Port et al.

Malaria Journal 2014, 13:454

http://www.malariajournal.com/content/13/1/454

METHODOLOGY Open Access

A reliable and rapid method for molecular

detection of malarial parasites using microwave
irradiation and loop mediated isothermal
amplification
Julia R Port1†, Christian Nguetse1†, Selorme Adukpo1 and Thirumalaisamy P Velavan1,2*

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.
Keywords: Malaria, Microwave irradiation, Plasmodium, Isothermal amplification, LAMP, Diagnostics, Point-of-care

Background diagnostic tests (RDT), using blood obtained from finger

Malaria remains a major public health problem in sub- pricks, are now widely used, especially where quick and
Saharan Africa. Approximately 3.4 billion people are at easy diagnosis of malaria is needed [2,3,5-9]. A major disad-
risk of malaria worldwide with an incidence of 207 million vantage of RDTs is that they are relatively expensive and
cases in 2012 and 627,000 reported deaths [1]. Microscopy unable to quantify the degree of parasitaemia. In some
of blood smears is still considered the gold standard cases, lack of sufficient sensitivity of RDTs causes, as does
for diagnosing malaria infections. Microscopy, however, microscopy, failure to detect low-density infections [10,11].
is frequently unable to detect low-density infections and Highest detection rates and specificity can currently be
it requires skilled expertise [2-4]. Consequently, rapid achieved only with polymerase chain reaction (PCR) or
real-time PCR (qPCR) assays. These techniques use expen-
* Correspondence: velavan@medizin.uni-tuebingen.de
†
sive equipment and reagents as well as functional molecular
Equal contributors
1
Institute of Tropical Medicine, University of Tübingen, Wilhelmstrasse 27,
biology laboratories and appropriate training of laboratory
72074 Tübingen, Germany staff. For field applications these conditions are often not
2
Fondation Congolaise pour la Recherche Medicale, Brazzaville, Republic of fulfilled. In addition, PCR reactions require a rather long
Congo

