方法文章

朊病毒安全实验室拭子检测

DOI：

10.3791/67889

⸱

2025年2月14日

 ,  , 
1Department of Medical Microbiology and Immunology, School of Medicine, Creighton University

本文内容

摘要

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目前尚缺乏一种用于评估实验室环境中朊病毒污染常见区域及有效去污方法的检测手段。本文所述方案为实施实验室朊病毒安全拭子检测提供了基本操作原理，且可根据不同实验室的具体需求进行简便调整。

摘要

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医源性朊病毒疾病的传播曾发生于使用受污染的神经外科器械、移植材料以及职业性接触受朊病毒污染的实验工具。朊病毒通过模板化机制使正常的细胞型朊蛋白 PrPC 发生错误折叠，转化为错误折叠且具有致病性的 PrPSc 形式，从而引发疾病，且此类疾病 invariably 致命。减少医源性和职业性朊病毒传播具有挑战性。首先，朊病毒可结合并长期存在于各种表面。其次，朊病毒对灭活处理具有高度抗性。因此，在去污不充分的情况下，表面可能长期保持感染性。这不仅可能对从事朊病毒研究的实验室工作人员构成潜在的职业风险，还可能污染使用高灵敏度朊病毒扩增技术的实验室实验。本文所述的朊病毒安全实验室拭子检测方案，包括识别和记录实验室高使用频率区域的步骤、推荐的拭子对照以确保结果有效性、针对表面拭子检测阳性位点采取适当应对措施的流程、典型的朊病毒拭子检测结果，以及可能出现的假象结果。总体而言，朊病毒安全实验室拭子检测可作为更广泛的朊病毒安全计划的一部分，用于评估表面去污效果、监测公共空间的朊病毒污染情况，并实现朊病毒去污状态的文件化记录。

引言

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朊病毒病是一类致命的神经退行性疾病，目前尚无已知的治疗方法或治愈手段。朊病毒病由PrPSc引起，它是正常细胞型朊病毒蛋白PrPC的错误折叠且具有致病性的形式1,2,3,4,5。已知朊病毒病可影响人类及多种其他动物物种。其中一种人类朊病毒病——克雅氏病（Creutzfeldt-Jakob Disease, CJD）具有三种已知的病因：散发型、遗传型和获得型。获得型CJD可因意外传播（医源性和职业性）而发生，据认为这也是巴布亚新几内亚福雷人（Fore people）中库鲁病（Kuru）发生的原因6。

朊病毒的传播与受朊病毒污染的医疗器械及移植材料有关7,8,9,10,11,12,13,14,15,16,17。医源性克雅氏病（CJD）可通过血液、组织或受朊病毒污染的表面传播18,19,20。例如，在接受过脑电图检查的患者中，若所使用的电极曾用于处于CJD临床前期的个体，之后该个体死于CJD，则该患者可能发展为医源性CJD21。更近期的实验室职业暴露传播案例中，一名实验室工作人员在使用镊子处理感染羊源适应性BSE动物的脑切片时，因皮肤被刺伤而感染了朊病毒病22,23。此类传播情形可能发生在临床、实验室以及诊断实验室等处理朊病毒样本的环境中。

朊病毒对常见的消毒技术具有抗性，并可在物体表面长期存留并保持感染性24,25,26,27,28,29。乙醇、酚类清洁剂、过氧化氢、各种形式的辐射以及甲醛等常用消毒方法均不足以灭活朊病毒，导致物体表面仍具有感染性30,31,32,33,34,35,36,37。这些特性促进了朊病毒在医源性暴露和职业暴露过程中的传播。

环境朊病毒的检测方法最近才得以开发。通过环境拭子采样结合实时震荡诱导转化（RT-QuIC）技术，可评估环境表面以及实验室常见表面在消毒不彻底后残留的朊病毒感染性38,39,40,41,42。本文描述了该技术如何整合到更广泛的朊病毒安全防控体系中。总体而言，该方法可用于监控实验室依赖的消毒方案，调查并准确记录污染状况，从而通过减少交叉污染来确保实验结果的有效性；同时可用于评估共用空间的朊病毒污染情况，并根据常见污染区域对工作人员进行有针对性的再培训。

方案

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All procedures involving animals were approved and in compliance with the Guide for the Care and Use of Laboratory Animals by the Creighton University Institutional Animal Care and Use Committee.

