Military Malaria Research Program, Malaria Vaccine Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
PLoS One. 2013 Aug 29;8(8):e71539. doi: 10.1371/journal.pone.0071539. eCollection 2013.
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 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl 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 <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.
我们描述了一种绝对多重实时定量 PCR 的开发，用于检测疟原虫 spp.、恶性疟原虫和间日疟原虫靶标，以生产一种适合高通量但成本降低的检测方法。研究了重要的 qPCR 实验细节和对检测结果性能和可靠性至关重要的信息。进行了抑制研究，以测试和比较从全血中提取的样品中 PCR 抑制剂的共纯化，使用手动或自动化方法。为了确定最佳的 qPCR 反应体积，从 10 µl 到 1 µl 反应混合液，每个反应中 1 µl 的模板 DNA，进行了反应混合液体积滴定。随着反应体积的减小，qPCR 检测变得更加高效，1 µl 反应混合液是最有效的。为了更准确地定量样品中的寄生虫，我们为所有三个检测靶点开发了质粒 DNA 进行绝对定量。所有绝对 qPCR 检测的效率都超过 94%，R(2) 值大于 0.99，每个重复的 STDEV 都小于 0.167。从绝对 qPCR 检测生成的线性回归图用于根据相对 qPCR 以寄生虫/µl 的形式估计相应的寄生虫密度。将一个质粒 DNA 拷贝定义为 Plasmodium spp. 检测的 0.1 个寄生虫/µl，恶性疟原虫检测的 0.281 个寄生虫，间日疟原虫检测的 0.127 个寄生虫/µl。本研究首次证明了质粒 DNA 在寄生虫绝对定量中的应用。在疟疾寄生虫定量中使用质粒 DNA 标准将是至关重要的，因为正在努力协调用于疟疾诊断的分子检测。
