Name: real-time quaking-induced conversion assay for detection of cwd prions in fecal material
Uploaded: 2017-09-29T15:00:00.000+00:00
Duration: 9 min 50 s
Description: Watch this Scientific Journal Video about Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material at JoVE.com

我们感谢 Dr. Caughey (NIH 岩石山实验室) 提供培训和 cervid PrP 细菌表达质粒。SG 得到加拿大研究主席计划的支持。我们承认这项研究的资金来自加拿大基因组、艾伯塔朊病毒研究所和艾伯塔省农业和林业, 通过基因组艾伯塔省和卡尔加里大学支持这项工作。我们承认来自玛格丽特的动物研究基金会的研究补助金。

1. 使用粪便材料的 RT QuIC <ol> <li> cervid 粪便提取物的制备 <ol> <li> 使粪便匀浆增加1克粪便物质到10毫升的粪便萃取缓冲液 (20 毫米磷酸钠, pH 7.1, 130 毫米氯化钠,0.05% 吐温 20, 1 毫米 PMSF 和1x 完整蛋白酶抑制剂, 无 EDTA), 以最后浓度为 10% (w/v)。均匀化缓冲器可在使用前准备, 并存储在-20 和 #176; C. </li>
		<li> 质粪丸 (1 克) 和缓冲 (10 毫升) 在管 (例如, gentleMACS M 管) 使用 dissociator 与预先设定的程序, 从制造商的蛋白质为1分钟室温。重复此步骤二至三次, 直到粪便样本完全均匀地在缓冲液中. </li>
		<li> 密封管与膜, 并把它们放在一个摇摆平台或旋转振动筛 1 h 孵化室温. </li>
		<li> 在室温下离心 1.8万 x g 的管子5分钟. </li>
		<li> 收集含有蛋白质提取物的清, 并将其分成1.5 毫升的管子。等分可存储在-80 和 #176; C 用于进一步的应用程序. </li>
	</ol> </li> <li> 在粪便提取物中的 PrP Sc 的浓度 注: 为了集中 CWD 朊在粪便提取样品中, 从而提高了 QuIC 检测的灵敏度, 增加了蛋白质沉淀步骤到协议。
	<ol> <li> NaPTA 降水方法 </li> <li> 添加250和 #181; 10% (w/v) 的 n-十二烷基酸 (sarkosyl) 到1毫升的粪便蛋白提取物, 以使 sarkosyl 的最终浓度为 2% (v/v). </li>
		<li> 用膜密封含有样品的管子, 在37和 #176 上孵育; C 为30分钟在恒定的震动下在 1400 rpm 在 thermomixer. </li>
		<li> 将粪便蛋白提取物和 sarkosyl 的混合物调整为含磷钨酸和170毫米氯化镁的 10% (w/v) 的股票溶液, pH 7.4, 以获得最终浓度为 0.3% (w/v) 钠磷钨酸的样品. </li>
		<li> 在37和 #176 中孵育样品; C 为2小时, 在 400 rpm 时持续晃动. </li>
		<li> 14 和 #176 离心样品; C 为30分钟在 15800 x g. 小心地移除清. </li>
		<li> 清洗颗粒溶液在洗涤缓冲器 (10 毫米三氯, pH 7.5, 100 毫米氯化钠, 0.5% 海卫一-X 100, 10 毫米 EDTA, 0.5% 钠胆酸 (w/v), 和 0.1% sarkosyl (w/v)). </li>
		<li> 14 和 #176 离心样品; C 为15分钟在 15800 x g. 小心地移除清. </li>
		<li> 重100和 #181 中的颗粒; L (粪便蛋白提取物的原体积 1/10) QuIC 稀释缓冲液. 
		注: RT-QuIC 稀释缓冲液 (20 毫米磷酸钠, pH 6.9, 130 毫米氯化钠, 0.1% SDS (瓦特/v) 和 1 X N2 补充) 是在使用前准备的。10毫升的总体积通过0.2 和 #181 过滤; m acrodisc 注射器过滤器使用注射器, 然后储存在-20 和 #176; C 直到使用. </li>
		<li> 将 NaPTA 处理过的样品保存在-20 和 #176 ° C, 直到使用 QuIC 测定. </li>
	</ol> </li> <li> RT QuIC 检测 <ol> <li> 准备一个新的 10 mM 的库存解决方案 (在10毫升的双蒸馏 H 2 O 中0.032 克 t). </li>
		<li> 将1:10 的日 t 库存溶液稀释为双蒸馏 H 2 O 以使其最终浓度为 1 mM, 通过0.2 和 #181 过滤它; m acrodisc 注射器过滤器使用注射器. </li>
		<li> 解冻鼠标重组 PrP (rPrP, aa 23-231) 基板 (10 和 #181; 每 RT QuIC 反应) 存储在-80 和 #176; C 在冰上. </li>
		<li> 加载500和 #181; rPrP 基板的 L, 一次为 100 kDa 大小排除过滤器管内和离心机在 1.4万 x g 为5分钟在室温 (一管/过滤器可以使用两次). </li>
		<li> 将洗基板转换为无菌1.5 毫升管, 并将其保持在4和 #176; C 直到使用. </li>
		<li> 解冻 RT-QuIC 稀释缓冲器, 存储在-20 和 #176; C. </li>
		<li> 在 RT QuIC 稀释缓冲区中稀释种子 (粪便蛋白提取物): 
		未稀释的样品: 使用未稀释的粪便蛋白提取物 稀释 2 x 10 -1 稀释 2 x 10 -2 稀释 2 x 10 -3 </li> 准备 QuIC 反应混合物的库存解决方案: <li> 磷酸盐缓冲盐水 (PBS): 5X 氯化钠: 2 M 双蒸馏制备的氯化钠库存溶液 (H 2 O. 
