Moriarity John R, Loftis Amanda D, Dasch Gregory A
Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
J Med Entomol. 2005 Nov;42(6):1063-7. doi: 10.1093/jmedent/42.6.1063.
To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 +/- 0.04 microg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-microl volumes with a reproducibility of +/- 0.038 microl when dispensing 1.0 microl. By using this process, 96 samples can be extracted and tested while reducing human labor to approximately 30 min.
为满足高通量样本检测的需求，已开发出基于96孔板形式的DNA提取试剂盒，用于血液和组织样本。这些方法尚未应用于蜱虫的DNA提取。为满足这一需求，我们开发了一种高通量的蜱虫样本DNA提取和聚合酶链反应（PCR）检测方法。使用液体处理机器人在带有真空歧管的96孔结合柱板中提取DNA。用这种方法从1821年肩突硬蜱若虫中回收的DNA的数量、纯度和质量具有可重复性，且与现有的手动DNA提取技术相当。五只若虫蜱虫样本池的DNA产量平均为0.432 +/- 0.04微克（95%置信区间）。该机器人还直接从提取的DNA在96孔板中制备实时PCR反应。对现有的P20工具进行改进后，在分配1.0微升时，能够准确移液1至2微升体积，重现性为+/- 0.038微升。通过使用该流程，可以提取和检测96个样本，同时将人工操作时间减少至约30分钟。
