movement of horses 6,7. Importation of carrier animals with no obvious signs of disease is a major risk factor for the introduction of EP into non-enzootic areas 1. Therefore, developing sensitive and specific diagnostic methods is essential for identifying asymptomatic equines carrying these parasites. Current diagnostic methods include aetiological diagnostics 8,9 , immunological diagnostics 10-14 and molecular diagnostics 9,15-33. Of these, molecular diagnostics is widely recognized due to its accuracy and sensitivity 9,34. Cortes et al. developed a multinested PCR assay for simultaneous detection of the equine piroplasmids T. equi and B. caballi by amplification of five genetic markers (18S rRNA, β-tubulin, cytB, ema-1 and rap-1) 23. To identify the species of piroplasmid in an infected horse, nested PCR assay is commonly used to amplify long fragments of 18S rRNA, followed by sequence analysis 22. However, PCR-based diagnosis requires a thermocycler, skilled personnel and a long detection time, which made it inappropriate for field diagnostic applications. As an alternative, recombinase polymerase amplification (RPA) has emerged as a novel isothermal technique for molecular diagnosis of various infectious diseases 35 , including the protozoan parasites 36 Theileria annulata 37 and Babesia gibsoni 38. Compared to PCR-based assay and loop-mediated isothermal amplification (LAMP), RPA is more rapid (<20 min), simpler to perform as it requires a lower temperature (37-42 °C) and has an acceptable sensitivity 39,40. RPA-lateral flow assay has been used for rapid detection of Trichinella 41 , Perkinsus beihaiensis 42 , Plasmodium knowlesi 43 , Babesia gibsoni 38 , Protozoan parasites 36 , Theileria annulata 37 , Fasciola hepatica 44 , Schistosoma japonicum 45 , Schistosoma haematobium 46 , Leishmania donovani 47 , Intestinal Protozoa 48 , Giardia 49 , Plasmodium falciparum 50. To avoid the "ghost band" and the false-positive results, the primers, probes and the detection procedure have to be carefully designed 39. To speed up the clearance of an imported horse at a port, or detect a piroplasmosis infection in the field or in low-resource underserved rural communities, we also adapted RPA to develop a duplex detection of both T. equi and B. caballi as an alternative to duplex qPCR 26,30. Various genomic sites have been used in species identification, phylogenetic and genotype studies of both T. equi and B. caballi with PCR-based molecular techniques, including the small subunit ribosomal RNA gene (18S rRNA) 11,22,51-54 ; and genomic sites targeted by qPCR assay have included the ema-1 gene of T. equi and the 48 kDa merozoite rhoptry protein (bc48) gene of B. caballi 30. These sites have been used to create a duplex real-time PCR for simultaneous detection of both parasites using the ema-1 gene of T. equi and the bc48 gene of B. caballi 30 ; or the 18S rRNA gene of B. caballi and the ema-1 gene of T. equi 26. In this study, we designed primers and fluorescent probes to target the 18S rRNA gene of both T. equi and B. ...
