LOOP-MEDIATED ISOTHERMAL AMPLIFICATION IN MICROCHAMBERS ON A CELL MICROARRAY CHIP FOR IDENTIFICATION OF PLASMODIUM SPECIES

Malaria is caused by Plasmodium spp., a parasitic protist that infects erythrocytes. A method that can detect the parasite with high sensitivity and that can identify the parasite species is urgently required for the control of malaria. The cell microarray chip was made using polystyrene with 200 cone-shaped frustum microchambers (800-mu m top diameter, 636-mu m bottom diameter, and 225 mu m deep). Approximately 3,000 erythrocytes could be accommodated in each microchamber with monolayer formation, there being 60,000 erythrocytes in total microchambers on a cell microarray. Plasmodium could be quantitatively detected with high sensitivity with the use of cell microarray chips. Plasmodium parasitizing in erythrocytes was labeled with a cell-permeant fluorescent nucleic acid stain (SYTO 21), which could be detected in erythrocytes in the microchambers. Next, we used loop-mediated isothermal amplification (LAMP) in the microchambers (on-chip LAMP) to identify the parasite species detected in the microchambers. LAMP was performed in the microchambers (in a reaction volume of 0.09 mu 1) using Plasmodium falciparum-infected erythrocytes as the template and specific primers targeting 18S rRNA. To avoid evaporation of the reaction buffer during heat treatment, mineral oil was overlaid on each microchamber and the cell microarray chips were heated at 63 C for 1 hr. The results of on-chip LAMP were assessed using a portable ultraviolet transilluminator. We showed that this method has the potential for detection of parasites in 600,000 erythrocytes and for identification of the parasite species on a cell microarray chip. In conclusion, the parasites can be detected quantitatively with high sensitivity, and the species can be identified with the use of cell microarray chips.