Visual Detection of Canine Monocytic Ehrlichiosis Using Polymerase Chain Reaction-Based Lateral Flow Biosensors

Abstract

A conventional PCR (cPCR) remains an effective molecular technique for the diagnosis of canine monocytic ehrlichiosis. However, agarose gel electrophoresis requires additional time after thermal cycling. In the present study, we developed a PCR-based lateral flow biosensor (PCR-LFB) to detect <i>Ehrlichia canis</i> (<i>E. canis</i>). Lateral flow strips allow for the simple and rapid detection of PCR products and provide an alternative to gel electrophoresis. The sensitivity, specificity, and detection limit of PCR-LFB were compared to those of TaqMan probe-based real-time PCRs (qPCRs). The PCR-LFB was performed with 5′ 6-FITC and biotin-labeled primers specific to <i>E. canis</i>, targeting the <i>dsb</i> gene. The detection limit of the PCR-LFB assay was 10⁻⁶ for the target DNA sequence in a 10-fold dilution of the recombinant plasmid, which is 10 times lower than that of qPCR. Among the confirmed qPCR results in the 30 dog samples, false-positive results were not detected by the PCR-LFB. Compared to qPCR, the sensitivity and specificity of PCR-LFB were 63.6% (95% CI; 42.9–80.2%) and 100% (95% CI; 67.5–100%), respectively. The Kappa value of the PCR-LFB is in moderate agreement with the qPCR (κ = 0.483). Perfect agreement (κ = 1) was observed between cPCR and PCR-LFB. Lower cost and shorter time consumption were demonstrated using PCR-LFB.