title: Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR.
abstract: Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies.
authors.affiliation: Centre hospitalier universitaire de Montpellier, Montpellier, France.|Maladies Infectieuses et Vecteurs, Ecologie, Génétique, Evolution et Contrôle, Institut de Recherche pour le Développement, Montpellier, France.|Centre for Advanced Studies for Agriculture and Food, Institute of Advanced Studies, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.
authors.forename: V
authors.initials: V
authors.lastname: Chaumeau
authors.affiliation: Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand.
authors.forename: C
authors.initials: C
authors.lastname: Andolina
authors.affiliation: Maladies Infectieuses et Vecteurs, Ecologie, Génétique, Evolution et Contrôle, Institut de Recherche pour le Développement, Montpellier, France.|Centre for Advanced Studies for Agriculture and Food, Institute of Advanced Studies, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.
authors.forename: B
authors.initials: B
authors.lastname: Fustec
authors.affiliation: Institut de Recherche pour le développement, UMR216, Mère et enfant face aux infections tropicales, Paris, France.|Communauté d'Universités et d'Etablissements Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
authors.forename: N
authors.initials: N
authors.lastname: Tuikue Ndam
authors.affiliation: Maladies Infectieuses et Vecteurs, Ecologie, Génétique, Evolution et Contrôle, Institut de Recherche pour le Développement, Montpellier, France.
authors.forename: C
authors.initials: C
authors.lastname: Brengues
authors.affiliation: UMR Intertryp, Institut de Recherche pour le Développement, Montpellier, France.|Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.
authors.forename: S
authors.initials: S
authors.lastname: Herder
authors.affiliation: Centre for Advanced Studies for Agriculture and Food, Institute of Advanced Studies, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.|Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand.
authors.forename: D
authors.initials: D
authors.lastname: Cerqueira
authors.affiliation: Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.
authors.forename: T
authors.initials: T
authors.lastname: Chareonviriyaphap
authors.affiliation: Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand.|Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: F
authors.initials: F
authors.lastname: Nosten
authors.affiliation: Maladies Infectieuses et Vecteurs, Ecologie, Génétique, Evolution et Contrôle, Institut de Recherche pour le Développement, Montpellier, France.|Centre for Advanced Studies for Agriculture and Food, Institute of Advanced Studies, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.|Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.
authors.forename: V
authors.initials: V
authors.lastname: Corbel
mesh_terms: D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D002138:Calibration; D017931:DNA Primers / Q000378:metabolism*; D007303:Insect Vectors / Q000469:parasitology*; D008018:Life Cycle Stages; D057230:Limit of Detection; D016778:Malaria, Falciparum / Q000175:diagnosis / Q000469:parasitology; D016780:Malaria, Vivax / Q000175:diagnosis / Q000469:parasitology; D002052:Myanmar; D009691:Nucleic Acid Denaturation; D010963:Plasmodium falciparum / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D010966:Plasmodium vivax / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D034101:Sporozoites / Q000502:physiology; D013785:Thailand
chemical_list: D017931:DNA Primers