Sample collection
The P. falciparum resistance genotype of those patients who presented a single malaria infection was analyzed as part of a prospective, observational, multicenter study, approved by the Medical and Health Research Ethics Committee (CEIC) of the Hospital Universitario 12 de Octubre (CEIm: 18/021).
The final selection was based on species-specific identification by nested-PCR [18, 19] and only samples with unique infection with P. falciparum were chosen for the study.
A total of 112 P. falciparum positive samples and 24 negative samples were selected for this assay. In addition, a strain, donated by the Parasitology Department of the Institute for Medical Research in Malaysia with the mutation C580Y of the gene that codes for the Kelch13 protein, was included.
Sample processing
DNA was extracted from 200 µl of whole blood, collected in ethylenediamine tetraacetic acid (EDTA) tubes, using the QIAamp DNA mini blood kit (QIAGEN®, Hilden, Germany) according to the manufacturer's instructions, resuspended in a final volume of 100 µl of distilled water. The DNA is stored at 4ºC until use, using 5 µl per reaction.
Plasmodium falciparum resistance genotyping
Genotyping of Pfmdr1, Pfcytb and PfK13 genes was carried by real time PCR modified from the original methods [20, 21, 22]. Reaction mixture consists of 1x QUANTIMIX HotSplit Probes kit (Biotools, Madrid, Spain) 0.25 μM of each primer (Table 1) and 1µM EvaGreen® dye (Biotium, Hayward, CA, USA) in a final volume of 20 µl. The thermal cycle was performed in a Qiagen Rotor Gene Q 5 Plex HRM (QIAGEN®, Hilden, Germany). The amplification conditions for the Pfmdr1 and Pfcytb genes were an initial cycle of denaturation of 10 minutes at 95°C, followed by 45 cycles of 15 seconds at 95°C, 15 seconds at 54°C and 30 seconds at 68°C, ending with a melting step (62°C to 92°C). In the case of the PfK13 gene, the conditions were an initial denaturation cycle of 7 minutes at 94°C, followed by 40 cycles of 30 seconds at 94°C, 1 minute at 60°C and 1 minute at 72°C, ending with a melting step (62°C to 92°C).
Table 1. Primers name, gene target, sequence and method for the P. falciparum resistance genotyping.
Target
|
Primer name/Reference
|
Method
|
Seq. 5'®3'
|
MDR
|
MDR1-pfmdr1-1FK76T21
|
RT-PCR MDR A & Sequencing
|
GTTGAACAAAAAGAGTACCGCTG
|
MDR2-pfmdr1-1RK76T21
|
TCGTACCAATTCCTGAACTCAC
|
MDR3-pfmdr1-2FK76T21
|
Sequencing
|
TTTCCGTTTAAATGTTTACCTGC
|
MDR4-pfmdr1-2RK76T21
|
CCATCTTGATAAAAAACACTTCTT
|
MDR1-1246-pfmdr1-1FD1246Y21
|
RT-PCR MDR B & Sequencing
|
ATGACAAATTTTCAAGATTA
|
MDR2-1246-pfmdr1-1FD1246Y21
|
ACTAACACGTTTAACATCTT
|
MDR3-1246-pfmdr1-1FD1246Y21
|
Sequencing
|
AATGTAAATGAATTTTCAAACC
|
MDR4-1246-pfmdr1-1FD1246Y21
|
CATCTTCTCTTCCAAATTTGATA
|
Cyt B
|
CytB1-1F22
|
RT-PCR / Sequencing
|
CTCTATTAATTTAGTTAAAGCACAC
|
CytB2-1R22
|
ACAGAATAATCTCTAGCACC
|
CytB3- 2F22
|
Sequencing
|
AGCAGTAATTTGGATATGTGGAGG
|
CytB4-2R22
|
ATTTTTAATGCTGTATCATACCCT
|
K13
|
Pfal-k13-2-2PCR20
|
RT-PCR / Sequencing
|
GCCAAGCTGCCATTCATTTG
|
Pfal-k13-3-2PCR20
|
GCCTTGTTGAAAGAAGCAGA
|
Specificity of amplification was determined by post reaction analysis using the melting temperature (Tm) curve of the amplified fragments (76.8°C and 76.5°C for Pfmdr1 fragment A and B respectively, 79.5°C for Pfcytb, and 82.0°C for PfK13).
