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Molecular Detection and Quantification of the Fish Pathogen Saprolegnia spp. Using qPCR and Loop Mediated Isothermal Amplification

Ghosh, Satyaki
http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1573814311236554

Abstract Details

2019, Doctor of Philosophy (Ph.D.), Bowling Green State University, Biological Sciences.
Saprolegniasis, caused by oomycete pathogens of the Saprolegnia genus is a serious emergent disease of fish causing losses worth approximately $40 million annually in USA. This study aims to analyze the efficacy of copper sulfate and peracetic acid as chemical agents to combat saprolegniasis; and to develop a molecular strategy for the rapid, sensitive and specific detection of Saprolegnia spp., suitable for on-site applications. The study indicates that copper sulfate and peracetic acid effectively reduced the various parameters of Saprolegnia spp. growth. Peracetic acid was effective against other oomycete pathogens to varying degrees, and its persistence in environmental water samples depends on the organic matter content of the water samples. Peracetic acid has been proposed as an effective, non-toxic, and eco-friendly approach to combat saprolegniasis. This study reports the isolation Saprolegnia spp. from various sources. Using cytochrome c oxidase subunit I (COI) and Internal Transcribed Spacer of rDNA (ITS) as molecular markers, these have been identified phylogenetically. Based on these markers, qPCR primers have been developed specific to the Saprolegnia genus and could detect as low as 2pg of Saprolegnia spp. genomic DNA. Also, qPCR based absolute quantification could be used as an approach to quantify the Saprolegnia spp. levels in environmental samples. Additionally, a LAMP assay was developed using the ITS marker. The established LAMP assay was specific to the Saprolegnia genus and could detect as low as 10 fg of Saprolegnia spp. genomic DNA within 30 min, thus making it significantly more sensitive compared to qPCR. Both qPCR and LAMP could also detect as low as 1 zoospore directly. The LAMP reactions could be performed using a simple equipment such as heat block, and results could be detected visually. Further, LAMP has the potential for direct on-field applications for detecting Saprolegnia spp. from water samples collected from Recirculating Aquaculture Systems (RAS). Based on the results, we propose the application of LAMP in conjunction with qPCR, as a rapid and cheap diagnostic technique for Saprolegnia spp. pathogens directly from water samples, which would enable fish farmers to make informed decisions regarding the timing and extent of chemical treatment.
Vipaporn Phuntumart, Dr. (Advisor)
Lubomir Popov, Dr. (Other)
Raymond Larsen, Dr. (Committee Member)
Paul Morris, Dr. (Committee Member)
Scott Rogers, Dr. (Committee Member)
David Straus, Dr. (Committee Member)
157 p.
Biology
Oomycetes; Saprolegnia; zoospores; PCR; qPCR; LAMP; Copper Sulfate; PAA; Aquaculture; ITS; COI

Recommended Citations

Citations

  • Ghosh, S. (2019). Molecular Detection and Quantification of the Fish Pathogen Saprolegnia spp. Using qPCR and Loop Mediated Isothermal Amplification [Doctoral dissertation, Bowling Green State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1573814311236554

    APA Style (7th edition)

  • Ghosh, Satyaki. Molecular Detection and Quantification of the Fish Pathogen Saprolegnia spp. Using qPCR and Loop Mediated Isothermal Amplification. 2019. Bowling Green State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1573814311236554.

    MLA Style (8th edition)

  • Ghosh, Satyaki. "Molecular Detection and Quantification of the Fish Pathogen Saprolegnia spp. Using qPCR and Loop Mediated Isothermal Amplification." Doctoral dissertation, Bowling Green State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1573814311236554

    Chicago Manual of Style (17th edition)

bgsu1573814311236554
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© 2019, some rights reserved.Creative Commons License Molecular Detection and Quantification of the Fish Pathogen Saprolegnia spp. Using qPCR and Loop Mediated Isothermal Amplification by Satyaki Ghosh is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Bowling Green State University and OhioLINK.