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

TL;DR

This paper introduces a new biosensor method for detecting a canine disease using a test strip, which is faster and safer than traditional methods.

Contribution

A PCR-based lateral flow biosensor for detecting Ehrlichia canis with improved speed and safety compared to agarose gel electrophoresis.

Findings

PCR-LFB detected Ehrlichia canis with a detection limit 10 times lower than qPCR.

PCR-LFB showed 100% specificity and no false positives in 30 dog samples.

PCR-LFB had moderate agreement with qPCR but perfect agreement with conventional PCR.

Abstract

Agarose gel electrophoresis (AGE) for the detection of PCR products faces several limitations, including low sensitivity for faint bands, time-intensive steps, the risk of contamination, resolution challenges for similar-sized DNA fragments, and health hazards from UV and carcinogenic dyes. The interpretation of the results requires highly experienced technicians. In addition, incompatibility with real-time analysis could reduce convenience utility in clinical settings. These drawbacks highlight the need for faster, more sensitive, and safer alternatives. This study developed the PCR lateral flow biosensor method for the detection of the Ehrlichia canis dsb gene, which allows for quicker detection by visually displaying PCR results on a simple test strip.

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 Ehrlichia canis (E. canis). 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 E. canis, targeting the dsb 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.

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