参见作者原研究论文

本实验方案简略版
Aug 2019

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Real-time Fluorescence Measurement of Enterovirus Uncoating
肠道病毒脱壳的实时荧光检测   

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Abstract

Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative staining, cryo electron microscopy, X-ray crystallography or gradient separation (reviewed in Tuthill et al., 2010, Myllynen et al., 2016, Ruokolainen et al., 2019). However, monitoring of uncoating has been limited by the lack of methods detecting dynamic changes of the virions. Here, we present a real-time fluorescence based protocol, which detects the viral genome (RNA) during various stages of uncoating in vitro, while RNA is still inside the particle that has been expanded before the actual RNA release, and when the RNA has been totally released from the viral particle. Our method allows to explore how various molecular factors may promote or inhibit virus uncoating.

Keywords: Picornavirus (小RNA病毒), Picornavirus (肠道病毒), Uncoating (脱壳), Genome (基因组), RNA (RNA), RNase (RNA酶), SYBR Green II (SYBR Green II), Fluorescence spectroscopy (荧光光谱)

Background

In our previous study, we found that infectious intermediate echovirus 1 particle allows SYBR Green II, a RNA intercalating dye, to enter the virus particle (Myllynen et al., 2016). This can be observed as an increase of fluorescence and the recorded fluorescence is not susceptible to RNase digestion (Myllynen et al., 2016). Using this information, we developed a real-time method to monitor virus opening using the SYBR Green II dye and RNase in fluorescence spectroscopy. We could follow the fenestration of the particles in real-time at +37 °C, or other temperature of interest, in a 96-well plate format by adding SYBR Green II and factors triggering the uncoating, and observing the increase of SYBR Green II fluorescence. Addition of RNase into parallel wells allowed us to monitor the extent of RNA release from the virions, as RNase readily degrades RNA from the solution, but not from inside of the virion (RNAse cannot enter through the small fenestrations inside to the virus particle, Myllynen et al., 2016). In case of intact virus particles, only very low amount of fluorescence was observed. As an example, in our previous study, a DPBS solution supplemented with 0.01% fatty acid free BSA produced high amounts of intermediate echovirus 1 particles. For more details see the original publication (Ruokolainen et al., 2019).

Materials and Reagents

  1. Pipette tips
  2. Sarstedt 96-well plate (Sarstedt, catalog number: 83.3924 ) (or similar)
  3. 1.5 ml tubes
  4. Purified virus stock (1 μg of virus per measured well; stored at -80 °C)
    Purification of enteroviruses can be done using either 5-20% or 10-40% sucrose gradient and is described in detail in the original publication (Ruokolainen et al., 2019). Also, CsCl purification may be used but it was observed by us to have more variation from batch-to-batch than sucrose purified virus. The protocol has been tested and observed to work with echovirus 1 and coxsackieviruses A9 and B3 suggesting its wide applicability for enteroviruses and probably for picornaviruses in general.
  5. Buffer/solution of your interest
    In our paper we used a wide spectrum of concentrations of different ions and albumin to study the virus priming and opening. As an example, DPBS solution supplemented with 0.01% BSA resulted in high amount of intermediate virus particles.
  6. SYBR Green II RNA gel stain (Invitrogen; ThermoFisher Scientific, catalog number S7564 ; stored at -20 °C)
  7. RNase A, 10 mg/ml (ThermoFisher Scientific catalog number EN0531 ; stored at -20 °C)
  8. 150 mM NaCl solution
  9. Ice

Equipment

  1. Pipette with a volume range including 100 µl
  2. 8-channel multipipette with a volume range including 50 µl, or similar (optional)
  3. Perkin Elmer 2030 Multilabel Reader Victor X4 (or similar fluorescence plate reader with suitable filter options)

Software

  1. Perkin Elmer 2030 Manager
  2. Microsoft Excel

Procedure

  1. Sample preparation
    1. Place the 96-well plate on ice to cool down.
      Note: You can use a metal plate on top of ice to make the surface even and the plate easy to handle.
    2. Cool down the solution(s) of interest on ice.
    3. Make 1:10 dilution from the 10,000x SYBR Green II stock solution. You can use ddH2O or the solution of interest to make the dilution. If the solution of interest is very exactly defined, then preferably use it for the dilution to keep the conditions unchanged (as ddH2O will change/dilute the solution). Avoid exposing the SYBR Green II to bright light.
    4. Pipette 450 µl of the solution of interest into two 1.5 ml tubes. You can have many different solutions of interest in one 96-well plate measurement. i.e., many pairs of 1.5 ml tubes. Up to seven different solutions of interest can be applied into one 96-well plate (see Figure 1 for example).


