DNA Diagnosis of Malaria Using PCR Techniques.

我们已经开发了三种DNA诊断系统，用于检测人体血液中的疟原虫，使用聚合酶链反应（PCR）技术。我们的系统很简单，既不需要DNA提取，也不需要放射性同位素。双重PCR是一种敏感性高、特异性强的两步法恶性疟原虫检测体系。双重PCR中使用的靶DNA序列是由恶性疟原虫的二氢叶酸还原酶-胸苷酸合酶（DHFR-TS）基因编码的序列。PCR产物琼脂糖凝胶电泳显示，10 μ IOTA血液中的10个寄生虫产生226个碱基对（bp）的阳性条带。在ED-PCR、PCR产物酶法检测中，我们选择了恶性疟原虫和间日疟原虫18 S核糖体RNA基因的一部分作为目的DNA。用二硝基苯基（DNP）标记的PCR产物用酶标抗DNP抗体检测。ED-PCR检测恶性疟原虫和间日疟原虫寄生虫没有区别。微孔板杂交是一种利用PCR产物分别检测恶性疟原虫和间日疟原虫特异性探针，从而实现对恶性疟原虫和间日疟原虫特异性检测的诊断系统。我们在疟疾高发区所罗门群岛的瓜达尔卡纳尔岛对这三种DNA诊断系统进行了研究。对98人的101份血样进行了DNA诊断系统和显微镜检查。本系统的结果与显微镜检查结果相一致。我们的系统也被证明是有用的治疗效果的判断。

We have developed three DNA diagnosis systems for detection of malaria parasites in human blood using the polymerase chain reaction (PCR) techniques. Our systems are simple and require neither DNA extraction nor radioisotopes. The double PCR is highly sensitive and specific detection system of Plasmodium falciparum parasites using two-step PCR. The target DNA sequence used in the double PCR was that encoded by the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of P. falciparum. As little as 10 parasites in 10 muIOTA of blood gave a positive band of 226 base pairs (bp) by agarose gel electrophoresis of the PCR products. In the ED-PCR, enzymatic detection of PCR products, we have chosen a part of 18S ribosomal RNA gene of P. falciparum and P. vivax parasites as the target DNA. The PCR products labeled with dinitrophenyl(DNP) were detected using enzyme-labeled anti-DNP antibody. Both P. falciparum and P. vivax parasites were detected without distinction by the ED-PCR. The microplate hybridization is a diagnosis system which can detect P. falciparum and P. vivax distinctively, using the P. falciparum-specific probe and the P. vivax-specific probe to detect the PCR products. In this method, the part of 18S rRNA gene was also used as the target DNA for the PCR amplification.We studied these three DNA diagnosis systems in the Guadalcanal of the Solomon lslands, where is a highly endemic area of malaria. 101 blood samples from 98 people were examined by the DNA diagnosis systems and microscopy. The results of our systems well accorded with that of microscopic examination. Our systems were also proved to be useful for a judgment of the effects of treatments.