Loop-mediated isothermal amplification for rapid detection of Cryptococcus neoformans

Objective To establish a method of loop-mediated isothermal amplification(LAMP) assay for rapid detecting Cryptococcus neoformans(C. neoformans). Methods Two pairs of primers were designed according to 18 S rRNA gene sequence of C. neoformans. Performing LAMP at different temperatures in order to ascertain the optimum temperature of LAMP method for detection of C. neoformans, other common pathogens of meningitis and some species of candida were all amplified through LAMP, to investigate the specificity of detecting C. neoformans by LAMP assay. Fresh cultured of C. neoformanswas diluted into different concentrations with non-cryptococcal infection cerebrospinal fluid, to build C. neoformans meningitis cerebrospinal fluid simulation environments. Sensitivity was evaluated through detecting C. neoformans of different concentrations in cerebrospinal fluid. Result LAMP assay could achieve good amplification between 60 ℃ and 65 ℃. In Specificity test, only C. neoformans showed amplification, no specific reactions were found in other meningitis pathogens nor candidas, LAMP showed a specificity of 100%. The minimum detection limit of LAMP for C. neoformans from cerebrospinal fluid is 102 CFU/ml. Detecting C. neoformans with LAMP assay could save time for culture and conventional identification. From DNA extraction to the end of the reaction could be completed within 2-3 hours, greatly reducing the time needed for diagnosis. Conclusion LAMP could be a very simple, rapid method for detecting C. neoformanswith high specificity and sensitivity.