      Figure 1. Example of 96-well plate with 7 different solutions of interest (highlighted with different colors) and three replicates of virus with a background control without virus, all measured with and without RNase. R ows A and H, and columns 1 and 12 are left empty.

    5. Add 4.5 µl of the previously diluted SYBR Green II into both 1.5 ml tubes.
    6. Add 4.5 µl of the RNase A into one of the 1.5 ml tube in a final concentration of 10 µg/ml. Preferably use high concentrated stock to negate the buffer effect to the solution of interest. We use pre-diluted 1 mg/ml RNase stock in 150 mM NaCl, i.e., 1 µl of RNase in 100 µl of solution of interest. Add 4.5 µl of 150 mM NaCl solution without RNase A into the other tube to keep the ion concentrations identical.
    7. Mix the 1.5 ml tubes well and pipette 100 µl into four adjacent wells of the 96-well plate. Keep the plate on ice. Do not use rows A and H and columns 1 and 12 since the proximity of the plate edge might affect the measurement. See Figure 1 for an example. Avoid bubbles. Easiest way to avoid them is to use reverse pipetting for mixing and placing the liquid into the wells. In reverse pipetting, press the pipette cylinder all the way down before taking the adjusted volume inside the tip, resulting in extra volume inside the tip when mixing the solution. Then the volume only up to the first step is pipetted out in the well before sucking in the next volume etc. (For further details, see for example Good Laboratory Pipetting Guide https://assets.thermofisher.com/TFS-Assets/LSG/brochures/D16542.pdf.)
    8. Add 1 µg of virus into each of the three first wells of a solution and leave the fourth well without the virus to measure the background fluorescence of the solution. Somewhat lower virus amount, at least down to 0.5 µg, may also be applicable. However, if the solution of interest contains molecules that cause background fluorescence (such as BSA in our case) the signal-to-noise ratio might be low and the results less reliable. Also, preferably use a concentrated virus stock so that adding of the virus in a buffer will not change the chemical concentrations too much. On the other hand, too concentrated stock may add to the pipetting error. Optimal virus stock concentration would be around 1 µg/µl, i.e., 1 µl of the virus stock would be added per well.
    9. Mix the wells. Avoid bubbles. Easiest way is to use 8-channel multipipette and reverse pipetting.
    10. Keep the plate on ice, cover with aluminium folio and transfer to the fluorescence plate reader.
    11. Sample preparation in short:
      1. Take 450 µl of solution of interest in two 1.5 ml tubes, tube A and tube B. Keep the tubes on ice.
      2. Add 4.5 µl of pre-diluted 1,000x SYBR Green II in both tubes A and B.
      3. Add 4.5 µl of RNase A from 1 mg/ml stock only into tube B to a final concentration of 10 µg/ml.
      4. Mix both tubes carefully and transfer 100 µl of the solution into four adjacent wells from each tube, i.e., 8 wells in total.
      5. Pipette 1 µg of virus into 3 first wells transferred from tube A and leave the fourth well without virus. Do the same for the wells transferred from tube B that has the RNase A.
      6. Mix well and avoid air bubbles. Protect from light, keep on ice and transfer to the measurement device.

