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Open Access Highly Accessed Methodology

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

Serge Y Ongagna-Yhombi15, Paul Corstjens2, Eran Geva1, William R Abrams1, Cheryl A Barber1, Daniel Malamud1* and Sungano Mharakurwa34

Author Affiliations

1 Department of Basic Science, NYU College of Dentistry, New York, NY, 10010, USA

2 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands

3 Johns Hopkins University, Bloomberg School of Public Health, W Harry Feinstone Department of Molecular Microbiology and Immunology, Baltimore, USA

4 The Malaria Institute at Macha, PO Box 630166, Choma, Zambia

5 Current address: University of Delaware, Department of Biological Sciences, Newark, DE, 19716, USA

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Malaria Journal 2013, 12:74  doi:10.1186/1475-2875-12-74

Published: 22 February 2013

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.

Keywords:
Malaria; Detection; Semi-nested PCR; PCR; Lateral flow; Plasmodium falciparum