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

Abstract

Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2-99.8% and 95.2-99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs.

Fig 1. P. ovale reticulocyte binding protein 2 (rbp-2) sequence alignment.

Both P. ovale curtisi and P. ovale wallikeri sequences were aligned using Geneious software program in order to select a conserved region for the two P. ovale subspecies. Cytosine is labelled purple, adenine pink, guanine yellow and thymine green. The forward (PoRBP2FWD) and reverse (PoRBP2REV) primers are denoted in dark and light green boxes, respectively.

Fig 2. Novel P. ovale primers only amplify P. ovale and not the other human-infecting species.

P. falciparum, P. vivax, P. malariae, P. knowlesi, P. ovale curtisi, P. ovale wallikeri DNA samples were utilized for primer specificity testing. Only the positive control (a known P. ovale sample), P. ovale curtisi and P. ovale wallikeri were amplified using our primers (amplification plots with Ct values of 25.57, 33.38 and 35.2 respectively). No amplification (flat lines) was noted for the other species and the no template control (NTC).