PCR-ELISA检测疟原虫DNA的研究

STUDY ON DETECTION OF MALARIA PARASITE DNA BY PCR ELISA *

目的：介绍一种诊断疟疾的新方法ＰＣＲ－ＥＬＩＳＡ。方法：根据业已报道的疟原虫ＳＳＵｒ－ＲＮＡ基因保守序列，设计并合成一对通用于恶性疟原虫和间日疟原虫的引物，其中一引物的５’端加以生物素标记。经ＰＣＲ扩增后，携带有生物素的扩增产物与先期包被于ＥＬＩＳＡ板上的亲和素结合，再经与恶性疟原虫、间日疟原虫特异、荧光素标记的寡核苷酸探针分别杂交，底物显色等步骤，使ＰＣＲ产物得以半定量地检出。结果：对于恶性疟原虫和间日疟原虫，本法检出最低原虫密度阈值分别为４和１０个原虫／μｌ血（取血２０μｌ），本法检测两种疟原虫未发现交叉反应。结论：本试验具有较高的敏感度和特异性。

AIM: To present a new malaria diagnostic method based on detection of malaria parasite DNA by PCR ELISA. METHODS: According to the conserved sequence of Plasmodium SSUrRNA genes reported, a pair of primers in which one primer was biotinylated and another was unbiotinylated, suitable for DNA amplification of both falciparum and vivax malaria parasites were designed and synthesized. After denaturation and washing, the incoporated biotinylated product with avidin coated on plates previously was hybridized with the fluorescein labelled oligonucleotide probes specific for Plasmodium falciparum or Plasmodium vivax. The color developed after adding POD conjugated with antibody to fluorescein and substrate can be semi quantitated spectrophotometrically. RESULTS: The thresholds of parasite density for the detection of Plasmodiun falciparum and Plasmodium vivax by this test were shown to be as low as 4 and 10 parasites per μl of blood, respectively and no cross reaction was seen in the detection of falciparun and vivax malaria parasites. CONCLUSION:With promising sensitivity and specificity, this test can be used in malaria survey.