Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis

Abstract

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. ...

Challenges in Tick-Borne Pathogen Detection: The Case for Babesia spp. Identification in the Tick Vector

“…The identification of B. caballi genotypes A and B in the equine blood samples and only B. caballi genotype B in H. marginatum ticks were achieved [109]. Other recently developed DNAbased diagnostic techniques are the loop-mediated isothermal amplification (LAMP) [110] and the rapid isothermal duplex real-time recombinase polymerase amplification (RPA) [25] assays for the diagnosis of equine piroplasmosis. However, the use of both the LAMP and the RPA assays for detection of the parasites in the vector ticks still needs to be explored.…”

“…The LAMP and RAP assays are detection methods also based on the molecular identification and have been implemented to Babesia detection, but mostly in its invertebrate host. Whereas, the serological methods implemented in vertebrate hosts include: Indirect Fluorescent Antibody Test (IFAT), Enzyme-Linked Immunosorbent Assay (ELISA), Complement Fixation Test (CFT) and Immunochromatography Test (ICT) [23][24][25][26][27][28][29][30]. Asexual reproduction is carried out in vertebrates: it begins when the infected tick feeds on the host and inoculates the infective phase of Babesia, the sporozoites (1).…”

Colorimetric loop-mediated isothermal amplification (LAMP) for cost-effective and quantitative detection of SARS-CoV-2: the change in color in LAMP-based assays quantitatively correlates with viral copy number

“…For instance, experimental evidence collected from a small number of COVID-19 patients (9 subjects) showed that 100% of them produced specic immunoglobulins G (IgGs) for SARS-CoV-2 within two weeks of infection, but only 50% of them did during the rst week post infection. 16 Nucleic acid amplication continues to be the gold standard for the detection of viral diseases in the early stages, [17][18][19][20][21] and very small viral loads present in symptomatic or asymptomatic patients can be reliably detected using amplication based methods, such as polymerase chain reaction (PCR), 22,23 recombinase polymerase amplication (RPA), 24 and loop-mediated isothermal amplication (LAMP). [25][26][27] During the last two pandemic events with inuenza A/H1N1/ 2009 and COVID-19, the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommended real-time quantitative PCR (RT-qPCR) methods as the gold standard for official detection of positive cases.…”

Development of a recombinase polymerase amplification assay for rapid detection of Haemophilus parasuis in tissue samples

“…Recombinase polymerase amplification (RPA), which is an isothermal DNA amplification technique, has experienced rapid development in the field of molecular diagnostic since it was firstly reported in 2006 (Daher, Stewart, Boissinot, & Bergeron, 2016;Lei et al, 2020;Li, Macdonald, & von Stetten, 2018;Liu et al, 2018;Miao et al, 2019;Piepenburg, Williams, Stemple, & Armes, 2006;Wang, Yuan, Han, Wang, & Liu, 2017). Some studies showed that RPA may be the most applicable approach for the on farm diagnosis due to its convenience and rapidness (Amer et al, 2013;Li et al, 2018).…”