A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification

Abstract

Background: Improved living conditions together with appropriate diagnosis can reduce avoidable malarial deaths substantially. Microscopy remains the gold standard in the diagnosis of malaria. However, rapid molecular diagnostic tests (RmDT) are becoming increasingly important and will, most likely, be the diagnostic techniques of choice in the next years.

Methods: In this study, a rapid and reliable nucleic acid extraction procedure from human blood and malarial parasites using microwave irradiation as a promising platform is described. In addition, a tailored loop mediated isothermal amplification (LAMP) methodology that utilizes hydroxynaphthol blue (HNB) and Bst 2.0 DNA polymerases in molecular detection of malarial parasites is described.

Results: Following microwave irradiation for DNA isolation, conventional PCR assays were able to detect up to five malaria parasites/μl. The LAMP methodology described here was capable to detect as low as one Plasmodium falciparum parasite/μl after DNA extraction by microwave irradiation. A turnover time of 45 minutes from nucleic acid extraction to final visual read-out was achieved.

Conclusions: The described procedure offers a cheap, simple and fast method of molecular detection of malaria parasites. This test can easily be performed in basic laboratories. The methodology has been validated as a proof of concept and has specifically be developed for use at low-resource settings. Such RmDTs may aid health providers to make timely therapeutic interventions in malaria endemic regions.

Figure 1

Microwave irradiated finger prick blood sample 10 μl fresh finger prick blood collected by capillary (A) before and (B) after microwave irradiation. Formation of vapour and condensed droplets can be observed on the walls and in the lid.

Figure 2

Amplified pfmdr1 gene products after nested PCR. Standard DNA extraction was carried out using the QIAamp DNA mini blood kit (Qiagen, Hilden, Germany). DNA extraction by microwave irradiation was performed using a microwave oven (MDA, model number: MW17M70G-AU, 230 V, 50 HZ, operated at 800 W). 1 μl of condensed droplets after microwave treatment were utilized for the PCR procedures. First lane: DNA ladder; NC: Negative Control; PC1 and PC2: Standard extraction from archived blood sample and pfmdr1 amplicons at expected sizes; PC3 and PC4: Standard extraction from 3D7 P. falciparum parasites in culture and pfmdr1 amplicons at expected sizes; ME1 and ME2: Microwave based extraction from archived blood sample and pfmdr1 amplicons at expected sizes; ME3 and ME4: Microwave based extraction in 3D7 culture parasites and pfmdr1 amplicons at expected sizes; ME5: Microwave based DNA extraction from fresh blood sample and pfmdr1 amplicons at expected sizes.

Figure 3

Limit of detection from standard extraction and microwave irradiation procedure. Repeated dilution series were made from 100,000 parasites/μl to one parasite/μl until negativity. Equal volumes of diluted culture were further used for DNA extraction and for standard PCR assays. A, Serial dilutions including dilutions from 100,000 parasites to negativitiy. The limit of detection was determined to be 1 parasite/μl culture applying the standard extraction procedure (QIAamp DNA mini blood kit); B, After microwave based DNA extraction the limit of detection was 5 parasites/μl.

Figure 4

Limit of detection of parasites extracted by microwave irradiation in tailored LAMP assays. Successful amplification was characterized by clear colour changes from violet to sky blue and the negative controls remained violet. NC: Negative control; A, Pure condensed vapour droplets were used as DNA templates. LAMP can detect 1 parasite/μl culture fluid. B, Vapour droplets diluted in PBS. LAMP detects 5 parasites/μl.

Figure 5

Visual assessment after 35 minutes following the LAMP assays. All amplicons were confirmed by gel electrophoresis. PC, positive control after standard DNA extraction; ME, clinical sample, LAMP assay after microwave DNA extraction; HDC, human DNA control; NC, negative control.