Abstract
BackgroundNucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing.MethodsA specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay.ResultsThe lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined.ConclusionsCombining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.
Highlights
Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum
Establishing the lateral flow recombinase polymerase amplification (RPA) In order to establish the RPA, initial experiments were undertaken in single tube reactions without the addition of the probe, to screen primers and test different reaction conditions
The use of a special probe structure and the nuclease enzyme results in a dual-tagged DNA amplicon in a single tube reaction, which can be detected on the lateral flow strip
Summary
Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. Like microscopy, often lack in sensitivity or clarity of results Molecular techniques such as polymerase chain reaction (PCR), which amplify a specific nucleic acid sequence, have been developed over the past years. These methods have the advantage that they can detect low level of parasitaemia [2] or give additional information on the infection in a single assay [3,4]. The continuous electricity dependency for the thermocycling process and the rather expensive equipment necessary for the PCR machinery makes it difficult to conduct this method in resource-limited settings, which might occur in some areas especially in developing countries and is restricted to be a confirmatory technique in laboratory diagnostics [5]
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