Chelex法从恶性疟原虫薄血涂片提取DNA的基因诊断

目的 建立从恶性疟原虫薄血涂片中提取DNA进行恶性疟原虫18S RNA基因套式PCR检测方法 ,并探讨其可行性.方法 利用螯合型的离子交换树脂Chelex-100作为介质,一步法分别提取患者染色和未染色薄血涂片恶性疟原虫DNA,行套式PCR扩增.以培养的不同浓度恶性疟原虫制备的染色和未染色薄血涂片提取恶性疟原虫DNA为模板进行PCR扩增,检测该方法的灵敏度.结果 用Chelex-100法提取恶性疟原虫患者染色与未染色薄血膜DNA,PCR扩增均出现205 bp特异扩增片段.染色及未染色薄血膜恶性疟原虫PCR扩增的最低虫体浓度分别为1.5×101/μL血和1.5×10-1/μL血.结论 Chelex-100法薄血涂片中提取微量DNA的套式PCR检测方法,可在基因水平检测存档的薄血涂片标本.为恶性疟疾的临床实验室诊断和分子流行病学研究提供了新方法.

Genetic detection of Plasmodium falciparum with Chelex-extracted DNA from thin blood smears

Objective To investigate the feasibility of Chelex DNA extraction from thin blood smears for genetic analysis, and to develop smear-based nested polymerase chain reaction (PCR) of the 18S RNA of Plasrnodium falciparum. Methods Chelex-100 which was chelating ion exchange resin was used to extract DNA from Giemsa-stained or unstained thin blood smears of different concentrations of Plasmodium falciparum. With the extracted DNA as the template, 18S RNA gene was amplified by nested PCR to test the susceptibility of Chelex method. Results Positive band of 205bp appeared in nested PCR with DNA extracted from Giemsa-stained or unstained thin blood smears of patient with falciparum malaria. Using the Chelex method, the detection limits of the smear-based nested PCR were 1.5 × 101 parasite/μL blood for Giemsa-stained and 1.5×10-1 parasite/μL blood for unstained thin blood smears. Conclusions Chelex DNA extraction is a simple and efficient method for extracting trace amount of DNA from thin blood smear. The smear-based nested PCR developed in this study is feasible to identify the gene from reserved thin blood smears and will provide a new approach for clinical diagnosis and study of molecular epidemiology.