聚合酶链反应检测恶性疟原虫的研究

STUDIES ON DETECTION PLASMODIUM FALCIPARUM IN BLOOD SAMPLES BY POLYMERASE CHAIN REACTION

通过５ 对根据恶性疟原虫不同基因序列设计的引物筛选及７ 种从滤纸干血滴中快速制备疟原虫ＤＮＡ模板的方法的比较，以建立一种敏感、特异、简便、快速的聚合酶链反应（ＰＣＲ） 检测恶性疟原虫的方法。结果表明，根据恶性疟原虫中度重复基因序列ｐＢＲＫ１１４ 设计的引物，可从恶性疟原虫ＤＮＡ 中扩增出２０６ ｂｐ 大小的ＤＮＡ片段，而间日疟原虫、人白细胞ＤＮＡ均无此扩增带出现，并至少可检测出原虫血症为５ ×１０－ ７ 的感染水平，且适用于我国不同地理株恶性疟原虫的检测；滤纸干血滴样本经生理盐水溶血煮沸法处理的ＰＣＲ 扩增效果最为理想，且简易、经济、重复性好；在云南疟疾流行区采集的３６０ 份血样中，ＰＣＲ 法与镜检法符合率为９７ ．５％ 。本研究建立的ＰＣＲ 检测恶性疟原虫方法具有敏感、特异、简易和快速的特点，便于现场推广应用。

On the basis of the former studies on the detection of Plasmodium falciparum(P.f.) by PCR, we chose 5 pairs of primers derived from different target sequences and 7 different methods to prepare malaria parasites DNA from dried blood spot specimens, so as to select and establish a PCR method which was rapid, sensitive and specific for P.f. .The result showed that the primer derived from the moderately repetitive DNA sequence pBRK1 14 was the most sensitive and specific in the 5 pairs of primers. With the primer, P.f. DNA was amplified yielding a 206 base pair(bp) fragment, while Plasmodium vivax and leucocyte DNA werent amplified. The PCR method using the optimal primer was sensitive enough to detect parasitemia as level as 5×10 -7 for P.f. and able to detect the different geographic strains. The whole blood samples collected on filter paper only required to be processed with normal saline prior to PCR. Among 360 samples collected from malaria endemic areas, 5 samles were P.f. positive by PCR, but negative by microscopy, 4 samples were negative by PCR, but P.f. positive by microscopy, the concordance between the two methods was 97.5%. The PCR method established in the study has the quality of sensitivity, specificity and simplicity, making it practical for field work.