Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria

Abstract

Background: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method.

Methodology and significant findings: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples.

Conclusion: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

Figure 1. Description of the RealAmp method.

The ESE-Quant Tube scanner equipped with temperature settings to amplify DNA isothermally and spectral devices to detect amplified product using fluorescence is shown (A). The tube scanner can hold 8,200 µL PCR tubes and is equipped with an LCD panel through which positive or negative results can be detected. If the tube scanner is connected to a computer with the appropriate software, the results are obtained in real-time as shown in B. The fluorescence units are shown on the Y-axis and the time to amplification on the x-axis. Amplification curves are observed (solid line) in case of a positive sample. No amplification curves (dotted line) indicate a negative sample.

Figure 2. Amplification of the four human-infecting Plasmodium species using the RealAmp method.

Plasmodium genus-specific primers were used to amplify the 18s ribosomal RNA gene in P. falciparum, P. vivax, P. malariae and P. ovale parasites. Amplification curves (positive) were observed for all the four species within 20 minutes (vertical dotted line). No amplification was seen with malaria-free human DNA (Human) or in the no template control (NTC).