Development of Rapid Detection of Candida auris Using Loop-Mediated Isothermal Amplification

Abstract

Objective. Candida auris is a multidrug resistant non-albicans Candida species, capable of causing nosocomial transmission in a susceptible hosts . Conventional laboratory diagnostic methods are not able to accurately identify C. auris from species close to it. Methods. This project was performed on 3 C. auris fungal isolates as a positive sample and 5 standard candida species as a negative control sample. After extracting DNA from positive and negative samples, using primers designed by Primer Explorer V software. the specificity of these primers was evaluated in both PCR and LAMP methods. The sensitivity of the LAMP assay was determined using different C. auris DNA concentrations in descending order by 10-fold serial dilution from 106 copy number to 1 copy number. LAMP products were identified by electrophoresis, turbidity, fluorescence and colorimetric using hydroxy naphthol blue dye. To finally confirm the correct amplification and specificity of the primers, the products obtained from the PCR reaction were sequenced. Results. The LOD (limit of detection) of the LAMP reaction in the identification of Candida auris was 10 times lower than the PCR reaction. LAMP and PCR correctly identified C. auris and provided 100% specificity. The homology analysis based on nBLAST results show the similarity of the sequences of the PCR products with the sequences registered in Gene Bank. Conclusions. In this study, the reproducibility and reliability of LAMP reaction in diagnosis of C. auris in isothermal conditions were investigated and finally confirmed, it was possible to differentiate this species from closely related species.