DNA diagnosis of malaria using microtiter plate-hybridization.
We have developed a new diagnostic method "microtiter plate-hybridization" (MPH) for the detection of human malaria parasite in which the target DNA sequence of the 18S ribosomal RNA gene is amplified by polymerase chain reaction and hybridized with the species-specific probes immobilized on a microtiter well. The PCR products bound on a well are visualized by the biotin-streptavidin system and the following chromogenic reaction. This method has allowed us to detect and identify the four species of human malaria parasites. We obtained blood samples by finger puncture from 435 donors in Japan, Solomon Island and Vietnam. The results of our method showed good correlation with the results of Giemsa staining microscopy. Furthermore, we developed a new system for the detection of new human malaria parasites. This system involves an acridine orange (AO) staining microscopic examination and "microtiter plate-hybridization". Using the system, we found a case of new variant of Plasmodium ovale whose PCR-amplified DNA did not hybridize with a probe for typical P. ovale in Vietnam. These results indicate that our new method can serve as a useful tool for the clinical management, epidemiological research of malaria, and investigation of the new type of malaria parasite.
Miyake
H
H
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Kim
H S
HS
Kawai
S
S
Yamane
A
A
Kimura
M
M
Wataya
Y
Y
D000165:Acridine Orange; D000818:Animals; D016054:DNA, Protozoan / Q000097:blood* / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D017125:Genes, Protozoan; D014644:Genetic Variation; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology; D008542:Melanesia / Q000453:epidemiology; D017720:Molecular Epidemiology; D009693:Nucleic Acid Hybridization; D010961:Plasmodium / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D013045:Species Specificity; D014744:Vietnam / Q000453:epidemiology
D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; D000165:Acridine Orange