The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots

Am J Trop Med Hyg. 2015 May;92(5):922-5. doi: 10.4269/ajtmh.14-0602. Epub 2015 Mar 9.

Abstract

Extraction and amplification of DNA from dried blood spots (DBS) collected in field studies is commonly used for detection of Plasmodium falciparum. However, there have been few systematic efforts to determine the effects of storage and extraction methods on the sensitivity of DNA amplification. We investigated the effects of storage conditions, length of storage, and DNA extraction methods on amplification via three PCR-based assays using field samples and laboratory controls. Samples stored as DBS for 2 or more years at ambient temperature showed a significant loss of sensitivity that increased with time; after 10 years only 10% samples with parasite densities > 1,000 parasites/μL were detectable by nested polymerase chain reaction (PCR). Conversely, DBS and extracted DNA stored at -20°C showed no loss of sensitivity with time. Samples with low parasite densities amplified more successfully with saponin/Chelex compared with spin-column-based extraction, though the latter method performed better on samples with higher parasite densities stored for 2 years at ambient temperature. DNA extracted via both methods was stable after 20 freeze-thaw cycles. Our results suggest that DBS should be stored at -20°C or extracted immediately, especially if anticipating 2 or more years of storage.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Protozoan / blood
  • DNA, Protozoan / isolation & purification*
  • Freezing
  • Malaria, Falciparum / diagnosis*
  • Malaria, Falciparum / parasitology
  • Models, Statistical
  • Plasmodium falciparum / genetics
  • Plasmodium falciparum / isolation & purification*
  • Polymerase Chain Reaction / methods
  • Polymerase Chain Reaction / standards
  • Sensitivity and Specificity
  • Specimen Handling / methods*
  • Temperature
  • Time Factors

Substances

  • DNA, Protozoan