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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.

Original publication

DOI

10.1371/journal.pone.0159160

Type

Journal article

Journal

PLoS One

Publication Date

2016

Volume

11

Keywords

Animals, Anopheles, Calibration, DNA Primers, Insect Vectors, Life Cycle Stages, Limit of Detection, Malaria, Falciparum, Malaria, Vivax, Myanmar, Nucleic Acid Denaturation, Plasmodium falciparum, Plasmodium vivax, Real-Time Polymerase Chain Reaction, Reference Standards, Sensitivity and Specificity, Sporozoites, Thailand