Methodology
Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis
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
Malaria Journal 2013, 12:74 doi:10.1186/1475-2875-12-74
Published: 22 February 2013Abstract
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.
