A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology

Michael Alifrangis; Sonia Enosse; Richard Pearce; Chris Drakeley; Cally Roper; Insaf F Khalil; Watoky Mmm Nkya; Anita M Rønn; Thor G Theander; Ib C Bygbjerg

A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology

Michael Alifrangis, Sonia Enosse, Richard Pearce, Chris Drakeley, Cally Roper, Insaf F Khalil, Watoky Mmm Nkya, Anita M Rønn, Thor G Theander, Ib C Bygbjerg

46 Citations (Scopus)

Abstract

Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA)-based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.

Original language	English
Journal	American Journal of Tropical Medicine and Hygiene
Volume	72
Issue number	2
Pages (from-to)	155-62
Number of pages	7
ISSN	0002-9637
Publication status	Published - 2005

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Alifrangis, M., Enosse, S., Pearce, R., Drakeley, C., Roper, C., Khalil, I. F., Nkya, W. M., Rønn, A. M., Theander, T. G., & Bygbjerg, I. C. (2005). A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology. American Journal of Tropical Medicine and Hygiene, 72(2), 155-62.

A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology. / Alifrangis, Michael; Enosse, Sonia; Pearce, Richard et al.
In: American Journal of Tropical Medicine and Hygiene, Vol. 72, No. 2, 2005, p. 155-62.

Research output: Contribution to journal › Journal article › Research › peer-review

Alifrangis, M, Enosse, S, Pearce, R, Drakeley, C, Roper, C, Khalil, IF, Nkya, WM, Rønn, AM, Theander, TG & Bygbjerg, IC 2005, 'A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology', American Journal of Tropical Medicine and Hygiene, vol. 72, no. 2, pp. 155-62.

Alifrangis M, Enosse S, Pearce R, Drakeley C, Roper C, Khalil IF et al. A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology. American Journal of Tropical Medicine and Hygiene. 2005;72(2):155-62.

Alifrangis, Michael ; Enosse, Sonia ; Pearce, Richard et al. / A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology. In: American Journal of Tropical Medicine and Hygiene. 2005 ; Vol. 72, No. 2. pp. 155-62.

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title = "A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology",

abstract = "Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA)-based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.",

author = "Michael Alifrangis and Sonia Enosse and Richard Pearce and Chris Drakeley and Cally Roper and Khalil, {Insaf F} and Nkya, {Watoky Mmm} and R{\o}nn, {Anita M} and Theander, {Thor G} and Bygbjerg, {Ib C}",

note = "Keywords: Animals; Antimalarials; Chloroquine; DNA Primers; DNA, Protozoan; Dihydropteroate Synthase; Drug Combinations; Drug Resistance; Enzyme-Linked Immunosorbent Assay; Humans; Malaria, Falciparum; Plasmodium falciparum; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Pyrimethamine; Sulfadoxine; Tanzania; Tetrahydrofolate Dehydrogenase",

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T1 - A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology

AU - Alifrangis, Michael

AU - Enosse, Sonia

AU - Pearce, Richard

AU - Drakeley, Chris

AU - Roper, Cally

AU - Khalil, Insaf F

AU - Nkya, Watoky Mmm

AU - Rønn, Anita M

AU - Theander, Thor G

AU - Bygbjerg, Ib C

N1 - Keywords: Animals; Antimalarials; Chloroquine; DNA Primers; DNA, Protozoan; Dihydropteroate Synthase; Drug Combinations; Drug Resistance; Enzyme-Linked Immunosorbent Assay; Humans; Malaria, Falciparum; Plasmodium falciparum; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Pyrimethamine; Sulfadoxine; Tanzania; Tetrahydrofolate Dehydrogenase

PY - 2005

Y1 - 2005

N2 - Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA)-based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.

AB - Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA)-based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.

M3 - Journal article

C2 - 15741552

SN - 0002-9637

VL - 72

SP - 155

EP - 162

JO - American Journal of Tropical Medicine and Hygiene

JF - American Journal of Tropical Medicine and Hygiene

IS - 2

ER -