环介导等温扩增技术检测恶性疟原虫的研究

目的环介导等温扩增(LAMP)技术检测恶性疟原虫。方法酚氯仿法提取恶性疟原虫基因组DNA,设计四条扩增恶性疟原虫环子孢子蛋白基因的LAMP引物,以间日疟原虫、弓形虫、卡氏肺孢子虫、人全血DNA为对照,进行LAMP反应。LAMP产物经显色、电泳鉴定。将原虫血症为1.5%的恶性疟原虫血样用正常人血按1∶10倍比稀释为1.5×10-3、1.5×10-4、1.5×10-5、1.5×10-6、1.5×10-7、1.5×10-86个浓度后进行LAMP,检测其敏感性。结果恶性疟原虫检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性)。恶性疟原虫LAMP产物经电泳后呈LAMP特征性梯状条带,对照组均无扩增产物。LAMP可检测恶性疟原虫的敏感度为1.5个疟原虫/107RBC。结论检测恶性疟原虫的LAMP方法特异、敏感及简便。

Detection of Plasmodium falciparum DNA by loop-mediated isothermal amplification

In order to detect the presence of Plasmodium falciparum simply and rapidly, the DNA from P. falciparum was extracted by phenol-chloroform extraction and amplified by loop-mediated isothermal amplification(LAMP) using 4 primers recognizing 6 distinct regions on the CSP gene of P.falciparum. The results of LAMP assay were to be judged by naked eyes and by electrophoretic analysis. To evaluate the specificity of this assay, P. vivax, Toxoplasma gondii, Pneumocystis carinii and human blood DNA were used as the negative controls, and its sensitivity was determined from the results in LAMP assay of 6 concentrations of blood samples with 1.5 % of protozoa in blood by 10 fold serial dilution with normal human serum. The result of experiment showed that the detection limit of LAMP assay for P. falciparum was 1.5 P. falciparum Per 107 RBC. It is clear the LAMP assay is a simple, sensitive and specific method of detecting P. falciparum in blood samples.