Loop-Mediated Isothermal Amplification for the Fast Detection of Bonamia ostreae and Bonamia exitiosa in Flat Oysters

PATHOGENS(2024)

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摘要
The haplosporidian parasites Bonamia ostreae (BO) and B. exitiosa (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 actin-1 and BE actin genes. A further generic assay was conceived on a conserved region of the 18S gene to detect both Bonamia species. The optimal reaction temperature varied from 65 to 67 degrees C depending on the test and instrument. Melting temperatures were 89.8-90.2 degrees C, 87.0-87.6 degrees C, and 86.2-86.6 degrees C for each of the BO, BE, and generic assays. The analytical sensitivity of these assays was 50 copies/mu L in a 30 min run. The BO and BE test sensitivity was similar to 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 Haplosporidium costale. 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.
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关键词
Bonamia ostreae,Bonamia exitiosa,bonamiosis,oyster,haplosporidia,LAMP,diagnostic,point-of-care test
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