生物素化质粒rep20探针检测恶性疟原虫感染的研究

STUDY ON DETECTION OF P. FALCIPARUM IN BLOOD SAMPLES WITH BIOTINYLATED PREP20 DNA PROBE

Bio—ll—dUTP经切口平移法标记于质粒rep20,自身杂交敏感度为100pg。检出云南株Pf(恶性疟原虫)培养血和病人血样中pf密度低限分别为1/10~5和1/10~4(虫/红细胞),病人血样中最低pf检出数为12000个。检测30份云南省恶性疟患者血样,28份阳性;检测20份云南省和海南省问日疟患者血样、29份正常人血样均为阴性;与三种动物疟原虫及人白细胞的DNA未出现杂交反应。表明质粒rep20对云南株pf具有高度同源性和特异性,该生物素化探针检测Pf具较高敏感性。

The prep20 DNA probe labeled with bio—11—dUTP by nick translation was evaluated for detection of P. falciparum in blood samples. The hybridization sensitivity of the biotinylated probe was at least 10pg, which was able to detect parasitemia levels of 0.001% in 30μ1 of RBC from culture of P. falciparum(Yunnan isolate)and 0.01% in 60μl whole blood from falciparum malaria patients of Yunnan Province. The smallest number of parasites detected was 12000 in blood samples. With this probe, 28 samples were obviously positive in detection of 30 blood samples from falciparum malaria patients of Yunnan Province, but 20 blood samples from vivax malaria patients of Yunnan Province and Hainan Province and 29 blood samples from donors of Xiann were all negative. In adition,this probe did not hybridize with the DNA of P. cynomolgi. P. knowlesi, p, yoelii and humann leucocytes. It is denanstrated that rep20 probe is specific to DNA of P. falciparum isolates of Yunnan Province.