Scientific Reports (Nov 2022)

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

  • Warunya Onchan,
  • Onchira Ritbamrung,
  • Phanupong Changtor,
  • Waranee Pradit,
  • Siriwadee Chomdej,
  • Korakot Nganvongpanit,
  • Puntita Siengdee,
  • Urasri Suyasunanont,
  • Kittisak Buddhachat

DOI
https://doi.org/10.1038/s41598-022-25165-7
Journal volume & issue
Vol. 12, no. 1
pp. 1 – 11

Abstract

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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.