实时 PCR 直接从全血和滤纸样本中检测疟原虫。
Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples.
机构信息
Department of Oncology, University of Alberta, Edmonton, Canada.
出版信息
Malar J. 2011 Aug 19;10:244. doi: 10.1186/1475-2875-10-244.
BACKGROUND
Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products.
METHODS
Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested.
RESULTS
Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA.
CONCLUSIONS
The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings.
背景
实时 PCR 是一种用于分析疟原虫 DNA 的敏感和特异方法。然而，由于血液中的 PCR 抑制剂和荧光淬灭会阻止 PCR 产物的有效扩增和检测，因此需要对血液中的基因组 DNA 进行预先纯化。
方法
专门设计用于克服 PCR 抑制和荧光淬灭的试剂，用于直接从血液中实时 PCR 扩增疟原虫 DNA。对来自哥伦比亚现场采集的临床样本全血和干血斑进行了测试。
结果
在 PCR 反应中，40×SYBR® Green 染料和 5%血液体积可实现最佳的扩增和荧光检测。可直接从临床样本的全血和干血斑中检测到疟原虫 DNA。与对纯化的疟原虫 DNA 进行 PCR 相比，该方法的灵敏度和特异性范围为 93-100%。
结论
所描述的方法学促进了现场采集的血液样本通过基于荧光的实时 PCR 进行高通量检测。该方法可应用于广泛的临床研究，具有即时样本检测、更低的实验成本和节省时间的优势。
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