A Simple and Cost-Effective Freeze-Thaw Based Method for Plasmodium DNA Extraction from Dried Blood Spot

Abstract

Background: Available DNA isolation methods for Plasmodium involve numerous processing steps, adding to the cost and conferring risk of contamination. Here we devise a simple and cost-effective method for direct extraction of Plasmodium DNA from dried filter paper spot (DBS), appropriate for resource-limited setups. Methods: The protocol involves simple freezing and thawing of DBS, neither involves any purification step nor any chemical reagent. The method was assessed in terms of DNA quantity, PCR detection sensitivity, time requirement, cost effectiveness, labor intensiveness and degree of shearing. The reliability of this method was confirmed by comparing it with other in use methods for Plasmodium DNA isolation. Results: Pure DNA was obtained with this method, as exemplified by the absorbance ratio (260nm /280nm) of 1.2. The protocol produced digestible, PCR-grade genomic DNA, also found to be suitable for sequencing. DNA isolated remained stable and retained its integrity after storage for one month at 4 0C. Conclusion: Our process substantiated as efficient, reproducible, simple, fast, and inexpensive. Development of this optimized freeze-thaw based DNA extraction method for malaria parasite may provide a valuable tool for molecular analysis in resource-limited setups. This is the first report of DNA extraction from DBS of Plasmodium utilizing freeze-thaw.