Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

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Abstract
Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions. Control of human African trypanosomiasis (HAT) or sleeping sickness relies on diagnosis and treatment of infected patients. However, the diagnostic tests in routine use have limited sensitivity, due to a characteristically low parasitaemia in infected individuals. Differentiation of infections by Trypanosoma brucei rhodesiense (causes acute disease) and T. b. gambiense (causes chronic disease) is essential, as the two forms of disease have different treatment regimens. In the present work, loop-mediated isothermal amplification (LAMP) of DNA was successfully used to detect T. b. rhodesiense, with a sensitivity of up to one trypanosome/ml of blood. The LAMP test was efficient and robust, and results were obtained within 35 min. Amplification was possible when a water bath was used to maintain the temperature at isothermal conditions (60–65°C), and results could be read by visual observation of colour change. These findings have increased the prospects for developing a simple molecular test for HAT that can be used with limited equipment at point of care in endemic rural areas.

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