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.

我们开发了一种DNA诊断方法--微滴定板杂交(MPH)，在该方法中，通过固定在微滴定孔上的物种特异性探针捕获PCR扩增产物，并通过随后的显色反应进行可视化。这一检测系统使我们能够以18S rRNA基因的DNA序列为靶标，检测和鉴定恶性疟原虫、间日疟原虫、卵形疟原虫和疟疾原虫这四种人类疟疾寄生虫。我们已经通过检测临床疟疾患者的一些血液样本来评估MPH的有效性。我们的研究证实，MPH方法在临床实践中对疟疾的管理是有价值的，特别是在诊断低级寄生虫血症、物种分化、复发预测和治疗评估方面。我们发现了一种变异型的卵圆假单胞菌寄生虫，在显微镜下无法与典型的卵圆假单胞菌区分开来，但对卵圆单胞菌特异的MPH检测结果为阴性。对扩增产物进行测序分析，发现探针区有一个碱基替换和两个碱基缺失。根据揭示的序列设计的新构建的探针可以检测到卵圆假单胞菌的变异体。对卵圆假单胞菌变种的18S rRNA基因进行了2.1kb的序列分析，发现了三种类型的序列。它们在序列上与典型的卵形拟青霉相差约4%，与其他原生生物相差约10%。我们还测定了卵圆假单胞菌变种在血液期的转录RNA序列。总之，MPH方法不仅对临床实践有价值，而且对于寻找人类疟疾寄生虫的新物种或变种也是有价值的。

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.