An improved nucleic acid extraction method from dried blood spots for amplification of Plasmodium falciparum kelch13 for detection of artemisinin resistance.

Published online

Journal Article

BACKGROUND: Mutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia. This problem can be overcome by collecting large volumes of venous blood. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance. METHODS: Methods for k13 amplification from standard Whatman 3MM DBS (stored for 14 days at 28 °C with 80% relative humidity) were optimized by systematically testing different extraction conditions. Conditions that improved parasite DNA recovery as assessed by quantitative polymerase chain reaction (PCR) of 18S rDNA were then tested for their impact on k13 PCR amplification. RESULTS: The optimized protocol for amplification of k13 from DBS is markedly more sensitive than standard methods using commercial kits. Using this method, k13 was successfully amplified from laboratory-created DBS samples with parasite densities as low as 500 parasites/mL. Importantly, the method recovers both DNA and RNA, making it compatible with RNA-based ultrasensitive techniques currently in use. CONCLUSIONS: The optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.

Full Text

Duke Authors

Cited Authors

  • Zainabadi, K; Nyunt, MM; Plowe, CV

Published Date

  • June 11, 2019

Published In

Volume / Issue

  • 18 / 1

Start / End Page

  • 192 -

PubMed ID

  • 31185976

Pubmed Central ID

  • 31185976

Electronic International Standard Serial Number (EISSN)

  • 1475-2875

Digital Object Identifier (DOI)

  • 10.1186/s12936-019-2817-8

Language

  • eng

Conference Location

  • England