Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR

PLoS One. 2013;8(2):e56677. doi: 10.1371/journal.pone.0056677. Epub 2013 Feb 20.

Abstract

There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. falciparum and the Plasmodium genus by real-time PCR. A total of 119 samples consisting of different malaria species and mixed infections were used to test the utility of the novel PET-PCR primers in the diagnosis of clinical samples. The sensitivity and specificity were calculated using a nested PCR as the gold standard and the novel primer sets demonstrated 100% sensitivity and specificity. The limits of detection for P. falciparum was shown to be 3.2 parasites/µl using both Plasmodium genus and P. falciparum-specific primers and 5.8 parasites/µl for P. ovale, 3.5 parasites/µl for P. malariae and 5 parasites/µl for P. vivax using the genus specific primer set. Moreover, the reaction can be duplexed to detect both Plasmodium spp. and P. falciparum in a single reaction. The PET-PCR assay does not require internal probes or intercalating dyes which makes it convenient to use and less expensive than other real-time PCR diagnostic formats. Further validation of this technique in the field will help to assess its utility for large scale screening in malaria control and elimination programs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers*
  • Fluorescent Dyes
  • Humans
  • Malaria / diagnosis*
  • Malaria / genetics
  • Malaria / parasitology
  • Plasmodium falciparum / genetics
  • Plasmodium falciparum / isolation & purification*
  • Polymerase Chain Reaction / methods*
  • RNA, Ribosomal, 18S / genetics
  • Sensitivity and Specificity

Substances

  • DNA Primers
  • Fluorescent Dyes
  • RNA, Ribosomal, 18S

Grant support

This study was supported by the Atlanta Research and Education Foundation, Atlanta, Georgia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.