  2. Fluorescence measurement
    1. Prepare a measuring protocol using appropriate filters to measure SYBR Green II. With the Perkin Elmer 2030 Multilabel Reader Victor X4 use CW-Lamp Filter F485 and emission filter F535. Use CW-Lamp energy of 14,592 (or similar value around 15,000) and counting time of 1 second. Use measurement height with “user defined” -option of 13 mm and Plate type of “Generic 8x12 size plate”. Save the protocol for later use.
    2. Mark the wells to measure, measure each plate 90 times (Using 90 repeats and two-minute gap between the repeats results in total of 3-hour measurement. To use some other total measuring time adjust the field of “measure each plate” accordingly) and delay between repeats 0 s. Pre-run the protocol and time how long one round of measurements takes. To measure each well every two min, subtract the time taken by one round of measurements from 120 s and place the result into “delay between repeats” -field. For example, if the time to measure the wells in your experiment takes 30 s, subtract this from 120 s and place the resulting 90 s into “delay between repeats”. In this way the plate reader waits 90 s after the first round of measurements before starting to take the next ones and, as a result, every well is measured exactly every 2 min. Note, that when changing the amount of wells in your measurement, the “delay between repeats” needs to be adjusted accordingly.
    3. Pre-heat the plate reader to +37 °C or to any other temperature you wish to measure in. The plate reader takes some time to heat up, so do this before starting to prepare the samples and the plate.
    4. Place the previously prepared plate inside the plate reader with the lid on, but without the aluminium folio.
    5. Start the protocol.

  3. Data handling
    1. Export the data into Excel.
    2. Subtract the fluorescence background of each solution at each time point from the fluorescence of the same solution with the virus. Do this for all three replicates.
    3. Calculate average of the three replicates. Calculate the standard deviation and the standard error of the mean.
    4. Plot the data into a graph.

Data analysis

After plotting the graphs, first of all the results with the same solution with and without RNase should be compared. Fluorescence without the RNase presents the fluorescence originating from the empty and expanded particles. Fluorescence with the RNase presents the fluorescence originating from the expanded particles and the difference between the conditions with and without RNase originates from the empty capsids. Also, a measurement in virus storage buffer, or in another stabile environment for the virus, should be performed to verify the stability status of the unmodified virus, which should result in low amount of fluorescence. After comparing the measurements with and without RNase in one solution, one can also compare the results between different solutions. At least the proportion of different virus states can be compared. See Figure 2 as an example of measurements monitoring stable virus (only small increase in fluorescence), virus priming (increased fluorescence and only small decrease of signal with RNase treatment) and virus opening (increased fluorescence but loss of fluorescence after RNase addition).
  When comparing the absolute fluorescence values, one must remember that the fluorescence potency of SYBR Green II might be somewhat affected by the surrounding conditions. In order to verify the proportional share of different states of the virions, other methods are recommended such as negative staining with TEM or cryo-EM.


Figure 2. Example of three cases: protective conditions where the virus stays more or less unchanged (black curves), priming conditions where the virus mainly converts to primed and expanded intermediate form (blue curves), and opening conditions where the virus releases its RNA (red curves). For each condition, the fluorescence below the dotted line originates from the expanded particles since the RNase cannot enter inside the primed or intact particles, and the fluorescence from between the solid and dotted lines originates from the externalized genomes as the RNase abolishes the fluorescence.

Notes

Depending on the reproducibility of your virus purification, different batches of purified viruses might show different degree of virus priming and opening. Especially, we observed CsCl purified virus batches to have more variability than sucrose purified. Unpurified culture supernatants contain several contaminants that probably add into background fluorescence and lower the quality of the outcome and they might have also too low a virus concentration. For the reasons mentioned above, always verify the usability of a new batch of virus with some well known controls.

Competing interests

There are no competing interests by the authors of this article.

References

  1. Myllynen, M., Kazmertsuk, A. and Marjomäki, V. (2016). A novel open and infectious form of echovirus 1. J Virol 90(15): 6759-6770.
  2. Ruokolainen, V., Domanska, A., Laajala, M., Pelliccia, M., Butcher, S. J. and Marjomaki, V. (2019). Extracellular albumin and endosomal ions prime enterovirus particles for uncoating that can be prevented by fatty acid saturation. J Virol 93(17).
  3. Tuthill, T. J., Groppelli, E., Hogle, J.M. and Rowlands, D. J. (2010). Picornaviruses. In: Johnson, J. (Ed.). In: Cell Entry by Non-Enveloped Viruses. Current Topics in Microbiology and Immunology, vol 343. Springer, Berlin, Heidelberg.

