Development and Dual-Format Recombinase Polymerase Amplification (RPA) Assay for Rapid Detection of Anaplasma Platys in Canine Blood

Abstract

Anaplasma platys, the etiological agent of canine cyclic thrombocytopenia, is an emerging tick-borne pathogen with limited diagnostic options in resource-poor settings. We developed and validated a recombinase polymerase amplification (RPA) assay for rapid detection of A. platys in canine blood, using both fluorescence-based real-time detection and lateral flow strip visualization. Primers and probe targeting the 16S rRNA gene were designed and optimized at 39 °C for 20 minutes. Analytical sensitivity was 10 fg DNA, surpassing conventional PCR (100 fg). Specificity testing showed no cross-reactivity with Ehrlichia canis, Babesia vogeli, Hepatozoon canis, or Mycoplasma haemocanis. Fluorescence RPA exhibited a strong correlation between threshold time and DNA concentration (R² = 0.981), while lateral flow RPA produced clear test bands within 30 minutes with high observer agreement (κ = 0.95). In 500 clinical canine blood samples from Chennai, India, A. platys prevalence was 8.6% (43/500), with perfect concordance between RPA (both formats) and PCR (κ = 1.000). The combined formats establish RPA as a rapid, versatile, and field-deployable diagnostic tool for veterinary practice and epidemiological surveillance.