Rapid and sensitive detection of Theileria equi using a novel integrated RPACRISPR/Cas13a lateral flow assay

IF 1.6 3区农林科学 Q2 VETERINARY SCIENCES

Journal of Equine Veterinary Science Pub Date : 2025-12-01 Epub Date: 2025-11-10 DOI:10.1016/j.jevs.2025.105732

Amjed Alsultan , Salah Mahdi Karim , Mohammed Al-Saadi , Dhama Alsallami , Mourad Ben Said , Hanène Belkahia

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引用次数: 0

Abstract

Background

Equine piroplasmosis (EP), caused by the intracellular protozoa Theileria equi, Babesia caballi, and Theileria haneyi, represents a major health and economic threat to the equine industry worldwide. Existing diagnostic methods, including PCR, serology, and microscopy, are constrained by their dependence on specialized equipment, lengthy protocols, and the requirement for skilled personnel.

Aim

This study aimed to develop a rapid, accurate, and field-deployable molecular diagnostic assay for T. equi.

Methods

A nucleic acid-based diagnostic platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas13-mediated detection and lateral flow device (LFD) readout was developed. The assay targets a conserved region of the erythrocyte merozoite antigen 1 (EMA-1) gene of T. equi. Validation was performed using 22 blood samples collected from horses, as well as specificity controls including B. caballi- and Anaplasma phagocytophilum-infected samples, synthetic EMA-1 DNA, and non-template controls. All assay steps were conducted at room temperature.

Results

The integrated RPA-CRISPR/Cas13-LFD assay generated clear visual results within 50 minutes. It demonstrated complete specificity with no false positives across all tested samples. The method effectively differentiated horses infected with T. equi, including both clinically affected and asymptomatic individuals, from healthy, uninfected animals, confirming its high accuracy and reliability.

Conclusion

The developed assay provides a rapid, precise, and equipment-free diagnostic platform suitable for both field and clinical environments. Although the current protocol relies on DNA extraction, future optimization will aim to enable direct detection from unprocessed blood samples, thereby further simplifying point-of-care diagnostics for equine piroplasmosis.

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利用新型集成RPACRISPR/Cas13a横向流动试验快速灵敏地检测马伊勒氏菌

背景：马螺浆虫病（EP）是由细胞内原生动物马伊勒氏菌、卡巴贝斯虫和哈尼伊伊勒氏菌引起的，是全球马业的主要健康和经济威胁。现有的诊断方法，包括聚合酶链反应（PCR）、血清学和显微镜，由于依赖专门的设备、冗长的协议和对熟练人员的要求而受到限制。目的：本研究旨在建立一种快速、准确、可在现场部署的马蹄疫分子诊断方法。方法：建立结合重组酶聚合酶扩增（RPA）、CRISPR/ cas13介导检测和侧流装置（LFD）读数的核酸诊断平台。该检测的目标是马蹄疫红细胞裂殖子抗原1 （EMA-1）基因的一个保守区域。使用从马身上采集的22份血液样本进行验证，以及特异性对照，包括caballi和无原体吞噬细胞感染样本、合成EMA-1 DNA和非模板对照。所有实验步骤均在室温下进行。结果：整合的RPA-CRISPR/Cas13-LFD检测在50分钟内产生了清晰的视觉结果。它显示出完全的特异性，在所有测试样本中没有假阳性。该方法有效地将感染马弓形虫的马匹（包括临床感染和无症状个体）与未感染的健康动物区分开来，证实了其较高的准确性和可靠性。结论：开发的检测方法提供了一个快速、精确、无设备的诊断平台，适用于现场和临床环境。虽然目前的方案依赖于DNA提取，但未来的优化将旨在实现从未处理的血液样本中直接检测，从而进一步简化马体浆虫病的即时诊断。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Equine Veterinary Science 农林科学-兽医学

CiteScore

2.70

自引率

7.70%

发文量

249

审稿时长

77 days

期刊介绍： Journal of Equine Veterinary Science (JEVS) is an international publication designed for the practicing equine veterinarian, equine researcher, and other equine health care specialist. Published monthly, each issue of JEVS includes original research, reviews, case reports, short communications, and clinical techniques from leaders in the equine veterinary field, covering such topics as laminitis, reproduction, infectious disease, parasitology, behavior, podology, internal medicine, surgery and nutrition.