title: 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.
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Division of Malaria Research, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Weiping
authors.initials: W
authors.lastname: Xu
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Division of Malaria Research, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Ulrika
authors.initials: U
authors.lastname: Morris
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Division of Malaria Research, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Berit
authors.initials: B
authors.lastname: Aydin-Schmidt
authors.affiliation: Zanzibar Malaria Elimination Programme, Zanzibar, Tanzania.
authors.forename: Mwinyi I
authors.initials: MI
authors.lastname: Msellem
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Division of Malaria Research, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Delér
authors.initials: D
authors.lastname: Shakely
authors.affiliation: Center for Applied Biostatistics, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Max
authors.initials: M
authors.lastname: Petzold
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Division of Malaria Research, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Björkman
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Division of Malaria Research, Karolinska Institutet, Stockholm, Sweden; Department of Public Health Sciences, Division of Global Health (IHCAR), Karolinska Institutet, Stockholm, Sweden; Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden.
authors.forename: Andreas
authors.initials: A
authors.lastname: Mårtensson
mesh_terms: D045303:Cytochromes b / Q000235:genetics*; D017125:Genes, Protozoan; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology*; D010961:Plasmodium / Q000145:classification / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D012337:RNA, Ribosomal, 18S; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D012337:RNA, Ribosomal, 18S; D045303:Cytochromes b