Abstract
The objective of this research was to establish loop-mediated isothermal amplifications (LAMP) that could be used to detect parasitic ciliate Ichthyophthirius multifiliis (I. multifiliis) in freshwater cyprinid fish. Primers were developed from the distinguishing fragments of 18S ribosomal RNA of I. multifiliis and the LAMP test was then used to evaluate and optimize various concentrations of chemicals, time and temperature. The results indicated that LAMP required 1.6 μM of FIP primers and BIP primers, 0.2 μM of F3 and B3, 2 mM of Mg2+, 1 M of Betaine, and 0.6 mM of dNTP. This assay was able to detect parasite DNA within a 40 min period of incubation and at a constant optimal temperature of 64oC. The positive sample appeared as a clear ladder like pattern on gel electrophoresis, while a yellowish green color appeared with SYBR Green I under ultraviolet light with the use of a heating block. The LAMP test was determined to be more sensitive than conventional PCR in the detection of I. multifiliis. In conclusion, we have presented a sensitive and specific rapid detection system for I. multifiliis based on isothermal DNA amplification. Importantly, this system could then be employed as an alternative and effective diagnostic method in place of other molecular techniques.
Highlights
Freshwater cyprinid fish are the most popular order of fish that are kept as pets worldwide
To confirm the specifications of the reaction, the loop-mediated isothermal amplifications (LAMP) product was analyzed by AluI restriction enzyme digestion and the results indicated predicted sizes of 95 bp on 1.5% of agarose gel, otherwise the BanI and ApaI restriction enzyme could not digest the LAMP product and resulted in a ladder-like form on gel electrophoresis (Figure 3)
To establish sensitivity or detection limits, the results of the LAMP assay were achieved at 5 pg of I. multifiliis DNA (Figure 4), whereas the results of the conventional PCR were achieved at 50 pg of the template DNA concentration
Summary
Freshwater cyprinid fish are the most popular order of fish that are kept as pets worldwide. A number of serious problems have been associated with the keeping of these fish These problems have resulted from inappropriate management practices, while disease outbreaks that occurred as a result of the presence of various pathogens such as bacterial infections (McDermott and Palmeiro 2020; Saengsitthisak et al, 2020), viruses (Pikulkaew et al, 2009; Pikulkaew et al, 2020) and parasitic infections (Saengsitthisak et al, 2021, Sirri et al, 2020). Ichthyophthirius multifiliis (I. multifiliis) is the known cause of freshwater white spot disease, or ichthyophthiriasis, and other serious ectoparasitic infestation/infections in freshwater cyprinid fish along with other freshwater species of fish worldwide (Dickerson and Clark, 1996). Ichthyophthiriasis infections frequently lead to high morbidity and mortality rates on fish farms (Noga, 2010)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
