Date Published: February 6, 2008
Publisher: Public Library of Science
Author(s): Zablon Kithinji Njiru, Andrew Stanislaw John Mikosza, Tanya Armstrong, John Charles Enyaru, Joseph Mathu Ndung’u, Andrew Richard Christopher Thompson, Serap Aksoy
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.
Partial Text: Human African trypanosomiasis is endemic in tropical Africa. In eastern and southern Africa the disease is caused by Trypanosoma brucei rhodesiense, while T. b. gambiense infections are common in central and West Africa. T. b. rhodesiense causes an acute form of disease, whereas T. b. gambiense causes a more chronic form. Moreover, the treatment regimen for the two infections is different, expressing the need for a specific diagnostic test for each trypanosome. The geographical demarcation of T. b. rhodesiense and T. b. gambiense to a large extent forms the basis of trypanosome identification and treatment. In East Africa the introduction of T. b. rhodesiense into the T. b. gambiense region is certain to occur due to the closeness of the two disease foci and continuous movement of the livestock-reservoir host for T. b. rhodesiense. This prospect further obligates the development of test kits that can differentiate the two parasites. The serum resistance-associated (SRA) gene , is conserved and specific to T. b. rhodesiense– and therefore provides unequivocal identification of this parasite. It is a low-copy gene, therefore the polymerase chain reaction (PCR) test is inadequate to amplify this target reliably in clinical samples without recourse to parasite multiplication in mice. Besides, available molecular methods of parasite detection require elaborate precision instruments –, which make their use under field conditions unfeasible. There is therefore a need for a simplified method of amplification and product detection that would compliment the available tests and make feasible molecular diagnosis for case detection and confirmation of cure in the regions that are endemic for sleeping sickness.
In the present study we were able to demonstrate the successful amplification of T. b. rhodesiense DNA within 20–25 min at 62°C using the SRA LAMP assay. However, we set the optimal time at 35 min to amplify DNA at low concentrations. The results of the SRA LAMP assay were identical when either a water bath or a thermocycler was used to maintain the temperature at 62°C, demonstrating its robustness. Preheating of the template increased the efficiency of the assay by shortening the duration (Figure 2) and increasing sensitivity of the test. DNA amplification is preceded by strand separation under isothermal conditions using betaine, which destabilises the DNA helix . It would appear that preheating of the sample produced a faster and/or a greater amount of strand separation, which translated into a far more rapid assay. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. This ability highlights another advantage of LAMP technique: the results of amplification can visually be observed through addition of a DNA intercalating dye (Figure 3), eliminating the need for gel electrophoresis and greatly reducing the time taken for result analysis.