Multiplex qPCR for Detection and Absolute Quantification of Malaria
Multiplex qPCR for Detection and Absolute Quantification of Malaria
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DOI：
10.1371/journal.pone.0071539
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发表时间：
2013-08-29
期刊：
影响因子：
3.7
通讯作者：
Ockenhouse, Christian F.
中科院分区：
文献类型：
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作者：
Kamau, Edwin;Alemayehu, Saba;Ockenhouse, Christian F.
我们描述了一种用于检测恶性疟原虫、恶性疟原虫和间日疟原虫靶标的绝对多重实时定量聚合酶链式反应的发展，以便生产一种高通量但成本较低的检测方法。研究了重要的定量聚合酶链式反应实验细节和对检测结果的性能和可靠性至关重要的信息。进行抑制研究，以测试和比较使用手动或自动方法从全血中提取的样本中的聚合酶链式反应抑制剂的共纯化。为了建立最适的qPCR反应体积，对反应母液进行体积滴定，从10亩L开始到1亩L反应母液，每次反应中加入1亩L模板DNA。随着反应体积的减小，定量聚合酶链式反应的效率提高，以1亩L反应母液效果最好。为了更准确地定量样本中的寄生虫，我们为所有三个绝对定量的检测目标开发了质粒DNA。所有的绝对定量聚合酶链式反应的效率都在94%以上，R-2值大于0.99，每个重复的标准偏差为
We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 mu l to 1 mu l reaction master mix with 1 mu l of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 mu l reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R-2 values greater than 0.99 and the STDEV of each replicate was