Bst polymerase

The cfDNA was subjected to library preparation as previously described [7 (link)] with modifications. In brief, initially blunt ending and 5′ phosphorylation was performed using T4 polymerase and T4 kinase respectively. Sequencing adaptors were then ligated at both ends using T4 Ligase (New England Biolabs, Ipswich, UK). Nicks were removed in a fill-in reaction using Bst polymerase (New England Biolabs). Library amplification was performed using Herculase II Fusion Polymerase (Agilent Technologies, Santa Clara, CA), and unique barcodes were assigned to all samples. At each step, products were purified using Ampure XP magnetic beads according to manufacturer’s instructions.
Prior to library construction of buccal swab, amniotic fluid and CVS samples, extracted genomic DNA was sheared to an average size of 250 bp using the Bioruptor Pico sonication system (Diagenode, Liege, Belgium). Blunt ending, adaptor ligation and adaptor fill-in reactions were performed without intermediate purification steps. A single purification step was performed following library amplification using Ampure XP magnetic beads according to manufacturer’s instructions.
All library preparation steps were run on Hamilton STAR (Hamilton, Bonaduz, Switzerland) or epMotion (Eppendorf) systems using in-house developed automated methods.

The MG-LAMP was performed at the health facilities by boil-and-spin method [19 ] using 50 µL of the collected blood. DNA was released from whole blood by boiling in a heat-block at 95 °C for 10 min. The samples were centrifuged for 3 min at 15,000 × g and the supernatant (containing the DNA) was collected and used for the MG-LAMP assay. Five µL of the supernatant was used in the MG-LAMP assay and the rest stored in the -80ºC freezer. The MG-LAMP assay was performed in a 20 μL total reaction volume containing 2X in-house buffer (40 mM Tris–HCL pH 8.8, 20 mM KCl, 16 mM MgSO₄, 20 mM (NH₄)SO₄, 0.2% Tween-20, 0.8 M Betaine, 2,8 mM of dNTPs each), 0.004% MG, 8 units of Bst polymerase (New England Biolabs, Ipswich, MA) and 5 μL of template DNA [20 (link)]. Mitochondria Plasmodium genus-specific primers were used to amplify the DNA at 63 °C for 60 min using a simple heat block. The samples were allowed to cool for 15 min before being scored by three independent readers by visual inspection of color change. Positive (known P. falciparum) and negative (no template/DNA) control samples were included in each run. Positive samples retained a light green/blue malachite green colour while negative samples were colourless. For the purpose of quality control, 20% of the samples were randomly selected and retested at KEMRI/CGHR Malaria Laboratories.

The RealAmp assay was performed at the NAMRU-6 laboratory in Lima following a previously reported method with some modifications[12 (link)]. Briefly, all reactions were carried out in a volume of 12.5 μl that contained 2X in-house reaction buffer (40mM Tris-HCl pH8.8, 20mM KCl, 16mM MgSO₄, 20mM (NH₄)₂SO₄, 0.2% Tween -20, 1.6 M Betaine, 2mM of dNTPs each), 0.25μL of 1:400 SYTO 9 dye, 8 units of Bst Polymerase (New England Biolabs, Ipswich, MA) and 2μL of template DNA. Genus and species-specific primers for P. vivax and P. falciparum were used for the amplification of the 18S ribosomal RNA. The RealAmp assay was performed on an Mx3005P qPCR system (Agilent technologies, Santa Clara, CA) with fluorescence measured at 1 minute intervals under the FAM fluorescence channel (λ 494nm absorption/ 518nm emission). The genus and P. falciparum reactions were performed at 63°C whereas the P. vivax reaction was run at 64°C. All samples were run in triplicates and amplification was performed for 1 hour.