Amplicons were purified using Illustra DNA and Gel Band Purification Kit (General Electric Healthcare, England), then sequenced, with its specific primers (Table 1), using Big Dye Terminator v3.1 Kit in an ABI PRISM® DNA Analyzer 3700. All amplified products were sequenced in both directions, twice. Blast tool from NCBI was used to confirm correct target amplification. Multiple nucleotide sequence alignments and analysis were performed using BioEdit version 7.0.5.3 [23] using sequences from 3D7 strain as wild-type for comparison.
Plasmodium falciparum genotyping
Plasmodium falciparum genotyping was performed by characterizing two merozoite surface membrane genes (MSP-1 and MSP-2) by PCR described elsewhere [24, 25]. Multiplicity of infection (MOI) defined as the number of genetically distinct parasite strains co-infecting a single host, was determined as the largest number of different alleles present at one of the two loci studied.
Kompetitive allele-specific PCR (KASP)
Sequences flanking SNPs were submitted for KASP ™ assay design to Biosearch Technologies (California, USA). KASP assay was carried out following the instructions of the manufacturer [6]. The reaction mix per reaction consists of 5.1 µl of the KASP Master Mix, which include the two allele specific primers and one reverse primer (Table 2), and 0.138 µl of the Assay Mix, containing universal fluorescent probes, Taq polymerase and dNTPs in an optimized buffer solution.
Table 2. Primers and probes used for the SNPs characterization by the KASP method.
ID
|
Primer_AlleleX & Allele Y
|
Primer_Common
|
Cyt b M133I
|
AATTACAGTTGCACCCCAATAACTC
GTAATTACAGTTGCACCCCAATAACTT
|
AACTGCTTTCGTTGGTTATGTCTTACCAT
|
K13 C580Y
|
ATACCCCTAGATCATCAGCTATGTG
AATACCCCTAGATCATCAGCTATGTA
|
CTCACCATTAGTTCCACCAATGACATAAA
|
MDR N86Y
|
GTGTTTGGTGTAATATTAAAGAACATGA
CTGTGTTTGGTGTAATATTAAAGAACATGT
|
GTACTAAACCTATAGATACTAATGATAATA
|
To five µl of this reaction mix, five µl of DNA from the sample to be analyzed, are added.
PCRs and fluorescent readings were performed in a Qiagen Rotor Gene Q 5 Plex HRM (QIAGEN®, Hilden, Germany) following the recommended thermal cycling conditions (Table 3).
Table 3. Thermal cycle conditions for KASP genotyping reactions
Step
|
Description
|
Temperature
|
Time
|
cycles
|
1
|
Activation
|
94°C
|
15 minutes
|
1 cycle
|
2
|
Denaturation
|
94°C
|
20 seconds
|
10 cycles
|
Annealing/ Elongation
|
61-55 °C
(drop 0.6°C per cycle)
|
60 seconds
|
3
|
Denaturation
|
94°C
|
20 seconds
|
35 cycles
|
Annealing / Elongation
|
55°C
|
60 seconds
|
4
|
Read stage
(FAM™, HEX™, ROX™)
|
30°C
|
60 seconds
|
|
Analysis of KASP genotyping data using cluster plots
To analyze and interpret genotypic data, an Excel sheet is used, although it can also be done using the Thermo Fisher Cloud Genotyping application [26]. In a KASP general assay, a sample that is homozygous for the allele reported by the X-signal oligonucleotide will only generate X-signal fluorescence during the end-point genotyping reaction. This data point will be plotted close to the X-axis, representing a high X-signal and no Y-signal. A sample that is homozygous for the allele reported by the Y-signal oligonucleotide will only generate Y-signal fluorescence during the end-point genotyping reaction. This data point will be plotted close to the Y-axis, representing a high Y-signal and no X-signal. A heterozygous sample will contain both the allele reported by the X-signal oligonucleotide and the allele reported by the Y-signal oligonucleotide. This sample will generate half as much X-signal fluorescence and half as much Y-signal fluorescence as the samples that are homozygous for these alleles. This data point will be plotted in the center of the plot, representing half X-signal and half Y-signal. In falciparum-malaria, the situation is more complicated, since, in the blood phase infection, Plasmodium is haploid, but P. falciparum infections are usually multi-infection so different levels of "heterozygosis" can occur depending on the number of populations present in each infection (MOI). Thus, the points are scattered on the plot but always close to the corresponding axis if only one allele is present, while infections with both alleles will be located in the center of the plot. Furthermore, to ensure the proper performance of the assay always is useful to include an end-point genotyping reaction without any template DNA as a negative control (referred to as no template control or NTC) that should appear near zero in the cluster plot.