SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination

Abstract

A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7-100%) when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.

Fig 1. Area chart showing the sequence alignments of target genes, primers and probes.

a) Five copies of the 18S rRNA gene in the P. falciparum genome. b) Five copies of the 18S rRNA gene in the P. vivax genome. c) Combined ten copies of the 18S rRNA gene in the P. falciparum and P. vivax genomes. d) The cytb genes of P. falciparum, P. vivax, P. malariae, and P. ovale. Identical sequences are shown in grey in the chart area. Nucleotide base pair position is shown on the X-axis and the proportion of sequences in consensus is shown on the Y-axis. Primer and probe positions of respective PCRs are indicated using white arrows along the dashed lines (1, 18S-nPCR pan-Plasmodium primers; 1’, 18S-nPCR species-specific primers; 2, 18S-qPCR-R pan-Plasmodium primers and probe; 2’, 18S-qPCR-R species-specific primers and probes; 3, 18S-qPCR-K; 4, cytb-nPCR; and 5, cytb-qPCR).

Fig 2. The cytb-qPCR products and RFLP assays for species determination.

a) The 430 bp cytb-qPCR products for each species. b-e) Four RFLP assays using FspBI, AluI, HpyCH4V and Csp6I restriction enzymes for Plasmodium species determination. f) Schematic diagram showing the RFLP product lengths. a-e) Lane 1, GeneRuler low range DNA ladder (Thermo Fisher); F, P. falciparum; V, P. vivax; M, P. malariae; O, P.ovale; NC, negative control.

Fig 3. The cytb-qPCR standard curves derived from tenfold dilution series of P. falciparum, P. vivax, P. malariae, and P. ovale samples.

The cytb-qPCR efficiency (E) and coefficient of determination (R²) of the standard curves were calculated for each species. The cytb qPCR was conducted in eight replicates (4 extractions × 2 cytb-qPCRs). Dashed lines represent 95% CI.

Fig 4. Parasite detection limits for each PCR method.

Solid dots represent PCR positive and hollow dots represent PCR negative samples. Detection limits are shaded in grey.

Fig 5. PCR and species results for the 65 ‘final positive’ field samples.

Each row represents one sample. Pan, Plasmodium spp.; F, P. falciparum; M, P. malariae; FM, P. falciparum and P. malariae mixed infection; +, positive; −, negative.