Inoue Juliana, Huber Nina, Fendel Rolf, Held Jana
Institute of Tropical Medicine, Eberhard Karls University of Tübingen, Tübingen, Germany.
German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany.
Malar J. 2024 Dec 23;23(1):398. doi: 10.1186/s12936-024-05227-z.
Molecular methods play an important role in clinical trials assessing anti-malarial drugs and vaccines, as well as in epidemiological studies aimed at detecting Plasmodium species, especially when dealing with large sample sizes. Molecular techniques are more sensitive and generally have a higher throughput compared to the gold standard microscopy. Further optimization can be achieved with automation of nucleic acid isolation, allowing for rapid and precise extraction. This study evaluated the isolation of total nucleic acids from Plasmodium falciparum mocked samples using an automated extraction method with a magnetic bead-based kit compared to a manual silica column-based kit. Additionally, two different RNA preservation solutions were compared.
Plasmodium falciparum Dd2 parasites were serially diluted and spiked into whole blood. The dilutions were stored in two different RNA preservation solutions and total nucleic acids extracted with an automated magnetic bead-based kit and a manual silica column-based kit. Subsequently, a reverse transcription (RT) qPCR for Plasmodium detection targeting Plasmodium 18S rRNA and DNA in a single reaction was performed and the quantification cycle (Cq) values across the different sample groups were compared.
Comparable Cq values across the various sample preparations were obtained, suggesting minimal influence from RNA preservation solutions (p = 0.686) or extraction methods (p = 0.119) on RT-qPCR outcomes. Automated nucleic acids extraction allowed processing numerous samples in a shorter timeframe and showed similar efficiency in detecting Plasmodium in blood samples by RT-qPCR as manual extraction.
The automated method for nucleic acid isolation is a valuable tool for the detection of Plasmodium infections in large-scale studies. It is efficient, reliable, and cost-effective. Its potential applications extend to other molecular surveillance studies to support malaria control measures.
分子方法在评估抗疟药物和疫苗的临床试验以及旨在检测疟原虫种类的流行病学研究中发挥着重要作用，尤其是在处理大量样本时。与金标准显微镜相比，分子技术更灵敏，通常通量更高。通过核酸分离自动化可实现进一步优化，从而实现快速、精确的提取。本研究评估了使用基于磁珠的试剂盒的自动化提取方法与基于手动硅胶柱的试剂盒从恶性疟原虫模拟样本中分离总核酸的情况。此外，还比较了两种不同的RNA保存溶液。
将恶性疟原虫Dd2寄生虫进行系列稀释并加入全血中。稀释液保存在两种不同的RNA保存溶液中，并用基于磁珠的自动化试剂盒和基于手动硅胶柱的试剂盒提取总核酸。随后，在单一反应中进行针对疟原虫检测的逆转录（RT）qPCR，以疟原虫18S rRNA和DNA为靶点，并比较不同样本组的定量循环（Cq）值。
在各种样本制备中获得了可比的Cq值，表明RNA保存溶液（p = 0.686）或提取方法（p = 0.119）对RT-qPCR结果的影响最小。自动化核酸提取能够在更短的时间内处理大量样本，并且在通过RT-qPCR检测血样中的疟原虫时显示出与手动提取相似的效率。
核酸分离的自动化方法是大规模研究中检测疟原虫感染的有价值工具。它高效、可靠且具有成本效益。其潜在应用扩展到其他分子监测研究，以支持疟疾控制措施。
