Development and Sensitivity Determination of Gene-specific Fast Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of .

Objective: , one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by . In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to 18S rRNA gene to detect more rapidly the presence of in clinical and environmental samples.

Methods: strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL.

Results: To determine the sensitivity of the test, positivity of genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%.

Conclusion: It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of in environmental and clinical samples.