简介


[摘要 ] 病毒需要打开,即,脱壳,以释放其基因组的有效复制和翻译。特别是对于诸如肠病毒之类的非包膜病毒,导致脱壳的线索鲜为人知。病毒的状态之前已经使用负染色主要见于通过透射电子显微镜,低温电子显微镜,X射线晶体学或梯度分离(在塔海尔综述等人。,2010,Myllynen 等人,2016年,Ruokolainen 等。,2019)。但是,监控您 由于缺乏检测病毒粒子动态变化的方法,涂层受到了限制。在这里,我们提出了一种实时基于荧光的协议,其中在脱壳的不同阶段检测到病毒基因组(RNA)在体外,而RNA仍然是颗粒内部的是已经实际RNA释放之前被扩展,并且当所述RNA具有从病毒颗粒中完全释放出来。我们的方法允许探索各种分子因素如何促进或抑制病毒的脱壳。

[背景 ] 在我们先前的研究中,我们发现感染性中间回声病毒1颗粒使RNA嵌入染料SYBR Green II进入病毒颗粒(Myllynen 等,2016)。可以观察到这是荧光的增加,并且记录的荧光不易被RNase消化(Myllynen 等,2016)。利用此信息,我们开发了一种实时方法,用于在荧光光谱中使用SYBR Green II染料和RNase监视病毒的打开。通过添加SYBR Green II和引发脱膜的因素,并观察SYBR Green II的增加,我们可以在+37°C或其他感兴趣的温度下,以96孔板的形式实时跟踪颗粒的开窗状态。荧光。将RNase加到平行孔中使我们能够监测RNA从病毒粒子中释放的程度,因为RNase可以轻易地从溶液中降解RNA,但不能从病毒粒子内部降解(RNAse 不能通过病毒颗粒内部的小孔进入,Myllynen et al。,2016)。对于完整的病毒颗粒,仅观察到非常少量的荧光。例如,在我们先前的研究中,补充有0.01%不含脂肪酸的BSA的DPBS溶液产生了大量的中间回声病毒1 颗粒。有关更多详细信息,请参见原始出版物(Ruokolainen 等,2019)。

关键字:小RNA病毒, 肠道病毒, 脱壳, 基因组, RNA, RNA酶, SYBR Green II, 荧光光谱

材料和试剂


 


1. P IP Ë TTE提示      


2. Sarstedt 96孔板(Sarstedt ,目录号:83.3924)(或类似产品)      


3. 1.5毫升管      


4. 纯化的病毒库存(1       μ克病毒的每个测量井; 储存在-80 °C)


肠病毒的纯化可以使用5-20%或10-40%的蔗糖梯度进行,并在原始出版物中有详细描述(Ruokolainen 等,2019)。同样,可以使用CsCl 纯化,但是我们观察到批次之间的差异比蔗糖纯化的病毒更大。已对该协议进行了测试,并观察到它可与回声病毒1以及柯萨奇病毒A9和B3一起使用,这表明该协议广泛适用于肠病毒,也可能适用于微小RNA病毒。


5. 您感兴趣的缓冲/解决方案      


在我们的论文中,我们使用了各种浓度的不同离子和白蛋白来研究病毒的启动和开启。例如,补充有0的DPBS溶液。01%BSA导致大量的中间病毒颗粒。


6. SYBR Green II RNA凝胶染料(Invitrogen;ThermoFisher Scientific,目录号S7564;存储在-20°C)      


7. RNase A,10 mg / ml(ThermoFisher Scientific目录号EN0531;在-20 °C 储存)      


8. 150 mM NaCl溶液      


9. 冰      


 


设备


 


移液器的体积范围包括100 µl
8通道多管移液器,体积范围包括50 µl或类似体积(可选)
Perkin Elmer 2030多标签读取器Victor X4(或类似的荧光板读取器,带有合适的过滤器选项)
 


软件


 


Perkin Elmer 2030经理
微软Excel
 


程序


 


样品制备
放置96 - 冰孔板降温。
注:哟u可以使用的金属板冰的顶部,让即使是表面与板容易处理。


在冰上冷却感兴趣的溶液。
用10,000x SYBR Green II储备溶液进行1:10稀释。您可以使用ddH 2 O或目标溶液进行稀释。如果目标溶液非常精确地定义,则最好将其用于稀释以保持条件不变(因为ddH 2 O将改变/稀释溶液)。避免将SYBR Green II暴露在强光下。
吸取450 µl目标溶液到两个1.5 ml试管中。一次96孔板测量中,您可以拥有许多感兴趣的解决方案。一世。e。,许多双1.5毫升试管。可以在一个96孔板中最多使用7种感兴趣的解决方案(例如,见图1)。
 


