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

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.

Comparative Detection of Trypanosomal DNA by Loop-Mediated Isothermal Amplification and PCR from Flinders Technology Associates Cards Spotted with Patient Blood

“…Furthermore, the high level of concordance (98.4%) and agreement (kappa value, 0.85; 95% CI, 0.64 to 1) between SRA-LAMP and RIME-LAMP provide the possibility that either test could be used to reliably diagnose HAT due to T. b. rhodesiense. The major advantages of LAMP were the need for just a single reaction to obtain results within 1 h of initiation by adding SYBR green (11,12). LAMP therefore has great potential application in resource-poor settings with basic facilities, since it can be done in a water bath, eliminating the need for a thermocycler.…”

“…For SRA-LAMP, we made use of primers recently described by Njiru et al (11), namely, SRA-F3, SRA-B3, SRA-FIP, SRA-BIP, SRA-LF, and SRA-LB. The total reaction volume was 25 l, into which the above-mentioned primers were added to final concentrations of 0.2 M for F3 and B3, 2 M for the forward inner primer (FIP) and the backward inner primer (BIP), and 0.8 M for the forward loop (LF) and the backward loop (LB).…”

“…These tests amplify 18S ribosomal DNA or RNA, respectively, and as such cannot be used to distinguish between the two HAT types. Recently, loop-mediated isothermal amplification of DNA (LAMP) has been used for detection of members of the Trypanozoon subgenus as well as T. b. rhodesiense (11,12) subsequent to earlier studies targeting various trypanosomal genes (8,16). The reportedly high sen-sitivity, coupled with no need for specialized equipment, increases the likelihood of its adoption in resource-poor health centers where HAT is endemic.…”

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

“…In DNA polymerization by DNA polymerase, pyrophosphate ions are released from dNTP as a by-product, which react with magnesium ions in the LAMP reaction buffer, yielding an insoluble white precipitate. 13,14 This method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, and the advantages are i) easy identification of positive reaction by visual inspection of turbidity, and ii) requirement of only a heating block or water bath. 13 In this study, we developed a LAMP reaction for detection of HP del with the aim of establishing a feasible detection method for HP del in clinical diagnostic laboratories.…”

The molecular epidemiology of parasite infections: Tools and applications

“…Loop-mediated isothermal DNA amplification (LAMP) is a newly developed, rapid, quantitative, highly sensitive and specific nucleic acid-based, non-PCR diagnostic tool [14,15,16], applicable to 'low-cost' laboratory settings. This simple molecular test can be carried out on a bench with a heating block instead of a thermal cycler and may prove to be an invaluable 'field friendly' tool for screening and quantifying infections in host populations while providing important genotypic information [17].…”