Journal Article

Molecular Diagnosis of Malaria by Photo-Induced Electron Transfer Fluorogenic Primers: PET-PCR

Lucchi, Naomi W., Kariuki, Simon, Narayanan, Jothikumar, Karell, Mara A., Hill, Vincent, Xayavong, Maniphet, DaSilva, Alexandre J., Udhayakumar, Venkatachalam

2013

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Biology / Deoxyribonucleic acid / Development and progression / Diagnosis / Diagnostic software / Diagnostic systems / Diagnostic tests / Disease control / Disease prevention / DNA / DNA Primers / Dyes / Electron transfer / Electron transport / Epidemiology / Fluorescent Dyes / Health aspects / Humans / Infection / Infectious diseases / Malaria / Malaria - diagnosis / Malaria - genetics / Malaria - parasitology / Medical diagnosis / Medicine / Microscopy / Parasites / Parasitic diseases / Plasmodium / Plasmodium falciparum / Plasmodium falciparum - genetics / Plasmodium falciparum - isolation & purification / Plasmodium vivax / Polymerase chain reaction / Polymerase Chain Reaction - methods / Primers / Quenching / Real time / RNA, Ribosomal, 18S - genetics / Screening / Sensitivity / Sensitivity and Specificity / Surveillance / Vector-borne diseases / Zoonoses