D:\ Reformatting \ 2020-2-7 \ 1902920--1327 Visa Ruokolainen 809471 \ Figs jpg \图1.jpg


图1 。96孔板的示例,其中有7种不同的目标溶液(以不同的颜色突出显示)和3次重复的病毒复制(带有无病毒的背景对照),均使用RNase和不使用RNase进行测量。ř OWS A和H,且将列1和12被保留为空。             


 


将4.5 µl先前稀释的SYBR Green II加入两个1.5 ml试管中。
添加4.5 微升的RNase A到1.5ml试管中的一个以10微克/毫升的最终浓度。最好使用高浓度原液,以消除对目标溶液的缓冲作用。我们在150 m M NaCl中使用预稀释的1 mg / ml RNase储备液。e。,在100 µl目标溶液中加入1 µl RNase。加入4.5微升150mM NaCl的溶液而不RN 一知的到另一个管,以保持离子浓度相同。
混合1.5 ml试管孔,然后将100 µl移液到96孔板的四个相邻孔中。将盘子放在冰上。请勿使用A和H行以及1和12列,因为板边缘的邻近可能会影响测量。见图1 FO 为r的例子。避免气泡。避免它们的最简单方法是使用反向移液进行混合,然后将液体放入孔中。在反向移液中,将移液器筒完全向下压,然后再将吸头内的体积调整为一定值,从而在混合溶液时在吸头内产生额外的体积。然后,体积仅达到第一个步骤中吸取了很好的下卷吸前等(有关详细信息,请参阅例如良好的实验室移液指南https://assets.thermofisher.com/TFS- 资产/ LSG /brochures/D16542.pdf。)
在溶液的三个第一孔中各加入1 µg病毒,并在第四孔中不加病毒,以测量溶液的背景荧光。较低的病毒量(至少低至0.5 µg)也可能适用。但是,如果感兴趣的溶液包含引起背景荧光的分子(例如本例中的BSA),则信噪比可能会很低,结果可靠性也会降低。另外,最好使用浓缩的病毒原液,以使将病毒添加到缓冲液中不会过多改变化学浓度。另一方面,过于集中的库存可能会增加移液误差。最佳病毒原种浓度约为1 µg / µl,即。e。,则每孔将添加1 µl病毒储备。
混合井。避免气泡。最简单的方法是使用8通道多移液器和反向移液器。
将板放在冰上,盖上铝制对开纸,然后转移到荧光板读取器上。
样品准备简述:
在两个1.5 ml试管,试管A和试管B中取450 µl目标溶液。将试管放在冰上。
在试管A和B中加入4.5 µl预稀释的1,000x SYBR Green II 。
从1 mg / ml的储备液中仅向管B中加入4.5 µl RNase A,至终浓度为10 µg / ml。
仔细混合两个试管,然后将100 µl溶液从每个试管中转移到四个相邻的孔中,即。e。,总共8口井。
吸取1 µg病毒到从试管A转移的3个第一个孔中,使第四个孔中没有病毒。对从具有RNase A的试管B转移的孔进行相同的操作。
充分混合,避免气泡。避光,放在冰上并转移到测量设备。
 


荧光测量
使用适当的过滤器准备测量规程以测量SYBR Green II。对于Perkin Elmer 2030多标签读取器Victor X4,使用CW灯泡过滤器F485和发射过滤器F535。使用14,592(或类似值约15,000)的CW-Lamp能量,计数时间为1秒。使用“用户定义”的测量高度-选项为13 mm,板类型为“通用8x12尺寸板”。保存该协议供以后使用。
标记要测量的孔,测量每个板90次(使用90次重复,两次重复之间的间隔为2分钟,总共需要3小时的测量。要使用其他总测量时间,请相应地调整“测量每个板”的字段)重复之间的延迟为0 s。预运行协议并确定一轮测量需要多长时间。要每两分钟测量一次孔,请从120 s中减去一轮测量所需的时间,然后将结果放入“重复之间的延迟”字段中。例如,如果您在实验中测量孔的时间需要30 s,请从120 s中减去该时间,并将得到的90 s放入“重复之间的延迟”。这样,读板器在第一轮测量后等待90 s,然后开始进行下一次测量,因此,每两分钟精确测量一次。请注意,在更改测量中的孔数时,需要相应地调整“重复之间的延迟”。
将读板器预热至+37°C或您要测量的任何其他温度。读板器需要一些时间进行加热,因此在开始准备样品和板之前先进行加热。
将先前准备好的印版放在有盖的印版读取器内部,但不要放入铝制折页。
启动协议。
 


