Improving detection of avian malaria from host blood: a step towards a standardised protocol for diagnostics

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• 作者：Chris N. Niebuhr ; Isabel Blasco-Costa
• 关键词：Plasmodium ; Avian malaria ; Extraction ; PCR ; Elution ; Qiagen
• 刊名：Parasitology Research
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：115
• 期：10
• 页码：3905-3911
• 全文大小：863 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Biomedicine
  Medical Microbiology
  Microbiology
  Immunology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-1955
• 卷排序：115

文摘

Avian malaria, caused by Plasmodium spp., has been linked to the mortality and population-level declines in native birds in some regions. While molecular diagnostic methods have greatly improved our ability to detect infections of both human and bird malaria, failing to identify false negatives remains an important handicap, particularly for avian malaria due to host DNA presence in the bird blood cells. In an attempt to improve the accuracy of diagnostics by PCR, we evaluated the performance of a commercial silica-membrane-based DNA extraction kit by modifying the protocol with four unpooled elution volume alternatives. Our results suggest that the best template is the DNA extract obtained from the second eluate of a first 50 μL elution step. In one case, the only band visible was from this second eluate and, thus, may not have been identified as positive for Plasmodium spp. if a different elution protocol had been followed. Our results are likely explained by the concept of size exclusion chromatography by which particles of different sizes will elute at different rates. Overall, first elution templates may consist of a lower ratio of parasite to host DNA, while second eluates may contain a higher parasite to host DNA ratio. A low ratio of parasite to host DNA is a concern in detecting chronic infections, in which birds typically carry low levels of parasitemia, making accurate diagnostics imperative when identifying reservoirs of disease that could lead to spillback events.