End-point detection of loop-mediated isothermal amplification (LAMP) on malaria by direct observation with colorimetric dyes

Abstract

In order to ascertain the results of the LAMP technique, different end-point detection methods can be employed. However, these methods require sophisticated equipment. To simplify current end-point detection methods for the diagnosis of malaria, we propose the incorporation of colorimetric dyes: malachite green (MG), phenol red (PR), and xylenol orange (XO) in the LAMP assay. To evaluate the optimum concentration of dyes, 5 different concentrations (50 μM, 75 μM, 100 μM, 125 μM, and 150 μM) were used with buffer pH 8.5 and pH 8.8, respectively. The results showed that 125 μM of MG at pH 8.8 produced the most obvious colour change. A total of 71 clinical blood samples of Plasmodium knowlesi, Plasmodium malariae, Plasmodium vivax, Plasmodium falciparum, and healthy donors were tested using MG-LAMP. It showed 100% sensitivity and specificity. The simplicity and affordability of this method make it ideal to be used as an end-point detection method for malaria diagnosis in resource limited settings.