Magnetic bead-based enhancement of qPCR detection rates in low-density Plasmodium falciparum samples: A comparative analysis of nucleic acid extraction methods.

Abstract

In post-elimination malaria settings, monitoring asymptomatic low-density infections following Artemisinin-based Combination Therapy (ACT) is critical for interrupting occult transmission and alerting to drug resistance. Real-time quantitative PCR (qPCR) is central to molecular surveillance, yet its sensitivity depends on extracted DNA quality. This study evaluated three DNA extraction methods to provide a basis for optimizing laboratory screening protocols for low-density Plasmodium falciparum (P. falciparum) infections. Nucleic acids were extracted from P. falciparum-positive whole blood samples using a silica membrane spin column method (M1), a modified red blood cell lysis method (M2), and a magnetic bead-based method (M3). DNA concentration and purity (A260/A280 ratio) were measured, and integrity assessed via agarose gel electrophoresis. Template yield and amplification efficiency were compared by qPCR. Furthermore, the detection sensitivity was compared between M1 and M3 via serial dilution experiments. Clinical performance was validated using 40 microscopy-negative or low-density (<150 parasites/µL) samples collected 48 hours post-treatment. M3 yielded significantly higher DNA concentration (39.0 ± 7.57 ng/µL) than M1 (10.49 ± 2.73 ng/µL) and M2 (7.48 ± 1.76 ng/µL) (P < 0.0001). Although M3 had a slightly lower A260/A280 ratio, electrophoresis showed it better preserved high-molecular-weight DNA. qPCR confirmed that the template yield from M3 was approximately 4.0-fold and 2.8-fold higher than that from M2 and M1, respectively. Serial dilution experiments demonstrated that M3 exhibited significantly higher detection sensitivity, with a lower limit of detection (LOD) than M1. Among the 40 clinical low-density samples, the positive detection rate for M3 (77.5%, 31/40) was significantly higher than for M1 (60.0%, 24/40) (P = 0.0082), with M1 showing a missed detection rate of 22.6% (7/31). The magnetic bead-based method (M3) exhibits marked advantages in DNA yield, DNA integrity and downstream qPCR sensitivity, which elevates the detection rate of low-density P. falciparum and reduces missed detection risk. For post-elimination surveillance and screening of asymptomatic infections, prioritizing magnetic bead-based nucleic acid extraction can provide more reliable technical support for molecular surveillance in post‑elimination settings.