DNA Diagnosis of Ovale Malaria and Malariae Malaria Using Microtiter Plate-Hybridization

Abstract

We have developed a colorimetric assay, "microtiter plate-hybridization", for the detection of human malaria parasite, Plasmodium ovule and Plasmodium malariae, 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.

A trial for a DNA diagnosis of Plasmodium vivax malaria recently reemerging in the Republic of Korea using microtiter plate hybridization assay.

“…As an alternative measure, the PCRbased MPH assay was tested for its practical utility and reported as a useful tool for the differential diagnosis of various species of malaria. [11][12][13] In the present study, we confirmed the usefulness and feasibility of the PCR-based MPH technique for the genetic diagnosis of the reemerging Korean P. vivax malaria. Using this technique it was possible to provide a specific diagnosis of human malaria.…”

“…It is well known that the plasmodial genes encoding 18S rRNA contain species-specific regions: P. vivax, 14 P. falciparum, 15 Plasmodium malariae, 16 Plasmodium ovale, 12 and Plasmodium ovalevariant (showing variations at the probe region, reported from Vietnam). 17 The oligonucleotide primer set (primer MPH-1; 5Ј-CAGATACCGTCGTAATCTTA-3Ј, and primer MPH-2; 5Ј-CCAAAGACTTTGATTT CTCAT-3Ј) cross-reactive for P. vivax, P. falciparum, and P. malariae was used as described previously.…”

“…17 The oligonucleotide primer set (primer MPH-1; 5Ј-CAGATACCGTCGTAATCTTA-3Ј, and primer MPH-2; 5Ј-CCAAAGACTTTGATTT CTCAT-3Ј) cross-reactive for P. vivax, P. falciparum, and P. malariae was used as described previously. 12,13 Species-specific probes (P. vivax; 14 5Ј-TAAACTCCGAA-GAGAAAATTC-3Ј, P. falciparum; 15 5Ј-GTCACCTCGAAA-GATGACTT-3Ј, P. malariae;…”

“…8,9 To overcome these difficulties, several researchers have developed DNA probe-based, RNA-probe based or polymerase chain reaction (PCR)-based procedures in order to detect malarial genes. [8][9][10][11][12][13] Among them, the PCR-based microtiter plate hybridization (MPH) was reported to be a simple but highly useful technique for a DNA diagnosis of different species of malaria. [11][12][13] However, more trials are needed to verify the usefulness and feasibility of the MPH technique.…”

“…[8][9][10][11][12][13] Among them, the PCR-based microtiter plate hybridization (MPH) was reported to be a simple but highly useful technique for a DNA diagnosis of different species of malaria. [11][12][13] However, more trials are needed to verify the usefulness and feasibility of the MPH technique. After the reemergence of P. vivax malaria in the Republic of Korea, the diagnosis of patients has usually been based on the examination of blood films, 3 and DNA diagnosis has never been tried.…”