Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection

Abstract

BackgroundLoop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions.MethodsA visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection.ResultsThe wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method.ConclusionsThis novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Malaria Infections in an Area of Endemicity in Thailand

“…The use of the simple boiling method for template preparation provides a good alternative to conventional (expensive and labor-intensive) DNA extraction methods, which might not always be possible in the field. In this study, we were able to successfully use heat-treated samples for LAMP amplification, similar to results reported by Sirichaisinthop et al, Lucchi et al, and Tao et al (28,33,34). As such, the rapid boiling method for extracting DNA from filter paper is favorable for LAMP.…”

Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Malaria Infections in an Area of Endemicity in Thailand

“…The visualization for the LAMP product was done by the naked eye using a microcrystalline wax dye capsule containing the DNA fluorescence dye SYBR green I (34). That study showed 98.3% sensitivity and 100% specificity of LAMP results compared to microscopy.…”

Novel Molecular Diagnostic Platform for Tropical Infectious Diseases

“…This technology enables the detection of LAMP reactions without the use of fluorescence detectors, which are usually expensive and difficult to manage in resource-limited settings. Other technologies for visual detection using LAMP have also been reported (Tao et al, 2011;Goto et al, 2009;Mori et al, 2006).…”