Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

Serge Y. Ongagna-Yhombi, Paul Corstjens, Eran Geva, William R. Abrams, Cheryl A. Barber, Daniel Malamud, Sungano Mharakurwa

Research output: Contribution to journalArticle

Abstract

Background: A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods. A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results: This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions: The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples.

Original languageEnglish (US)
Article number74
JournalMalaria Journal
Volume12
Issue number1
DOIs
StatePublished - 2013

Keywords

  • Detection
  • Lateral flow
  • Malaria
  • PCR
  • Plasmodium falciparum
  • Semi-nested PCR

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

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