Development, Optimization, and Validation of a Quantitative PCR Assay for Borrelia burgdorferi Detection in Tick, Wildlife, and Human Samples

Tick-borne pathogens are growing in importance for human and veterinary research worldwide. We developed, optimized, and validated a reliable quantitative PCR (qPCR; real-time PCR) assay to assess Borrelia burgdorferi infection by targeting two B. burgdorferi genes, <i>ospA</i> and <i>flaB</i>. When assessing previously tested tick samples, its performance surpassed the nested PCR in efficiency, sensitivity, and specificity. Since the detection of <i>Borrelia</i> is more difficult in mammalian samples, the qPCR assay was also assessed using wildlife tissues. For wildlife samples, the sensitivity and specificity of <i>ospA</i> primers, with the incorporation of a pre-amplification step, was equivalent or superior to the nested PCR. For human samples, no primer set was successful with human tissue without culture, but we detected Borrelia with <i>ospA</i> and <i>flaB</i> primers in 50% of the Lyme culture samples, corresponding to 60% of the participants with a Lyme disease diagnosis or suspicion. The specificity of amplification was confirmed by Sanger sequencing. The healthy participant culture samples were negative. This PCR-based direct detection assay performs well for the detection of <i>Borrelia</i> in different biological samples. Advancements in detection methods lead to a better surveillance of <i>Borrelia</i> in vectors and hosts, and, ultimately, enhance human and animal health.