Loop-Mediated Isothermal Amplification for the Fast Detection of and in Flat Oysters.

The haplosporidian parasites (BO) and (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 and BE genes. A further generic assay was conceived on a conserved region of the gene to detect both 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 . 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.