Mtambo Jupiter, Van Bortel Wim, Madder Maxime, Roelants Patricia, Backeljau Thierry
Department of Veterinary and Livestock Development, P.O. Box 670050, Mazabuka, Zambia.
Exp Appl Acarol. 2006;38(2-3):189-99. doi: 10.1007/s10493-006-0004-4.
Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 degrees C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.
对五组保存方式不同的成年微小扇头蜱标本进行了DNA提取质量的比较。使用了三种方法从标本中提取DNA，即两种经过验证的简单蚊虫DNA提取方法和一种经过验证的蜱虫提取方法。尝试从蜱虫腿部提取DNA。通过ITS2基因和线粒体COI基因片段的PCR扩增成功率来评估提取的DNA质量。新鲜标本（即提取前刚处死）的DNA扩增成功率最高，其次是在乙醇中处死并随后保存在冰箱（4摄氏度）中的标本。冷冻保存和70%乙醇保存的标本在扩增成功率上没有显著差异。从蜱虫腿部扩增DNA是可行的。从蜱虫腿部获得的模板的ITS2扩增子测序结果与从整个蜱虫提取物中获得的结果一样清晰可读。两种经过验证的蚊虫DNA提取方法的扩增成功率明显低于经过验证的蜱虫提取方案。
