An improved nucleic acid extraction method from dried blood spots for amplification of Plasmodium falciparum kelch13 for detection of artemisinin resistance
PublisherBioMed Central Ltd.
MetadataShow full item record
AbstractBackground: 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. Copyright 2019 The Author(s).
SponsorsThis work was supported by Howard Hughes Medical Institute (PI: Plowe CV), the National Institute of Allergy and Infectious Diseases, International Centers of Excellence for Malaria Research network (Grant U19AI129386; PIs Plowe CV and Nyunt MM) and the Bill and Melinda Gates Foundation (Grant OPP1109551; PI: Nyunt MM).
Dried blood spot
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85067264191&doi=10.1186%2fs12936-019-2817-8&partnerID=40&md5=a95c4294298e3e004be21143832ba654; http://hdl.handle.net/10713/10218
- Limited polymorphisms in k13 gene in Plasmodium falciparum isolates from Dakar, Senegal in 2012-2013.
- Authors: Torrentino-Madamet M, Fall B, Benoit N, Camara C, Amalvict R, Fall M, Dionne P, Ba Fall K, Nakoulima A, Diatta B, Diemé Y, Ménard D, Wade B, Pradines B
- Issue date: 2014 Dec 4
- A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections.
- Authors: Zainabadi K, Adams M, Han ZY, Lwin HW, Han KT, Ouattara A, Thura S, Plowe CV, Nyunt MM
- Issue date: 2017 Sep 18
- Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker.
- Authors: Tun KM, Imwong M, Lwin KM, Win AA, Hlaing TM, Hlaing T, Lin K, Kyaw MP, Plewes K, Faiz MA, Dhorda M, Cheah PY, Pukrittayakamee S, Ashley EA, Anderson TJ, Nair S, McDew-White M, Flegg JA, Grist EP, Guerin P, Maude RJ, Smithuis F, Dondorp AM, Day NP, Nosten F, White NJ, Woodrow CJ
- Issue date: 2015 Apr
- Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia.
- Authors: Takala-Harrison S, Jacob CG, Arze C, Cummings MP, Silva JC, Dondorp AM, Fukuda MM, Hien TT, Mayxay M, Noedl H, Nosten F, Kyaw MP, Nhien NT, Imwong M, Bethell D, Se Y, Lon C, Tyner SD, Saunders DL, Ariey F, Mercereau-Puijalon O, Menard D, Newton PN, Khanthavong M, Hongvanthong B, Starzengruber P, Fuehrer HP, Swoboda P, Khan WA, Phyo AP, Nyunt MM, Nyunt MH, Brown TS, Adams M, Pepin CS, Bailey J, Tan JC, Ferdig MT, Clark TG, Miotto O, MacInnis B, Kwiatkowski DP, White NJ, Ringwald P, Plowe CV
- Issue date: 2015 Mar 1
- No evidence of amplified Plasmodium falciparum plasmepsin II gene copy number in an area with artemisinin-resistant malaria along the China-Myanmar border.
- Authors: Huang F, Shrestha B, Liu H, Tang LH, Zhou SS, Zhou XN, Takala-Harrison S, Ringwald P, Nyunt MM, Plowe CV
- Issue date: 2020 Sep 14