NOTE: A schematic overview of the prion safety laboratory swipe test is shown in Figure 1.

1. Selection of swabbing sites and preparation for surface swabbing

  1. Identify and label appropriate surveillance areas for swabbing. Refer to the template provided for documentation and reference (Figure 2 and Supplementary Figure 1).
  2. Prepare two 1.5 mL microcentrifuge tubes for each of the identified areas. Add 250 µL of Dulbecco's phosphate-buffered saline to the first set of microcentrifuge tubes for use in step 3.2. Let the second set of microcentrifuge tubes remain empty for use in step 4.3.
  3. Retrieve foam-tipped swabs from storage in an area free of prion contamination.
  4. Prepare a squeeze bottle with Milli-Q water (MQ H2O) for use in step 2.1.

2. Positive and negative control swab preparation

  1. Prepare dilutions for positive and negative swab controls using relevant control types. Example: For positive control, prepare a 1% dilution of prion-infected brain homogenate in Dulbecco's phosphate-buffered saline (DPBS). For negative control, prepare a 0.1% dilution of uninfected (UN) brain homogenate (BH) from the same species as the positive control in DPBS.
  2. Using clean gloves, retrieve the appropriate number of swabs from the clean packaging and place the handle side down into a tube rack, taking care to space out foam swabs so that the tips do not contact other swabs or surfaces. For each control sample, prepare three swabs. Change gloves between each sample.
  3. Holding a clean foam swab by the handle, apply 50 µL of respective positive and negative control samples to foam swab tips. Make sure to apply to both sides of the swab tip to ensure complete absorption.
    NOTE: Always change gloves prior to removing a clean swab from the tube rack.
  4. Using scissors, cut off the excess handle of the swab (approximately ½ of the length) and place the swab into the preloaded microcentrifuge tube with the foam tip portion pointed down. Ensure that the foam tip is submerged in the DPBS and that the handle is cut adequately to close the lid of the microcentrifuge tube completely.
  5. Continue applying control samples after changing gloves until all samples have been applied.

3. Surface swabbing

  1. Holding a clean foam tip swab by the handle, prewet the foam tip with MQ H2O and shake off the excess. Place the moistened foam tip of the swab onto the area chosen for surveillance and swab the area back and forth approximately ten times while simultaneously rotating the tip of the swab on the surface.
  2. Using scissors, cut off the excess handle of the swab (approximately ½ of the length) and place the swab into the preloaded microcentrifuge tube with the foam tip portion pointed down. Ensure that the foam tip is submerged in the DPBS and that the handle is cut adequately to close the lid of the microcentrifuge tube completely.
  3. Discard gloves and place new gloves on before each respective swabbing site to minimize the probability of cross contamination.
  4. Repeat the procedure until all areas chosen for surveillance have been swabbed.

4. Swab extraction and vacuum concentration

NOTE: Turn on the vacuum concentrator 30 min prior to use to allow the instrument to warm up.