© 2014 Port et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Port et al. Malaria Journal 2014, 13:454 Page 2 of 8
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time from DNA extraction to visual read-out by electro- blood is difficult to achieve. Therefore, the DNA extrac-
phoresis or other confirmatory techniques (~5 hours, if per- tion and purification steps are crucial for subsequent
formed in a well equipped laboratory) and are as such not applications. As LAMP is more robust than are standard
appropriate when rapid diagnoses are needed. PCR assays, the extraction step is not that important.
In the last 10 years, isothermal amplifications, espe- Cheap and easy methods have been described using heat
cially loop-mediated isothermal amplification (LAMP) denaturation or chemical lysis. In combination with the
[12] has revolutionized the field of molecular diagnostics Eiken LAMP kit, a “boil and spin” method and the “PURE”
by offering rapid and reliable approaches at highest sen- method, i.e. a simple DNA isolation technique by water
sitivity levels. In 2006, a study described a first set of bath or thermoblock, are advised for DNA isolation under
LAMP primers for malaria [13] and LAMP specific field applications. An alternative is offered by employing
primers for four Plasmodium species infecting humans microwave irradiation, which was first described in 1991
were reported in 2007 [14], all targeting the 18 s rRNA [22] and 1995 for hepatitis B virus (HBV) DNA from
region of the Plasmodium species. Primers were also de- serum [23], and for extraction of Toxoplasma gondii de-
signed for the genus Plasmodium and for Plasmodium rived DNA in 2010 [24]. Downstream applications may
falciparum, targeting mitochondrial DNA [15]. A com- directly be performed without prior purification measures.
mercial kit for malaria LAMP is currently available from Here, a microwave extraction method was established,
Eiken Chemical Co., Ltd., also targeting this genetic re- along with a tailored LAMP approach for diagnosing
gion of plasmodia. This LAMP assay has successfully malaria. The microwave extraction method was tested
been tested under field conditions for both the18s rRNA for parasite and human genes derived from clinical sam-
[16] and the mtDNA primers [17,18] and been successful ples. Sensitivity was tested in repeated serial dilutions
in clinical evaluation of symptomatic and asymptomatic and the quality was compared to reference extraction
infections [10]. Under field conditions, the easy endpoint methods using a commercial kit.
visualization of results such as colourimetric change is
most advantageous and, thus, desirable. Due to the nature Methods
of LAMP reactions, the method offers unique opportun- Study samples
ities. LAMP amplification causes production of magne- Whole venous blood samples were collected either into
sium pyro-phosphate precipitates, which may be recorded heparinized tubes (5 mls) or by finger prick (5–10 μl) with
as a rise in turbidity. Such observations are difficult to blood stored on sterile Whatman filter paper at admission
make for untrained staff and an extra step of visualization to the Albert Schweitzer Hospital, Lambaréné, Gabon,
would be advantageous. Similar to gel electrophoresis, from patients suffering from severe P. falciparum infec-
where LAMP results are visualized by intercalating sub- tions [25]. Heparinized samples were kept at −80°C until
stances such as SYBR Green© or ethidium-bromide, shipment to Germany at −20°C, while blood spots were
hydroxynaphtholblue (HNB) can be applied as a potent air-dried and stored in clean, sealed plastic bags at room
detection system. As discussed previously [19], SYBR temperature. Equal volumes of blood were also collected
Green© and HNB, usually applied in metal ion titration from healthy adult volunteers in Lambaréné.
[20], show higher sensitivity than calcein, which may also
be used in LAMP reactions. HNB has been described to Parasite cultures and repeated dilution series
be superior to SYBR Green©, as no further equipment is Plasmodium falciparum parasite strain 3D7 were kept in
needed for the final read-out and the risk of contamin- culture, synchronized with D-sorbitol in their ring stages.
ation is minimized as the dye solution is added directly to Culture-adapted parasites were then used for extraction
the reaction mix before amplification [19]. Calcein, a procedures and LAMP assays, applying serial dilutions. In
chemical dye quenched with manganese ions may also brief, following the determination of the parasitaemia by
serve as a fluorescent dye in LAMP reactions. During microscopy, the culture was centrifuged to obtain packed
amplification, the manganese ions are released by pyro- cells which contained 107 cells/μl. Fresh whole blood was
phosphate ions generated, resulting in fluorescence. In subsequently used to spike the culture and dilution series
addition, the free calcein combines with magnesium ions were repeatedly made, ranging from calculated 500,000
in the LAMP reaction mixture to enhance fluorescent parasites/μl to 1 parasite/μl and eventual negativity. Equal
emissions. Compared to SYBR Green© and calcein, HNB volumes of these dilution series were further used for gen-
is the cheapest dye and successful use of HNB LAMP has omic DNA extraction by microwave irradiation and, sub-
recently been demonstrated to be effective in detecting sequently, for standard PCR and tailored LAMP assays.
amplified DNA from malaria parasites [21].
The first critical step in standard molecular diagnostics DNA extraction: microwave irradiation
of infectious agents is extraction of DNA. Due to interfer- First, DNA was extracted from whole blood samples as
ence with haem, direct DNA amplification from whole well as from the cultured parasites of the dilution series
Port et al. Malaria Journal 2014, 13:454 Page 3 of 8
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using the conventional QIAamp DNA mini blood kit containing 1× Coral load PCR buffer, 0.25 mM of each
based extraction procedure (Qiagen, Hilden, Germany) deoxynucleotide-triphosphate (dNTP), 1 U Taq Poly-
following the manufacturer’s instructions. Next, three merase (Qiagen) and 0.2 μM of each primer. The tem-
standard operation procedures (SOPs) were established plate was 1 μl of the condensed watery solution after
for microwave irradiation based DNA extraction (MDA DNA extraction by microwave irradiation. Amplification
oven, model number: MW17M70G-AU, 230 V, 50 HZ). was performed as follows: 35 cycles with an initial de-
The three SOPs were designed to apply in processing (i) naturation step at 94°C for 5 min, denaturation at 94°C
fresh whole blood, (ii) archived blood samples in hepa- for 30 sec, annealing at 55°C (outer primers) or 60°C
rinized tubes and for (iii) small amounts of blood stored (nested primers) for 30 sec, extension at 65°C for 1 min
at ambient temperature and eluted from filter paper in and final extension at 65°C for 5 min. Amplicons were
30 μl PBS. Volumes of 10 μl of blood were transferred subjected to electrophoresis on a 1.2% agarose gel
into 0.5 ml tubes and treated at 800 W for 2 minutes stained with SYBR green I in 1× Tris-buffered electro-
until precipitated and condensed droplets were visible phoresis buffer (90 mM Tris-acetate, pH 8.0, 90 mM
on and retrievable from the tube walls. One μl of the boric acid, 2.5 mM EDTA). In addition and for confirm-
clear precipitated watery solution containing DNA was ation of the specificity of the amplification process, 1 μl
taken from the walls or the lid of the tube for further of the purified product was directly used as template for
use (Figure 1). Alternatively, for enduring storage, 30 μl direct sequencing, using the BigDye terminator v.1.1 cycle
of sterile phosphate buffer saline (PBS) were added to sequencing kit (Applied Biosystems, Foster City, CA,
the irradiated sample. Notably, smaller tubes than those USA) on an ABI 3130XL DNA sequencer. Results were
of 0.5 ml volumes should not be used in the irradiation confirmed visually from the sequencing electrophero-
procedure as they might break and be destroyed due to grams. Notably, both the DNA extraction procedure has
air expansion and, thus, might bear the danger of con- also be performed to extract human DNA for standard
tamination hazards. PCR reactions.

