Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)
Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)
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DOI：
10.1186/1475-2875-8-78
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发表时间：
2009-04-23
期刊：
影响因子：
3
通讯作者：
Farnert, Anna
中科院分区：
文献类型：
--
作者：
Liljander, Anne;Wiklund, Lisa;Farnert, Anna
背景：通过聚合酶链式反应扩增裂殖子表面蛋白1和2(msp1和msp2)的多态基因对恶性疟原虫进行基因分型，在疟疾研究领域已被广泛应用，以确定感染中并发克隆的数量和类型。在抗疟疾药物试验中，基因分型被认为是通过区分复发寄生虫和新感染来确定治疗结果的关键。由于大多数基因分型分析中用于片段分析的凝胶电泳法在特异性和分辨率方面的局限性，因此有必要改进方法学。另一种片段分析技术是使用自动DNA测序仪进行的毛细管电泳法。在这里，最广泛使用的恶性疟原虫msp1和msp2基因分型方法之一已被修改为CE技术。报告了该技术在分子流行病学研究和抗疟疾药物试验中的方法和优化过程，以及该技术在分子流行病学研究和抗疟疾药物试验中的潜力和局限性。方法：通过荧光标记巢式聚合酶链式反应中的msp1和msp2等位基因类型特异性引物，并在DNA测序仪中对最终的PCR产物进行分析，对原始的基因分型方法进行了改进。对荧光分析方法进行了实质性的优化。用已知的加纳和坦桑尼亚的实验室品系和现场样品的混合物验证了CE方法，并与原始的凝胶电泳法进行了比较。结果：基于CE的方法具有较高的精密度和重复性(
Background: Genotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported.Methods: The original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis.Results: The CE-based method showed high precision and reproducibility in determining fragment size (