数据处理
将数据导出到Excel。
从每个相同时间的溶液的荧光减去病毒在每个时间点的荧光背景。对所有三个重复执行此操作。
计算三个重复的平均值。计算平均值的标准偏差和标准误差。
将数据绘制到图形中。
 


数据分析


 


绘制图表后,首先应比较使用和不使用RNase的相同溶液的结果。没有RNase的荧光呈现出源自空的和膨胀的颗粒的荧光。带有RNase的荧光表示源自膨胀颗粒的荧光,带有和不带有RNase的条件之间的差异源自空衣壳。另外,应在病毒存储缓冲液中或在该病毒的另一个稳定环境中进行测量,以验证未修饰病毒的稳定性状态,这将导致荧光量降低。在一种溶液中比较有无RNase的测量结果后,还可以比较不同溶液之间的结果。至少可以比较不同病毒状态的比例。参见图2所示为监测稳定病毒(仅少量增加荧光),病毒引发(增加荧光并且仅使用RNase处理仅减少信号)和病毒开放(增加荧光但添加RNase后荧光损失)的测量示例。


  在比较绝对荧光值时,必须记住,SYBR Green II的荧光强度可能会受到周围条件的影响。为了验证病毒体不同状态的比例份额,建议使用其他方法,例如用TEM或cryo-EM进行负染色。


D:\ Reformatting \ 2020-2-7 \ 1902920--1327 Visa Ruokolainen 809471 \ Figs jpg \图2.jpg


图2.三种情况的示例:病毒或多或少保持不变的保护性条件(黑色曲线),病毒主要转化为引发和扩展的中间形式的引发条件(蓝色曲线)以及病毒释放RNA的开放条件(红色曲线)。对于每种情况,由于RNase不能进入已涂底漆的或完整的颗粒内部,因此虚线下方的荧光来自膨胀的粒子,而当RNase消除荧光时,来自实线和虚线之间的荧光源自外部基因组。


 


笔记


 


根据病毒纯化的可重复性,不同批次的纯化病毒可能会显示不同程度的病毒启动和打开。特别是,我们观察到CsCl 纯化的病毒批次比蔗糖纯化的变异性更大。未经纯化的培养物上清液包含几种污染物,这些污染物可能会增加背景荧光并降低结果质量,并且它们的病毒浓度也可能太低。由于上述原因,请始终使用一些众所周知的控件来验证新一批病毒的可用性。


 


致谢


 


我们感谢于韦斯屈莱大学的Jane和Aatos Erkko 基金会和博士学校的资助,以及原始论文的作者(Ruokolainen et al。,2019)为引入该协议的工作做出了贡献。我们还要感谢Marjomäki 组的所有成员为工作中使用的不同肠病毒的生产和纯化做出的贡献。


 


利益争夺


 


本文作者没有相互竞争的利益。


 


参考文献


 


Myllynen,M.,Kazmertsuk,A。和Marjom ä KI,V。(2016)。回声病毒的一种新颖的开放性和感染性形式1. J Virol 90(15):6759-6770。
Ruokolainen,V.,Domanska,A.,Laajala,M.,Pelliccia,M.,Butcher,SJ和Marjomaki,V.(2019)。细胞外白蛋白和内体离子会引发肠道病毒颗粒脱膜,可通过脂肪酸饱和来预防。J Virol 93(17)。
塔海尔,T. J.,Groppelli ,E.,霍格尔,JM和罗兰兹,D. J.(2010) 。 小核糖核酸病毒。于:Johnson ,J.(E d。)。在:非包膜病毒进入细胞。《微生物学和免疫学的最新话题》,第343卷。施普林格,柏林,海德堡。
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引用:Ruokolainen, V., Laajala, M. and Marjomäki, V. (2020). Real-time Fluorescence Measurement of Enterovirus Uncoating. Bio-protocol 10(7): e3582. DOI: 10.21769/BioProtoc.3582.
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