Amplification using standard PCR Tailored LAMP assays

As an example, a nested PCR assay targeting the pfmdr1 Primer design and reagents
gene of P. falciparum was applied as described elsewhere LAMP primer pairs for P. falciparum, as published [14],
[4]. For each PCR reaction, positive and negative con- were provided by Eurofins MWG Synthesis GmbH,
trols were included. Reaction volumes consisted of 20 μl Ebersberg, Germany (Table 1). Bst 2.0 WarmStart™ DNA

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.
Port et al. Malaria Journal 2014, 13:454 Page 4 of 8
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Table 1 LAMP Primers targeting Plasmodium 18 s A change from violet to light sky blue was considered a
ribosomal RNA positive result of amplification. If the reaction remained
Plasmodium spp. Primers Sequence 5′ - 3′ violet, the sample was assessed as being negative. This as-
Core primers FIP TCGAACTCTAATTCCCCGTTACCTAT sessment was performed at daylight. A positive amplifica-
CAGCTTTTGATGTTAGGGT tion was determined by a colour change occurring after a
BIP CGGAGAGGGAGCCTGAGAAATAGA minimum of 35 minutes. Absence of colour changes was
ATTGGGTAATTTACGCG assigned after 45 minutes. Accordingly, 45 minutes were
F3 GTATCAATCGAGTTTCTGACC set as the time needed to achieve a firm result.
B3c CTTGTCACTACCTCTCTTCT
Loop primers LPF CGTCATAGCCATGTTAGGCC Results
DNA extraction by microwave irradiation, standard PCR
LPB AGCTACCACATCTAAGGAAGGCAG
and LAMP assays
Either condensed vapour droplets or blood remains after
P. falciparum Primers Sequence 5′ - 3′ microwave irradiation diluted in a 30 μl volume of PBS
Core primers FIP AGCTGGAATTACCGCGGCTG GGTT proved to be appropriate templates for further processing
CCTAGAGAAACAATTGG
in both standard PCR and LAMP assays. DNA extraction
BIP TGTTGCAGTTAAAACGTTCGTAGCC was achieved from various sources, including fresh venous
CAAACCAGTTTAAATGAAAC
blood, heparinized and archived samples and blood eluted
F3 TGTAATTGGAATGATAGGAATTTA
from Whatman filter paper. A 0.5 ml tube containing
B3c GAAAACCTTATTTTGAACAAAGC 10 μl of blood inside a microwave oven led to boiling and
Loop primers LPF GCACCAGACTTGCCCT partial desiccation of the sample and to the formation of
LPB TTGAATATTAAAGAA vapour, which condenses and may be retrieved from the
walls and lids of the tubes (Figure 1). This haem-free con-
densed vapour contains the nucleic acid.
A nested PCR targeting the pfmdr1 gene of P. falciparum
Polymerase (New England BioLabs GmbH, Frankfurt am was applied and successful amplification of DNA extracted
Main, Germany) was used. This cheap polymerase was from fresh and archived samples as well as from filter pa-
specifically designed for high throughput assays and field pers by microwave irradiation was achieved (Figure 2). This
applications. The polymerase may be stored under up applied to both, DNA containing vapour droplets and
to 45°C without requiring refrigeration. HNB (Fluka, blood remains diluted in PBS. For confirmation of specifi-
Sigma-Aldrich, St. Luis, USA) was used as the visualization city of the amplicon, all PCR products were sequenced
dye. In addition, a 3 mM stock solution of HNB was pre- and aligned with reference sequences. Correct sequences
pared and stored at room temperature [21,26]. Isothermal of amplification targets were confirmed in all cases. The
reaction buffer was prepared as a 2× working solution. amplicons could successfully be applied to SNP analyses of
After thawing, 2.8 mM of dNTPs were added. Betaine solu- drug resistance markers. The total time of the entire diag-
tion for enhancing the polymerase activity and reducing the nostic procedure was reduced to less than one hour after
formation of secondary structures in GC-rich regions was sample collection. While maintaining high sensitivity
purchased from Sigma Aldrich, Munich, Germany. levels, expensive equipment or reagents are not required.

