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Loop-Mediated Isothermal Amplification for the Fast Detection of <i>Bonamia ostreae</i> and <i>Bonamia exitiosa</i> in Flat Oysters
oleh: Irene Cano, Gareth Wood, David Stone, Mathilde Noyer, Lydie Canier, Isabelle Arzul
| Format: | Article |
|---|---|
| Diterbitkan: | MDPI AG 2024-01-01 |
Deskripsi
The haplosporidian parasites <i>Bonamia ostreae</i> (BO) and <i>B. exitiosa</i> (BE) are serious oyster pathogens. Two independent laboratories evaluated fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for rapidly detecting these parasites. Specific LAMP assays were designed on the BO <i>actin-1</i> and BE <i>actin</i> genes. A further generic assay was conceived on a conserved region of the <i>18S</i> gene to detect both <i>Bonamia</i> species. The optimal reaction temperature varied from 65 to 67 °C depending on the test and instrument. Melting temperatures were 89.8–90.2 °C, 87.0–87.6 °C, and 86.2–86.6 °C for each of the BO, BE, and generic assays. The analytical sensitivity of these assays was 50 copies/µL in a 30 min run. The BO and BE test sensitivity was ~1 log lower than a real-time PCR, while the generic test sensitivity was similar to the real-time PCR. Both the BO and BE assays were shown to be specific; however, the generic assay potentially cross-reacts with <i>Haplosporidium costale</i>. The performance of the LAMP assays evaluated on samples of known status detected positives within 7–20 min with a test accuracy of 100% for the BO and generic tests and a 95.8% accuracy for BE. The ease of use, rapidity and affordability of these tests allow for field deployment.