Establishment of loop-mediated isothermal amplification assay for detection of parasitic ciliate ichthyophthirius multifiliis in cyprinid fish

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.