Reliable interpretation and long-term stability using SYBRTM safe fluorescent assay for loop-mediated isothermal...

Leishmaniasis, a vector-borne disease caused by Leishmania, is the second leading mortality after malaria. Continuously increasing cases of cutaneous and visceral leishmaniasis (CL/VL) have been documented in Thailand. Recently, loop-mediated isothermal amplification (LAMP) based on malachite green (MG) colorimetric assay that detects Leishmania DNA was developed to facilitate epidemiological studies of leishmaniasis in affected areas. However, ambiguous reading interpretation sometimes occurred using the MG-LAMP assay. In this study, the efficiency and effectiveness of the SYBR^TM Safe fluorescent assay for LAMP detection of Leishmania siamensis (MON-324) and Leishmania martiniquensis (MON-229) were compared under two different light sources, i.e., blue light and ultraviolet light transilluminators. Regarding the SYBR^TM-LAMP assay, the detection limit of DNA of both L. siamensis and L. martiniquensis was 10³ parasites/mL. The assay exhibited consistency and reproducibility without requiring any post-reaction preparations. The dye is generally available, affordable and safe while reliable interpretation can be easily visualized under both blue light and ultraviolet light transilluminators. Using buffy coat of VL patients, the SYBR^TM-LAMP offers an alternative method for screening samples with high sensitivity and specificity. This cost effective SYBR^TM Safe fluorescent assay is simple to use without ambiguous evaluation which could provide another suitable choice of a standard LAMP assay in molecular laboratories as well as further development in field studies.