Reliable interpretation and long-term stability using SYBRTM safe fluorescent assay for loop-mediated isothermal amplification (LAMP) detection of Leishmania spp.

摘要

Leishmaniasis, a vector-borne disease caused by Leishmania, is the secondleading mortality after malaria. Continuously increasing cases of cutaneous and visceralleishmaniasis (CL/VL) have been documented in Thailand. Recently, loop-mediatedisothermal amplification (LAMP) based on malachite green (MG) colorimetric assay thatdetects Leishmania DNA was developed to facilitate epidemiological studies ofleishmaniasis in affected areas. However, ambiguous reading interpretation sometimesoccurred using the MG-LAMP assay. In this study, the efficiency and effectiveness of theSYBRTM Safe fluorescent assay for LAMP detection of Leishmania siamensis (MON-324)and Leishmania martiniquensis (MON-229) were compared under two different lightsources, i.e., blue light and ultraviolet light transilluminators. Regarding the SYBRTM-LAMPassay, the detection limit of DNA of both L. siamensis and L. martiniquensis was 103parasites/mL. The assay exhibited consistency and reproducibility without requiring anypost-reaction preparations. The dye is generally available, affordable and safe while reliableinterpretation can be easily visualized under both blue light and ultraviolet lighttransilluminators. Using buffy coat of VL patients, the SYBRTM-LAMP offers an alternativemethod for screening samples with high sensitivity and specificity. This cost effectiveSYBRTM Safe fluorescent assay is simple to use without ambiguous evaluation which couldprovide another suitable choice of a standard LAMP assay in molecular laboratories aswell as further development in field studies.