Development of a new DNA diagnostic system for the species-specific detection of human malaria parasites using specific nucleotide sequences of the 18S ribosomal RNA gene.

我们已经开发了一种DNA诊断方法，“微滴板杂交（MPH）”，其中pcr扩增产物被固定在微滴孔上的物种特异性探针捕获，并通过随后的显色反应可视化。该检测系统以18S rRNA基因的DNA序列为靶点，可检测和鉴定恶性疟原虫、间日疟原虫、卵形疟原虫和疟疾疟原虫四种人类疟原虫。我们通过检查临床疟疾患者的一些血液样本来评估MPH的有用性。我们的研究证实了MPH方法在临床实践中对疟疾管理的价值，特别是在低级别寄生虫病的诊断、物种分化、复发预测和治疗评估方面。我们发现了一种卵形p.a vvale寄生虫的变异型，在显微镜下与典型的卵形p.a vval寄生虫无法区分，但p.a vval特异性MPH检测结果为阴性。pcr扩增产物序列分析显示，探针区存在1个碱基置换和2个碱基缺失。根据所揭示的序列设计了一种新的探针，可以检测p.o oval变异。我们对p.p ovale-variant的18S rRNA基因进行了2.1千碱基的序列分析，发现了三种类型的序列。它们的序列与典型卵形疟原虫的序列差异约4%，与其他疟原虫的序列差异约10%。我们还测定了卵形p.o vale变异血分期的转录RNA序列。总之，该方法不仅具有临床应用价值，而且对寻找人类疟原虫的新种或变异也有一定的应用价值。

We have developed a DNA diagnostic method, "microtiter plate-hybridization (MPH)", in which the PCR-amplified products are captured by species-specific probes immobilized on a microtiter well, and visualized by a subsequent chromogenic reaction. This assay system allows us to detect and identify all four species of human malaria parasites, Plasmodium falciparum, P.vivax, P.ovale, and P.malariae, using the DNA sequence of the 18S rRNA gene as the target. We have evaluated the usefulness of the MPH by examining a number of blood samples from patients with clinical malaria. Our study confirmed that the MPH method is valuable for the management of malaria in clinical practice, particularly in the diagnosis of low-grade parasitemia, species differentiation, prediction of recrudescence, and assessment of treatment. We have found a variant type of P.ovale parasite, which was indistinguishable from typical P.ovale by microscopy but which gave negative results by P.ovale-specific MPH.Sequence analysis of the PCR-amplified product revealed that there are one base substitution and two base deletions in the probe region. A newly constructed probe which was designed based on the revealed sequence could detect the P.ovale-variant. We performed a sequence analysis of 2.1 kilobase of 18S rRNA gene of P.ovale-variant and found three types of sequences. They differs about 4% in sequence from typical P.ovale and about 10% from other plasmodial species. We also determined the transcribed RNA sequence in blood stages of P.ovale-variant. In conclusion, the MPH method is valuable not only for clinical practice, but also for searching for a new species or variant of human malaria parasite.