Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum

Zhiyong Zhou, Amanda C. Poe, Josef Limor, Katharine K. Grady, Ira Goldman, Andrea M. McCollum, Ananias A. Escalante, John W. Barnwell, Venkatachalam Udhayakumar

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

A pyrosequencing protocol was developed as a rapid and reliable method to identify the mutations of the dhfr and dhps genes of Plasmodium falciparum that are associated with antifolate resistance. The accuracy and specificity of this method were tested using six laboratory-cultured P. falciparum isolates harboring known single nucleotide polymorphisms (SNPs) in the genes dhfr (codons 50, 51, 59, 108, and 164) and dhps (codons 436, 437, 540, 581, and 613). The lowest threshold for detection of all the SNPs tested by pyrosequencing was the equivalent of two to four parasite genomes. Also, this method was highly specific for P. falciparum, as it did not amplify any DNA products from the other species of human malaria parasites. We also mixed wild-type and mutant-type parasite DNAs in various proportions to determine how pyrosequencing, restriction fragment length polymorphism (RFLP), and direct conventional sequencing (for dhfr) compared with each other in detecting different SNPs in the mixture. In general, pyrosequencing and RFLP showed comparable sensitivities in detecting most of the SNPs in dhfr except for the 164L imitation, which required at least twice the amount of DNA for pyroseqencing as for RFLP. For detecting SNPs in dhps, pyrosequencing was slightly more sensitive than RFLP and direct sequencing. Overall, pyrosequencing was faster and less expensive than either RFLP or direct sequencing. Thus, pyrosequencing is a practical alternative method that can be used in a high-throughput format for molecular surveillance of antimalarial-drug resistance.

Original languageEnglish (US)
Pages (from-to)3900-3910
Number of pages11
JournalJournal of Clinical Microbiology
Volume44
Issue number11
DOIs
StatePublished - Nov 2006

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dihydrofolate synthetase
Dihydropteroate Synthase
Tetrahydrofolate Dehydrogenase
Plasmodium falciparum
Restriction Fragment Length Polymorphisms
Single Nucleotide Polymorphism
Parasites
Genes
Codon
DNA
Folic Acid Antagonists
Antimalarials
Drug Resistance
Malaria
Genome
Mutation

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

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Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum. / Zhou, Zhiyong; Poe, Amanda C.; Limor, Josef; Grady, Katharine K.; Goldman, Ira; McCollum, Andrea M.; Escalante, Ananias A.; Barnwell, John W.; Udhayakumar, Venkatachalam.

In: Journal of Clinical Microbiology, Vol. 44, No. 11, 11.2006, p. 3900-3910.

Research output: Contribution to journalArticle

Zhou, Zhiyong ; Poe, Amanda C. ; Limor, Josef ; Grady, Katharine K. ; Goldman, Ira ; McCollum, Andrea M. ; Escalante, Ananias A. ; Barnwell, John W. ; Udhayakumar, Venkatachalam. / Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum. In: Journal of Clinical Microbiology. 2006 ; Vol. 44, No. 11. pp. 3900-3910.
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