The c-MYC fragment previously PCR-amplified was used as a template for the LAMP reaction, performed as described by Veigas et al. [41 (link)]. This LAMP reaction requires four specific primers: a forward outer primer (FP), a backward outer primer (BP), a forward inner primer (FIP), and a backward inner primer (BIP) (see Table B1 for primers). LAMP primers for c-MYC were designed using Primer Explorer V4 (http://primerexplorer.jp/elamp4.0.0/). The reaction was carried out in a 10 µL reaction mixture containing 1.6 µM of FIP and BIP primers, 0.2 µM of B3 and F3 primers, 0.45 mM of dNTPs, 3 mM of MgCl₂, 0.8 M of Betaine, 700 µM of dNTPs, 1 X Bst Buffer, 1 X Evagreen dye, 1.8 U of Bst polymerase (New England Biolabs, Beverly, MA), and 1 µL (5–500,000 copies/µL) of template DNA; sterile water was used in place of DNA for the non-template control (NTC). The reaction was incubated at 65 °C for 60 min in a Bio-Rad MyCycler Thermocycler.

The Malachite Green (MG) malaria LAMP (MG-LAMP) assay was performed in a 20 μL total reaction volume containing 2X in-house buffer (40 mM Tris-HCL pH 8.8, 20 mM KCl, 16 mM MgSO₄, 20 mM (NH₄)SO₄, 0.2% Tween-20, 0.8 M Betaine, 2,8 mM of dNTPs each), 0.004% MG, 8 units of Bst polymerase (New England Biolabs, Ipswich, MA) and 5 μL of template DNA. Mitochondria Plasmodium genus-specific primers [6 (link)] were used to amplify the DNA. The amplification was carried out at 63°C for 60 minutes using a commercially available mini heat block (GeneMate, BioExpress, Utah, US). Samples were allowed to cool for 15 minutes before being scored by three independent human readers. Positive samples retained a light green/blue malachite green color while negative samples turned colorless, Fig 1.

Two thin and thick smears were prepared for each participant and stained with Giemsa. Slides were read by two microscopists whereas a third microscopist reviewed slides with discordant results.
DNA from whole blood was extracted using the Qiagen DNA extraction kit according to the manufacturer’s protocol. Malaria-positive cases were detected by Malachite green LAMP or real-time PCR as previously described^{20 (link)–22 (link)}. For MG-LAMP, the reaction was performed in a 20 µL reaction volume that contained 5 µL of template DNA in 2X in-house reaction buffer (40 mM Tris–HCl pH 8.8, 20 mM KCl, 16 mM MgSO₄, 20 mM (NH₄)₂SO₄, 0.2% Tween -20, 1.6 M Betaine, 2 mM of dNTP's each), 0.25µL of 1:400 SYTO 9 dye, 8 units of Bst Polymerase (New England Biolabs, Ipswich, MA) and 0.004% Malachite Green dye. DNA amplification was carried out at 63 °C for 60 min and two independent readers visually inspected the results after 15 min post amplification. For the real-time PCR, the reaction was performed in a 25 µL reaction volume that contained 5 µL of template DNA in a 1X master mix of PerfeCTa SYBR® Green Fastmix, 0.3 μM of each primer PL1473F18 5′-TAACGAACGAGATCTTAA-3′ and PL1679R18 5′-GTTCCTCTAAGAAGCTTT-3′. The real-time PCR conditions consisted of an initial denaturation step at 95 °C for 2 min, followed by 45 cycles of 20 s at 95 °C, 20 s at 50 °C, and 20 s at 68 °C.

LAMP reactions were carried out under different conditions including variable incubation temperature, time, and concentrations of dNTPs, MgSO₄, primer ratio, betaine, and Bst polymerase. The effects of incubation temperatures were evaluated at 59, 61, 63, 65, and 67 °C. Incubation times were 45, 60, and 75 min. The dNTP concentrations were 1.2, 1.4, 1.6, and 2 mM. The MgSO₄ concentrations were 4, 6, 8, and 10 mM. The outer-to-inner primer ratios were 1:3, 1:4, and 1:5. The betaine concentrations (Sigma-Aldrich, Germany) were 0.4, 0.6, and 0.8 M. Bst polymerase (NEB, USA) was adjusted to 4 and 8 U. All reactions were reproduced at least 3 times.