Location: Harry K. Dupree Stuttgart National Aquaculture Research CntrTitle: Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp
|GHOSH, SATYAKI - Bowling Green State University|
|Straus, David - Dave|
|GOOD, CHRISTOPHER - Freshwater Institute|
|PHUNTUMART, VIPAPORN - Bowling Green State University|
Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/2021
Publication Date: 12/13/2021
Citation: Ghosh, S., Straus, D.L., Good, C., Phuntumart, V. 2021. Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp. PLoS ONE. 16(12):e0250808. https://doi.org/10.1371/journal.pone.0250808.
Interpretive Summary: A typical type of water mold (or fungus) called Saprolegnia can cause significant problems in the aquaculture industry. Diagnosing this disease on fish or eggs can take time with existing analyses. Here we describe developing a new procedure that can detect extremely low levels of this disease within 30 minutes and this allows for earlier and more exact treatments. We compare this new procedure called Loop Mediated Isothermal Amplification (LAMP) to quantitative PCR analyses which is routinely used for this kind of disease diagnosis.
Technical Abstract: Saprolegniasis is estimated to cause economic losses of 10% in salmon production and 30% in fish production worldwide. Early detection of Saprolegnia spp. pathogens can significantly reduce losses by allowing for earlier treatments. Current culture-based detection techniques require one to two days to achieve results. Therefore, a molecular diagnostic procedure for rapid and sensitive detection of this pathogen that is suitable for onsite application would be welcomed by the aquaculture industry. This study establishes Loop Mediated Isothermal Amplification (LAMP) as a rapid and sensitive molecular tool for onsite detection of Saprolegnia spp. from water samples. This diagnostic procedure will allow informed decisions about the timing and extent of disease treatment. LAMP reactions are performed at a constant temperature and results can be estimated visually within 30 minutes which makes it suitable for field diagnostics. This LAMP procedure was specific to the Saprolegnia genus and will detect as low as 10fg of Saprolegnia salmonis genomic DNA, or just one zoospore of Saprolegnia spp. directly. As a comparison, we also developed a qPCR strategy for the detection of Saprolegnia spp. which has a detection limit of 2pg of Saprolegnia salmonis genomic DNA, or a minimum of 1 zoospore directly. The LAMP procedure was more sensitive and faster compared to qPCR and was suitable for on-site applications. The advantage to using qPCR is that it can provide quantitative information regarding Saprolegnia spp. loads in water samples.