  1. Place microcentrifuge tubes into a circular tube rack. Place the microfuge tube rack into the cup horn sonicator water bath. Ensure the foam swabs in DPBS within the microcentrifuge tubes are below the surface of the water in the cup horn (the handle portion does not have to be submerged). Apply the following settings: 15 s total run time (5 s on, 5 s off) at ~75-85 watts.
  2. Following sonication, centrifuge the tubes for approximately 15 s to collect DPBS in the bottom of the tube prior to transfer.
  3. Using a P1000 pipette set to 250 µL, carefully collect all liquid (swab extract) from the bottom of the microcentrifuge tube and transfer into the corresponding microcentrifuge tube from the second, empty prelabeled set. Use the pipette tip to squeeze any excess liquid from the foam tip. Discard empty tubes containing swabs.
  4. Set the following settings on the vacuum concentrator: Temperature: 45 °C, Heat time: 15 min, Run time: 2 h, Vacuum: 5.1.
  5. Place the microcentrifuge tubes containing swab extracts into the vacuum concentrator, ensuring the tubes are balanced and all tube caps are open.
  6. Upon cycle completion, ensure that samples are completely concentrated (only the pellet remains). Store the pellets at -80 °C until utilized for RT-QuIC.
    NOTE: In some instances, additional vacuum concentration time may be required to ensure complete concentration. Remove all concentrated samples, leaving only the tubes that still contain liquid. Rebalance the remaining tubes within the concentrator and run at additional 1-h increments until samples are completely concentrated.

5. Preparation of swabbing controls for use in RT-QuIC

NOTE: The RT-QuIC controls should be performed prior to the assay of environmental swab extracts to ensure that contamination has not been introduced during the swabbing, extraction, or concentration procedures. For example layouts, see Figure 3 and Figure 4.

  1. Prepare an appropriate negative RT-QuIC plate control (e.g., uninfected brain homogenate [UN BH]) by diluting to the appropriate concentration in tissue dilution solution (N2-0.1%SDS/PBS). Prepare a positive plate control dilution by diluting prion-infected brain homogenate to a dilution of 10-3 in tissue dilution solution.
  2. Remove previously stored control swab extract pellets from -80 °C and resuspend each with 50 µL of MQ H2O by pipetting up and down approximately 10 times, followed by vortexing briefly. Allow samples to sit at room temperature (RT) while performing the remaining steps.
  3. Load 2 µL of specified plate controls, including negative plate controls (tissue dilution solution alone and uninfected brain homogenate) and a positive plate control (infected brain homogenate).
  4. Load 2 µL of each positive and negative swab extract technical replicate into a minimum of four replicate RT-QuIC plate wells.
    NOTE: If negative swabbing controls exhibit RT-QuIC seeding above a laboratory's standard for a positive determination, this would indicate that contamination had been introduced during the swabbing, extraction, and concentration process and that the swabbing experiment should be performed again

6. Preparation of samples for use in RT-QuIC

  1. Prepare an appropriate negative plate control (e.g., uninfected brain homogenate, uninfected lymph node, etc.) by diluting to the appropriate concentration in tissue dilution solution (N2-0.1%SDS/PBS). Prepare positive plate controls by diluting infected brain homogenate to a dilution of 10-3 in tissue dilution solution.
  2. Remove previously stored swab extract pellets from -80 °C and resuspend with 20-50 µL of tissue dilution solution (depending on the suspected amount of contamination) by pipetting up and down approximately ten times, followed by vortexing briefly. Allow samples to sit at RT while performing the remaining steps.
  3. Load 2 µL of specified plate controls, including negative plate controls (tissue dilution solution alone and uninfected brain homogenate) and a positive plate control (infected brain homogenate) into the RT-QuIC plate wells.
  4. Load 2 µL of each swab extract into four replicate wells.

7. RT-QuIC analysis and results

  1. Perform RT-QuIC according to individual laboratory protocols (optional protocols38,40).
  2. Define the parameters required for determining the positivity of a sample (see representative results, Figure 5).
    NOTE: These parameters are defined by each lab.
  3. Record results in the provided table and take appropriate action based on laboratory best practices (Table 1 and Supplementary Figure 2).

