Sensitive and rapid detection of Babesia species in dogs by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD)

Abstract

Canine babesiosis is a tick-borne disease caused by Babesia spp., which infects and destroys healthy erythrocytes, leading to mortality and morbidity in dogs. The diagnosis of babesiosis is tedious and time-consuming, especially in latent and chronic infections. Here, a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay was developed for rapid and accurate detection of Babesia spp. in canine blood specimens based on the 18S rRNA region. The RPA-LFD assay using rpaBab264 gave specificity to Babesia spp. in dogs (B. vogeli and B. gibsoni) without cross-amplification to other parasites (apicomplexans and non-apicomplexans), with detection limit of at least 22.5 copies/μl (0.1 fg/µl) at 40 °C for at least 10 min. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. To determine the performance of the RPA-LFD assay, a total of 30 clinical samples was examined and compared with conventional PCR (cPCR) and multiplex HRM (mHRM). Eight dogs (26.67%) were detected as positive by RPA-LFD, while seven and six were found positive by cPCR and mHRM, respectively. RPA-LFD and cPCR showed high agreement with Babesia spp. detection with kappa > 0.9. We confirmed that the dogs were infected by B. vogeli from sequences of positive PCR results. Our findings suggested that RPA-LFD using the rpaBab264 assay offered a rapid, accurate, cost-effective and simple method for Babesia spp. detection that is feasibly applicable to be rapid kit at a pet hospital or point-of-care testing.

Recombinase polymerase amplification combined with lateral flow dipstick assay for rapid visual detection of A.simplex (s. s.) and A.pegreffii in sea foods

“…This difference may have resulted from the primer specificity under room temperature being lower than that of PCR, resulting in non-specific amplification and primer dimer. This disadvantage can be made up by optimizing the conditions for RPA to minimize false-positive results, as suggested in other reports [ 41 , 43 ]. Furthermore, the quality of DNA extracted methods may also affect the results of the RPA-LFD assay, and the high quality and concentration of pure DNA can improve its sensitivity and efficiency.…”

“…The RPA reaction system is typically sufficient for 10–20 min detection at 37 °C. RPA has been widely used for detecting bacteria [ 36 , 37 ], fungi [ 38 , 39 ], viruses [ 40 , 41 ], and parasites [ 42 , 43 ], with the aid of fluorescently labeled probes or the lateral flow dipstick (LFD) for visual analysis of the amplified products. Additionally, the RPA reaction system has advantages in identifying specific genes, e.g., those linked with drug-resistant genes, transfected genes, and novel coronaviruses-specific genes [ 7 , 35 , 44 ].…”

A novel detection method based on MIRA-CRISPR/Cas13a-LFD targeting the repeated DNA sequence of Trichomonas vaginalis

“…It simplifies the amplification of target genes in direct scenarios, eliminating the need for expensive equipment, and has been developed for diagnosing T. vaginalis [ 34 ]. The integration of IAT with LFD for visualizing detection results has been successfully reported in detecting viruses [ 35 ], bacteria [ 36 ], and parasites [ 37 ]. However, its application in T. vaginalis detection remains unreported, likely due to challenges in ensuring adequate specificity.…”

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries

“…When compared to other methods of detection, LFD's portability, ease of use, visualization, low cost, and quick setup time make it an attractive option [28]. In addition, DNA amplification using RPA and LFD readouts can be completed in under 30 min [41,42]. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) is currently widely employed in many testing domains, including clinical research [38], food safety research [42], plant disease detection [43], algae detection [44], and others [45].…”