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dc.contributor.authorZainabadi, K.
dc.contributor.authorNyunt, M.M.
dc.contributor.authorPlowe, C.V.
dc.date.accessioned2019-08-05T17:00:30Z
dc.date.available2019-08-05T17:00:30Z
dc.date.issued2019
dc.identifier.urihttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85067264191&doi=10.1186%2fs12936-019-2817-8&partnerID=40&md5=a95c4294298e3e004be21143832ba654
dc.identifier.urihttp://hdl.handle.net/10713/10218
dc.description.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).en_US
dc.description.sponsorshipThis 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).en_US
dc.description.urihttps://doi.org/10.1186/s12936-019-2817-8en_US
dc.language.isoen-USen_US
dc.publisherBioMed Central Ltd.en_US
dc.relation.ispartofMalaria Journal
dc.subjectArtemisinin resistanceen_US
dc.subjectAsymptomatic infectionen_US
dc.subjectDBSen_US
dc.subjectDiagnosticsen_US
dc.subjectDried blood spoten_US
dc.subjectDrug resistanceen_US
dc.subjectK13en_US
dc.subjectKelch13en_US
dc.subjectLow parasitaemiaen_US
dc.subjectLow transmissionen_US
dc.subjectMalariaen_US
dc.subjectMolecular surveillanceen_US
dc.subjectPCRen_US
dc.subjectPlasmodium falciparumen_US
dc.subjectSoutheast Asiaen_US
dc.titleAn improved nucleic acid extraction method from dried blood spots for amplification of Plasmodium falciparum kelch13 for detection of artemisinin resistanceen_US
dc.typearticleen_US
dc.identifier.doi10.1186/s12936-019-2817-8
dc.identifier.pmid31185976


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