LAMP Assays Analytical sensitivity

Reactions were performed at 60°C in volumes of 25 μl The sensitivity of the PCR assays after microwave-based
containing 1.6 μM FIP, 1 μM BIP, 0.2 μM F3, 0.2 μM B3c, extraction was established based on repeated dilution
0,2 μM LPB, 0.4 μM LPF, 120 μM HNB, 2× isothermal re- series. Heparin blood was spiked with ring stage parasites
action buffer, 1.4 mM dNTPs, 0.4 mM Betaine solution, from 3D7 cultures and serially diluted from 500,000 to
8U Bst polymerase and 1 μL of watery DNA solution as one parasite/μl and negativity. DNA was extracted from
template, following the STARD guidelines for diagnostics. all samples applying both standard reference methods and
Two heat blocks (Block Thermostat BT200, Kleinfeld microwave irradiation. When using standard DNA isola-
Labortechnik, Gehrden, Germany) were used and pre- tion methods, one parasite/μl could be reliably detected
heated to 60°C for DNA amplification for to 45 minutes, (Figure 3A). After microwave extraction, five parasites/μl
and enzyme inactivation for 2 minutes at 80°C. were detected (Figure 3B).

Visual detection of amplification/endpoint analysis Loop mediated isothermal amplification

The amplicon was visualized through a distinct colour No differences in the amplification sensitivity was ob-
change and subsequently confirmed by gel electrophoresis. served when comparing results obtained by a standard
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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.

thermocycler or a heating block as proposed here. 3D7 DNA extraction kits are not always available. Alterna-
parasites, starting at a dilution of 100,000 parasites/μl to tively, intensive heat treatment or shock freezing can
one parasite/μl were subjected to tailored LAMP assays also lyse cells for DNA release. Microwave irradiation
using the DNA extracted by microwave irradiation. The has been shown in this study to successfully extract
parasite content of the diluted culture was assessed by DNA from whole blood samples in less than 3 minutes.
microscopy, counting 100 oil immersion fields. When No further chemicals are required for isolation or purifi-
direct condensed vapour droplets were used in LAMP, cation. DNA isolated by irradiation could be subjected
positive amplification was observed with a limit of detec- to both standard PCR amplification and to the LAMP
tion of one parasite/μl (Figure 4A). When blood remains procedure. The use of PBS, although causing a certain
were diluted in PBS, positive amplification was observed decrease of sensitivity due to the dilution, was beneficial,
at a limit of detection of five parasites/μl (Figure 4B). as larger volumes of DNA could be stored. Sensitivity
With HNB used as appropriate dye, negative controls levels observed after microwave irradiation were close to
remained violet, while successful LAMP was character- those obtained after extraction with commercial kits.
ized by a colour change to sky blue. Gel electrophoresis Thus, microwave irradiation offers a cheap, fast and
was applied to verify HNB positive results (Figure 5). Re- easy technique that can be applied readily for isothermal
peated tests allowed to set the endpoint of a final deci- amplification and that may be applied in field settings.
sion on test positivity or negativity at a maximum of The nucleic acid extraction by microwave irradiation al-
45 minutes. lows to isolate human DNA and nucleic acid materials
from pathogens in blood. Our study demonstrates that
Discussion DNA may be extracted from fresh blood samples and
Multiple techniques to extract and purify DNA have from archived blood samples, either heparinized or
been described and applied to date. Problems arising stored on filter papers even after more than a decade.
from interference with haem, as occurring in extraction LAMP for malaria has been shown to detect low parasite
of DNA from venous blood, can easily be circumvented concentrations [17] and to be successfully applicable in
in laboratories when using commercially available ex- asymptomatic Plasmodium infections. When aiming at
traction kits, while, in low resource settings, commercial improving malaria control, simple and easy to perform

A 1000bp
500bp
5x10 4

5x10 3

1000

500
10 5

10 4

100

NC
50

10

1
5

B 1000bp
500bp
5x10 4

5x10 3

1000

500
10 5

10 4

100

NC
50

10

1
5

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.
Port et al. Malaria Journal 2014, 13:454 Page 6 of 8
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1000bp
A 500bp

100 bp

5x10 4

5x10 3

1000

500

100
10 5

10 4

NC
50

10

1
B 1000bp

500bp

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.
Ladder

PC ME HDC NC

PC ME HDC NC
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.
Port et al. Malaria Journal 2014, 13:454 Page 7 of 8
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doi:10.1186/1475-2875-13-454
Cite this article as: Port et al.: A reliable and rapid method for molecular
detection of malarial parasites using microwave irradiation and loop
mediated isothermal amplification. Malaria Journal 2014 13:454.

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