Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification.
With an aim to develop a quick and simple method to survey pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the identification of Plasmodium-carrying mosquitoes, specifically a Plasmodium-transmitting experimental model using rodent malaria parasite (Plasmodium berghei) and anopheline mosquitoes (Anopheles stephensi). The detection sensitivity limit of the LAMP reaction amplifying the SPECT2 gene was determined to be 1 x 10(2) purified Plasmodium parasites, estimated to be sufficient for reliable identification of infectious mosquitoes. The robustness of the LAMP reaction was revealed by its ability to detect both Plasmodium oocysts and sporozoites from an "all-in-one" template using whole mosquito bodies. Moreover, LAMP successfully identified an infectious mosquito carrying just a single oocyst in its midgut, a level that can be easily overlooked in conventional microscopic analysis. These observations suggest that LAMP is more reliable and useful for routine diagnosis of vector mosquitoes in regions where vector-borne diseases such as malaria are endemic.
Aonuma
Hiroka
H
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Suzuki
Moemi
M
Iseki
Hiroshi
H
Perera
Namal
N
Nelson
Bryce
B
Igarashi
Ikuo
I
Yagi
Takeshi
T
Kanuka
Hirotaka
H
Fukumoto
Shinya
S
D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D001483:Base Sequence; D016054:DNA, Protozoan / Q000032:analysis* / Q000096:biosynthesis; D005260:Female; D007303:Insect Vectors / Q000235:genetics; D008288:Malaria / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology; D008297:Male; D008969:Molecular Sequence Data; D010962:Plasmodium berghei / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity
D016054:DNA, Protozoan; D015800:Protozoan Proteins; C503442:spect2 protein, Plasmodium berghei