A Novel Method for the Rapid and Efficient Preparation of Polymerase Chain Reaction Template DNA from Filter Paper...

Filter paper blood samples (i.e., dry blood spots [DBSs]) have been widely used for pathogen detection, especially in malaria control and elimination. Dried blood spot-based polymerase chain reaction (PCR) testing has also been increasingly used for diagnosis and gene monitoring. The current method of DNA extraction from DBSs is limited in practical application because of its low efficiency, time-consuming procedure, and high cost. Accordingly, a hemolysis preservation (HP) method was developed and compared with the most commonly used Tween-Chelex method and spin-column method kit (QIAamp DNA Mini) under equivalent conditions by extracting template DNA from the same batch of Plasmodium falciparum density-gradient DBSs. The HP method yielded the highest DNA recovery volume (557.32 ng/µL of blood) and the lowest limit of detection (LOD; equivalent to 0.1 parasites/µL) from DBSs with the minimum blood volume (4-5 µL) using the simplest reagent. Additionally, it had the shortest time (∼50 minutes) and the lowest cost ($0.27 per sample). The LOD of the direct PCR method also reached a value of 0.11 parasites/µL. Therefore, the DBS PCR test conducted using the HP method can detect asymptomatic low-density Plasmodium infection and is a cost-effective option in resource-deficient areas.