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

Overview

• This research article presents the development of a rapid and sensitive diagnostic test for detecting Theileria equi, a parasite responsible for equine piroplasmosis, using an integrated assay based on recombinase polymerase amplification (RPA), CRISPR/Cas13a technology, and lateral flow strips.

Background and Importance

• Equine piroplasmosis (EP) is a significant disease impacting horses globally, caused by parasites including Theileria equi, Babesia caballi, and Theileria haneyi.
• The disease poses serious health risks to horses and leads to economic losses in the equine industry.
• Current diagnostic techniques such as PCR, serology, and microscopy require specialized equipment, trained personnel, and lengthy procedures, limiting their use in field conditions.

Research Objective

• The aim was to develop a rapid, accurate, and easy-to-use molecular diagnostic assay for T. equi that could be used in both laboratory and field settings without complex equipment.

Methodology

• Design:
  • The assay combines RPA, a technique for amplifying nucleic acids at a constant temperature, with CRISPR/Cas13a, an enzyme system that makes it possible to detect specific RNA sequences with high sensitivity and specificity.
  • Detection involves a lateral flow device (LFD), which provides a simple visual result similar to a pregnancy test strip, allowing easy interpretation without instruments.
  • The target for detection is a conserved genetic region called erythrocyte merozoite antigen 1 (EMA-1) gene from T. equi, ensuring specificity.
• Sample Validation:
  • 22 horse blood samples were tested, including clinically affected, asymptomatic infected, and healthy animals.
  • Specificity controls included samples infected with Babesia caballi and Anaplasma phagocytophilum, synthetic EMA-1 DNA as positive control, and non-template controls as negative checks.
• Operational Conditions:
  • All assay steps were performed at room temperature, negating the need for specialized incubation or thermal cycling equipment.

Results

• The assay produced visible results on the lateral flow strips in under 50 minutes, making it quite rapid compared to traditional methods.
• It exhibited 100% specificity, with no false positive detections noted from non-target parasites or negative controls.
• The test effectively distinguished horses infected with T. equi — whether exhibiting clinical symptoms or asymptomatic — from uninfected healthy animals, confirming its high accuracy and reliability.

Conclusions and Future Directions

• The developed RPA-CRISPR/Cas13a lateral flow assay is a fast, sensitive, specific, and user-friendly diagnostic platform that operates at ambient temperature, making it especially suitable for field use or low-resource environments.
• While the current protocol requires prior DNA extraction from blood samples, future improvements aim to allow direct testing from unprocessed blood, which would further simplify and accelerate point-of-care diagnosis for equine piroplasmosis.
• Such advancements could enhance disease surveillance, timely treatment, and control strategies, benefiting animal health and the equine industry worldwide.