct regions on the target gene. Both wet LAMP assays demonstrated the ability to amplify DNA at levels as low as 10 ng (0.1 pg), corresponding to a parasitaemia level of 0.0012 % and exhibited higher detection abilities than PCR. The assay also demonstrated high specificity, with no amplification observed for DNA template from other haemoprotozoans. Positive LAMP products were identified by a distinct colour change from violet to blue and pink to yellow for HNB and phenol red dyes, respectively. Results were confirmed using agarose gel electrophoresis, showing characteristic ladder patterns. The LAMP assays detected T. orientalis in 62.8 % of samples, outperforming PCR (60 %) and microscopy (52.8 %). With a sensitivity of 100 %, specificity of 93 %, positive predictive value of 95.54 % and negative predictive value of 100 %, the wet LAMP assay demonstrated diagnostic efficacy for T. orientalis.
东方泰勒虫是东方泰勒虫病的病原体，是一种全球分布的原生动物寄生虫，可感染家畜。快速准确地检测寄生虫对于有效的疾病管理和评估治疗干预措施至关重要。印度南部喀拉拉邦东方泰勒虫的高流行率要求开发一种用于特异性检测的灵敏现场工具。环介导等温扩增（LAMP）是一种在等温条件下进行的快速且高度灵敏的核酸扩增技术。在本研究中，开发了一种LAMP检测方法，以东方泰勒虫的主要梨形虫表面蛋白（MPSP）基因为靶点，使用比色染料（羟基萘酚蓝（HNB）和酚红）来改善扩增的视觉检测。该检测方法使用了一组六个专门设计的引物，识别目标基因上的八个不同区域。两种湿LAMP检测方法都证明能够扩增低至10 ng（0.1 pg）的DNA，对应于0.0012%的寄生虫血症水平，并且比PCR表现出更高的检测能力。该检测方法还表现出高特异性，未观察到来自其他血原虫的DNA模板的扩增。对于HNB和酚红染料，分别通过从紫色到蓝色以及从粉红色到黄色的明显颜色变化来鉴定阳性LAMP产物。使用琼脂糖凝胶电泳确认结果，显示出特征性的梯状条带模式。LAMP检测方法在62.8%的样本中检测到东方泰勒虫，优于PCR（60%）和显微镜检查（52.8%）。湿LAMP检测方法具有100%的灵敏度、93%的特异性、95.54%的阳性预测值和100%的阴性预测值，证明了对东方泰勒虫具有诊断效力。
