Schwartz Alanna, Baidjoe Amrish, Rosenthal Philip J, Dorsey Grant, Bousema Teun, Greenhouse Bryan
Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, California; Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands; Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, United Kingdom
Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, California; Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands; Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, United Kingdom.
Am J Trop Med Hyg. 2015 May;92(5):922-5. doi: 10.4269/ajtmh.14-0602. Epub 2015 Mar 9.
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.
在现场研究中，从干血斑（DBS）中提取和扩增DNA常用于检测恶性疟原虫。然而，很少有系统的研究来确定储存和提取方法对DNA扩增敏感性的影响。我们通过使用现场样本和实验室对照的三种基于PCR的检测方法，研究了储存条件、储存时间和DNA提取方法对扩增的影响。在室温下作为DBS储存2年或更长时间的样本显示出显著的敏感性丧失，且随时间增加；10年后，通过巢式聚合酶链反应（PCR）仅能检测到10%寄生虫密度>1000个寄生虫/微升的样本。相反，储存在-20°C的DBS和提取的DNA未显示出随时间的敏感性丧失。与基于离心柱的提取方法相比，低寄生虫密度的样本用皂角苷/螯合树脂提取时扩增更成功，尽管后一种方法对在室温下储存2年的高寄生虫密度样本效果更好。通过两种方法提取的DNA在20次冻融循环后仍保持稳定。我们的结果表明，DBS应储存在-20°C或立即提取，特别是如果预计储存2年或更长时间。
