DNA Diagnosis of Malaria Using PCR Techniques.

我们开发了三种DNA诊断系统，利用聚合酶链反应（PCR）技术检测人血液中的疟原虫。我们的系统很简单，既不需要提取DNA，也不需要放射性同位素。双PCR是一种灵敏、特异的恶性疟原虫双步PCR检测系统。双PCR的目的DNA序列为恶性疟原虫二氢叶酸还原酶-胸腺苷酸合成酶（DHFR-TS）基因编码的DNA序列。琼脂糖凝胶电泳结果显示，在10个样本中，仅有10只寄生虫出现226个碱基对（bp）的阳性带。在PCR产物的酶促检测ED-PCR中，我们选择了恶性疟原虫和间日疟原虫18S核糖体RNA基因的一部分作为靶DNA。二硝基苯（DNP）标记PCR产物用酶标抗DNP抗体检测。恶性疟原虫和间日疟原虫经ED-PCR检测均无明显差异。微孔板杂交是一种能够对恶性疟原虫和间日疟原虫进行特异性检测的诊断系统，采用恶性疟原虫特异性探针和间日疟原虫特异性探针检测PCR产物。在本方法中，18S rRNA基因部分也作为PCR扩增的靶DNA。我们在所罗门群岛的瓜达尔卡纳尔岛研究了这三种DNA诊断系统，那里是疟疾高度流行的地区。通过DNA诊断系统和显微镜检查了来自98人的101份血液样本。我们的系统的结果与显微镜检查的结果很吻合。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.