Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> 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...
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MDPI AG
2024-11-01
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| author | Julie Lewis Vett K. Lloyd Gilles A. Robichaud |
| author_facet | Julie Lewis Vett K. Lloyd Gilles A. Robichaud |
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| description | 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. |
| format | Article |
| id | doaj-art-b40acd7cc2c845a78a37379a37bec042 |
| institution | DOAJ |
| issn | 2076-0817 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
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| series | Pathogens |
| spelling | doaj-art-b40acd7cc2c845a78a37379a37bec0422025-08-20T02:57:17ZengMDPI AGPathogens2076-08172024-11-011312103410.3390/pathogens13121034Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> Detection in Tick, Wildlife, and Human SamplesJulie Lewis0Vett K. Lloyd1Gilles A. Robichaud2Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB E1A 3E9, CanadaDepartment of Biology, Mount Allison University, Sackville, NB E4L 1G7, CanadaDepartment of Chemistry and Biochemistry, Université de Moncton, Moncton, NB E1A 3E9, CanadaTick-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.https://www.mdpi.com/2076-0817/13/12/1034<i>Borrelia burgdorferi</i>Lyme diseasetickswildlife<i>Borrelia</i> cultureqPCR |
| spellingShingle | Julie Lewis Vett K. Lloyd Gilles A. Robichaud Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> Detection in Tick, Wildlife, and Human Samples Pathogens <i>Borrelia burgdorferi</i> Lyme disease ticks wildlife <i>Borrelia</i> culture qPCR |
| title | Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> Detection in Tick, Wildlife, and Human Samples |
| title_full | Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> Detection in Tick, Wildlife, and Human Samples |
| title_fullStr | Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> Detection in Tick, Wildlife, and Human Samples |
| title_full_unstemmed | Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> Detection in Tick, Wildlife, and Human Samples |
| title_short | Development, Optimization, and Validation of a Quantitative PCR Assay for <i>Borrelia burgdorferi</i> Detection in Tick, Wildlife, and Human Samples |
| title_sort | development optimization and validation of a quantitative pcr assay for i borrelia burgdorferi i detection in tick wildlife and human samples |
| topic | <i>Borrelia burgdorferi</i> Lyme disease ticks wildlife <i>Borrelia</i> culture qPCR |
| url | https://www.mdpi.com/2076-0817/13/12/1034 |
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