结果

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阳性与阴性结果的文字描述（包括阳性和阴性培养板及拭子对照）

在监测拭子采样过程中，需包含阴性对照拭子，以监测在拭子采样、提取和浓缩过程中可能引入的朊病毒污染。针对特定月份监测所进行的第一块RT-QuIC板，应包含阳性和阴性拭子对照。成功的阴性对照不会跨越阳性荧光阈值（图6A），该结果表明实验操作过程中未引入污染。成功的阳性对照拭子提取物在给定样本的所有重复孔中均应显示阳性接种信号（阳性对照拭子被仓鼠适应性传染性水貂脑病高毒株（HY TME）脑组织匀浆污染）。包含阳性对照的系列稀释样本，可用于确定特定实验中朊病毒检测的灵敏度（图6A）。

在检测表面拭子提取样本时，若表面未显示出高于预设阳性荧光阈值的扩增信号，则可视为不含朊病毒（图6B）。相反，被朊病毒污染的表面拭子提取物会表现出高于阳性荧光阈值的扩增能力，尽管其最大扩增点比率（MPR）和达到荧光信号的时间可能与同批实验中的阳性对照孔存在差异（图6B）。该方法评估消毒效果的能力可通过经漂白剂处理的朊病毒污染表面得到体现：这些表面样本不再引发RT-QuIC扩增反应（图6B）。

重要的是，尽管本实验室将阳性样本定义为在至少一半的重复孔中达到设定的阳性荧光阈值的样本，但每个实验室都必须建立自身的标准。淀粉样蛋白形成速率（RAF）和荧光出现时间也可用于帮助建立实验室特有的阳性判定阈值。

我们观察到一些表面污染物的检测结果，其荧光信号超过了阳性荧光阈值，但动力学曲线发生改变，且荧光出现时间明显长于阳性对照样本（图6C）。这些结果应谨慎解读，因其可能由表面存在的灰尘或残留化学物质引起。这些发现凸显了保持实验室整体清洁的重要性，以及区分真正阳性与假阳性信号的标准的必要性。

固体和液体生物危害性废物的处置应遵循相关机构现行的生物危害物处理指南。常见的朊病毒消毒方法包括使用氢氧化钠、次氯酸钠（漂白剂）处理，或在134 °C下高压灭菌18分钟43,44,45,46。

朊病毒安全检测流程：位点识别、拭子取样/超声处理、RT-QuIC分析、记录保存。
图1：朊病毒安全实验室拭子检测示意图。 请点击此处查看此图的放大版本。

实验室布局示意图；关键设备位置和工作台面，对实验室工作流程至关重要。
图2：样本拭子采样点布局。 请点击此处查看此图的放大版本。

稀释系列和平行对照实验的板布局，显示样本制备及拭子对照。
图3：包含朊病毒安全实验室擦拭检测的拭子对照样本实验设计。 （A）阴性与阳性拭子提取物对照的样本布局。（B）阴性和阳性拭子提取物对照应使用 50 µL H2O 重悬。将重悬后的拭子提取物加入组织稀释液中，制备 10 倍稀释液。请点击此处查看该图的放大版本。

微生物实验平板示意图；表面拭子样本布局；重悬说明。
图 4：朊病毒安全实验室擦拭测试中表面拭子提取物的示例实验设计。 （A）表面拭子提取物的样本布局。（B）表面拭子提取物样本应使用 20 µL 组织稀释液进行重悬。请点击此处查看该图的放大版本。

朊病毒实验室安全检测流程图；RT-QuIC结果，去污决策流程。
图5：每月一次的朊病毒安全实验室拭子检测。用于解读检测结果并确定适当应对措施的流程图。请点击此处查看此图的放大版本。

微量滴定板检测稀释结果，蛋白质聚集的MPR和ThT荧光图谱。
图6：典型的表面拭子采样实验。（A）表面拭子对照板，包含三份阴性拭子提取物对照（DPBS、未感染仓鼠脑匀浆 10-4 和 10-10）和三份阳性拭子提取物对照（HY 脑匀浆 10-3）。（B）代表性的拭子提取物结果，来自实验台面、玻璃和不锈钢（S.S.）表面，在污染前采样、接种 HY 10-3 后采样，以及污染表面经漂白剂处理后采样。（C）未感染脑匀浆 10-4、HY 脑匀浆 10-3 以及覆盖有一层细尘膜的实验台面拭子提取物的荧光轨迹比较。A 和 B 面板的阴性对照板包括空白对照（组织稀释液）和未感染仓鼠脑匀浆 10-4。B 面板额外增加了未感染脑匀浆 10-4 和阳性对照板 HY 脑匀浆 10-3。阳性荧光阈值（以红色虚线表示）设定为 2。报告的最大点比率（MPR）为微孔板读数仪测得的最大荧光值除以初始荧光读数。每个数据点代表特定样本类型的单个技术重复孔。数据以均值 ± 标准差表示。请点击此处查看该图的放大版本。

