Automated total nucleic acid extraction with magnetic beads for the detection of Plasmodium falciparum in large study cohorts.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.