适配器-超速离心法提高电镜检测病毒的灵敏度

通过专用超速离心机将病毒直接离心至载网是重要的提高诊断电镜灵敏度的方法,作者尝试建立适配器-超速离心法实现相同目的。设计制造了外形类似离心管的载网适配器,其内部有一平底的凹槽,可以放置电镜载网,添加病毒悬液后,经过超速离心,能够将病毒粒子直接离心到载网上。利用重组腺病毒和重组腺相关病毒为模式病毒,适配器-超速离心法较悬滴法的病毒检测灵敏度提高了50～200倍;对人腺病毒41型（Ad41）细胞培养物进行电镜检测,灵敏度提高了50倍。如果将免疫凝集法（immune clumping）与适配器-超速离心法相联合进行电镜制样,检测灵敏度较常规免疫凝集法提高1～2个数量级。这些结果说明适配器-超速离心法能够高效地富集病毒,可以应用于病毒的电镜检测工作。

Adaptor-ultracentrifugation to improve the sensitivity of virus detection using electron microscopy

Direct ultracentrifugation of virus particles to specimen grid is one of the important techniques for diagnostic electron microscopy（EM）.In this study,we attempt to establish a method of adaptor-ultracentrifugation to achieve the same aim of enriching viruses directly on the EM grid.Adaptor with a flat-bottomed cavity was designed and produced,which has an ultracentrifugation tubelike shape.Formvar-coated grid can be loaded on the cavity bottom.Virus-containing samples are added to the cavities,then the adaptors can be installed on the swing rotor as ultracentrifugation tubes.After ultracentrifugation,grids are picked out and subjected to negative staining,followed by EM examination.Recombinant adenovirus（Ad） and recombinant adenovirus-associated virus（AAV） were chosen as the models to compare the routine droplet method with the adaptorultracentrifugation.The results showed that the adaptor-ultracentrifugation method were about 50 to 200 times more sensitive than the droplet procedure.The similar effectiveness was also observed when the adapter-ultracentrifugation was used to detect the human diarrhea Ad（Ad41） cultivated in cells.If adapter-ultracentrifugation was combined with the immune clumping method to prepare EM specimen,the sensitivity could increase about 1 to 2 magnitudes compared to the conventional immune clumping.These data demonstrated that adapter-ultracentrifugation can enrich virus particles very effectively and can be utilized to detect viruses in diagnostic EM.