Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related micro-organisms
Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related micro-organisms
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DOI：
10.1111/j.1365-2672.2010.04737.x
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发表时间：
2010-10-01
影响因子：
4
通讯作者：
Bannert, N.
中科院分区：
文献类型：
--
作者：
Laue, M.;Bannert, N.
目的：为了确定诊断负染色电子显微镜的检测限的诊断病原体，可用于bioterrorism.Methods和结果：牛痘病毒和枯草芽孢杆菌的内生孢子的悬浮液中使用的定义浓度作为模型的痘病毒和炭疽孢子（炭疽芽孢杆菌），这两种病原体，可用于生物恐怖袭击。负染色电子显微镜检查直接进行，或在这些悬浮液沉降到样品支持物上后使用离心超离心法进行。对于病毒和孢子，使用10 μ l样品体积直接吸附到样品载体上的检测限为每ml 106个颗粒。用80 μ l的样品体积进行离心超离心，孢子的检测限可降至105个/ml，痘病毒的检测限可降至5 × 104个/ml。孵育时间，洗涤和吸附程序对颗粒检测的影响进行了investigated.Conclusions：该方法的重现性和灵敏度是可以接受的，特别是考虑到小的样品体积和低的颗粒数应用到样品support.Significance和影响的研究：诊断负染色电子显微镜用于诊断病原体在紧急情况下，因为它可以快速检查所有的颗粒物下降到纳米级。本研究为该方法提供了精确的检测限，这是验证和改进该技术的重要因素。
Aims:To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism.Methods and Results:Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10-mu l sample volume onto the sample support was 106 particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 mu l, the detection limit could be reduced to 105 particles per ml for spores and to 5 x 104 particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated.Conclusions:The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support.Significance and Impact of the Study:Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.