Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID(50) Assays.
Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID(50) Assays.
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DOI：
10.3390/microorganisms9010156
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发表时间：
2021-01-12
期刊：
影响因子：
4.5
通讯作者：
Davey RA
中科院分区：
文献类型：
--
作者：
Keiser PT;Anantpadma M;Staples H;Carrion R;Davey RA
针对丝状病毒开发有效疗法的持续努力在不同程度上依赖于通过空斑、半数组织培养感染剂量（TCID50）和聚焦试验对样本中活病毒的数量进行定量。不幸的是，这些技术存在固有差异，而且实验室特定的偏好使得数据难以直接比较。此外，人为错误，如操作人员失误和主观偏差，会进一步加剧结果的差异。为了克服这些偏差，我们基于开源的CellProfiler软件平台开发了一种用于聚焦试验的基于计算机的自动图像处理方法，该方法能够一次性对许多处理样本进行高通量筛选。我们使用3种主要丝状病毒（扎伊尔埃博拉病毒、苏丹埃博拉病毒和马尔堡马尔堡病毒）的通用病毒株，由多名操作人员在多天内分别进行空斑、TCID50和聚焦试验，并将使用该平台计算出的病毒滴度与空斑和TCID50试验进行比较。我们表明空斑试验得出的结果具有可比性，差异小于3倍。由于聚焦形成单位（FFU）试验敏感性较高，TCID50试验敏感性较低，FFU和TCID50试验与空斑试验的差异在10倍或更小。然而，每种试验的重复性和准确性存在显著差异，中性红琼脂糖覆盖空斑试验和TCID50由于主观分析和操作人员误差重复性最低。结晶紫甲基纤维素覆盖空斑试验和聚焦试验在准确性方面表现最佳，而聚焦试验在速度和通量方面表现最佳。
Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID50, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer-based automated image-processing method for a focus assay based on the open-source CellProfiler software platform, which enables high-throughput screening of many treatment samples at one time. We compared virus titers calculated using this platform to plaque and TCID50 assays using common stocks of virus for 3 major Filovirus species, Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus with each assay performed by multiple operators on multiple days. We show that plaque assays give comparable findings that differ by less than 3-fold. Focus-forming unit (FFU) and TCID50 assays differ by 10-fold or less from the plaque assays due a higher (FFU) and lower (TCID50) sensitivity. However, reproducibility and accuracy of each assay differs significantly with Neutral Red Agarose Overlay plaque assays and TCID50 with the lowest reproducibility due to subjective analysis and operator error. Both crystal violet methylcellulose overlay plaque assay and focus assays perform best for accuracy and the focus assay performs best for speed and throughput.
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影响因子：
8.8
作者：
Leung DW;Borek D;Luthra P;Binning JM;Anantpadma M;Liu G;Harvey IB;Su Z;Endlich-Frazier A;Pan J;Shabman RS;Chiu W;Davey RA;Otwinowski Z;Basler CF;Amarasinghe GK
通讯作者：
Amarasinghe GK
DOI：
10.3390/v8040113
发表时间：
2016-04-21
期刊：
Viruses
影响因子：
--
作者：
Shurtleff AC;Bloomfield HA;Mort S;Orr SA;Audet B;Whitaker T;Richards MJ;Bavari S
通讯作者：
Bavari S
影响因子：
12.3
作者：
Carpenter AE;Jones TR;Lamprecht MR;Clarke C;Kang IH;Friman O;Guertin DA;Chang JH;Lindquist RA;Moffat J;Golland P;Sabatini DM
通讯作者：
Sabatini DM
DOI：
10.1073/pnas.38.8.747
发表时间：
1952-01-01
影响因子：
11.1
作者：
DULBECCO, R
通讯作者：
DULBECCO, R
影响因子：
3.1
作者：
Smither, Sophie J.;Lear-Rooney, Calli;Olinger, Gene G., Jr.
通讯作者：
Olinger, Gene G., Jr.