表1：样本拭子采集位点月度记录表。 请点击此处下载该表格。

补充图1：拭子采样位点布局模板。 请点击此处下载该文件。

补充图2：拭子采样点月度记录表。 请点击此处下载该文件。

讨论

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所述的朊病毒安全拭子检测方法可用于加强现有的朊病毒安全措施。该方法可用于监测朊病毒实验室空间和设备，以及共用的实验室区域，以发现潜在的朊病毒污染。重要的是，该方法可被调整用于测试实验室特定的消毒技术，以验证被朊病毒污染表面的去污效果。由于不同朊病毒株对消毒方法的敏感性不同，该方法可确认这些消毒技术对当前实验室实验的有效性，例如氢氧化钠或次氯酸钠（漂白剂）处理43,44。

该方法的关键步骤包括确定合适的拭子采样位点，以获取高频率接触区域的样本，同时监测可能涉及实验下游交叉污染的区域。此外，实验室应使用与特定实验室或诊所常规操作相匹配的阳性和阴性对照。例如，若实验涉及啮齿类动物朊病毒，则阳性和阴性对照应与该物种一致。最后，应持续更新并系统整理监测数据，以便易于识别阳性结果的变化趋势，从而在特定区域持续出现阳性结果时，及时采取干预措施并对实验室人员进行再培训。

本方案的一个局限性是可能产生错误的RT-QuIC结果。鉴于RT-QuIC具有高度敏感性，实验台面上残留的去垢剂、盐类及其他物质可能会影响反应结果39。因此，考虑到这一现象，保持实验表面不含有干扰RT-QuIC的物质（如残留盐分、去垢剂和灰尘）将是有利的。随着RT-QuIC技术方法的不断改进，我们预计当前RT-QuIC的许多局限性将得到解决。因此，我们建议最终使用者密切关注相关文献，以充分利用RT-QuIC技术的最新进展。

对于研究朊病毒的实验室而言，该方法的一个关键优势在于能够最大限度地减少可能影响敏感扩增检测结果的污染。剖检工具在重复用于后续剖检前通常会进行消毒处理。本文所述的拭子取样方法可用来评估剖检工具上是否残留有朊病毒传染性，从而可能影响后续实验结果。该方法可为实验提供额外的严谨性，以排除组织中检测到朊病毒是源于剖检工具污染的可能性。

披露

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J.C.B. 和 Q.Y. 是一项关于朊病毒表面拭子技术的专利申请的发明人。

致谢

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本工作由克雅氏病基金会的资助项目支持。资助方在研究设计、数据收集和解释，以及决定提交论文发表等过程中均未发挥任何作用。

材料

本文使用的材料清单
姓名公司目录编号评论
Fisherbrand  PurSwab 泡沫拭子Fisher brand目录号 #14-960-3E
Milli-Q IQ 7005 超纯水系统MilliporeSigmaQ7005T0C
微型离心机，6000 rpmSouthern LabwareMLX-306
Omega 酶标仪BMG LabtechFLUOstar Omega 酶标仪
Q700 超声破碎仪QSonicaQ700-110
重组仓鼠朊病毒蛋白MNPROMNPROtein-Hamster叙利亚仓鼠，氨基酸 90-231
Savant speedvac Thermo ScientificSPD1030-230
二氧化硅纳米球（50 nm）nano Composix SISN50-25M

参考文献

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