		edta: 100 毫米的 edta 库存溶液, 用双蒸馏 H 2 O. </li>
		<li> 在使用所有解决方案之前 (步骤 1.3.8), 请通过0.2 和 #181 分别筛选每个解决方案; acrodisc 注射器用注射器过滤, 并在室温下保持不育. </li>
		<li> 根据样品的数量 (98 和 #181, 每个样品的反应量和每个样品的四次复制) 准备一个总体积的 RT-QuIC 反应混合物, 以确保有足够的混合物为所有的反应。
		<ol> <li> 使用15毫升管来制备 RT-QuIC 反应混合物 (20 毫米磷酸钠, pH 值 6.9, 300 毫米氯化钠, 1 毫米 EDTA, 10 和 #181; M T, 10 和 #181; rPrP 基板, 和双蒸馏水, 以调整 rPrP 最终浓度的反应) 使用股票溶胶utions. </li>
			<li> 将所有的解决方案混合在一起使用过滤器提示的吸管移上下。尽量避免使用涡流. </li>
			<li> 将混合物倒入50毫升一次性移水库. </li>
			<li> 使用多通道吸管将98和 #181 的 QuIC 反应混合物注入到96井光学底板的每一个井中. </li>
		</ol> </li> <li> 添加到每个 RT-QuIC 反应2和 #181; 未稀释或10倍稀释的粪便蛋白提取物。在四复制中测试每个示例. 
		注意: 在每个实验中, 粪便样本的序列稀释 (从未稀释到 2 x 10 -3 ) 从 CWD 阴性和经验证的 CWD 阳性动物分别用作阴性和阳性对照. </li>
		<li> 在42和 #176 的平板读卡器上进行孵化前, 用密封胶带将板材封住; C 为 25 h, 重复循环1分钟双轨道晃动 (700 rpm), 在整个孵化过程中休息1分钟. </li>
		<li> 定义荧光测量设置: 
		励磁: 450 nm 发射: 480 nm 底部读取, 闪烁次数:20 手动增益: 1000 (取决于机器) 集成时间:20 和 #181; s </li> <li> 在 25 h 的末尾从板读取器中卸下板. </li>
		<li> 在 3000 x g 处向下旋转3分钟以避免井间的潜在污染. </li>
		<li> 从盘中取下封条. </li>
		<li> 通过加入90和 #181, 制备新的96井板; QuIC 反应混合物中含有新鲜基质和新鲜荧光染料的 1.3.1, 如1.3.6 节所述. </li>
		<li> 传输10和 #181; 从第一个板块的每一个井到新准备的96井板. </li>
		<li> 在步骤1.3.13 中描述的步骤后, 将新板密封并继续进行 RT QuIC 检测, 以增加50小时。50 h 的这一附加步骤将使总反应时间为 75 h. 
		注: 所有的 QuIC 检测程序均在生物安全柜下进行. </li>
		<li> 收集数据。
		<ol> <li> 读取并记录每个井的 Th T 荧光信号每15分钟. </li>
			<li> 使用数据分析软件收集总周期数据, 并将份反应的平均值转移到电子表格中. </li>
			<li> 绘制数据的平均值。x 轴对应于 RT-QuIC (h) 的反应时间, y-axis 对应于相对荧光单位 (RFU)。每个曲线对应于一个已知样本的稀释. </li>
			<li> 在每个实验中, 通过计算负控制的最高平均值和当前出版的文献中描述的5标准偏差来标定阈值 41 . 
			注意:所有跨越定义阈值的复制都被认为是正数。如果四复制中至少有两个跨越阈值, 则该示例被视为正值. </li>
		</ol> </li> </ol> </li> </ol> 2。重组朊蛋白的纯化 (rPrP) <ol> <li> 细菌种群的生长及 rPrP 的表达 注: 大肠杆菌 ( 大肠杆菌 ) 菌株罗塞塔 (DE3) 转换为矢量 pET-24 编码用小鼠 PrP (残留物 23-231) 制备 rPrP。
	<ol> <li> 解冻在冰上与 pEt-24 革命 (23-231) 转换的罗塞塔 (DE3) 甘油库存. </li>
		<li> 条纹 LB (Luria Bertani) 板, 其最终浓度为50和 #181; g/毫升, 在一夜之间孵育37和 #176; C 孵化器。确保盘子是倒置的, 以避免在含有细菌的固体培养基中产生湿气凝结。准备两个板块, 以确保至少有一个两个板块的增长. </li>
		<li> 准备1升的 LB 和高压釜, 使其无菌. </li>
		<li> 用1毫升卡那霉素 (50 毫克/毫升) 和1毫升氯霉素 (34 毫克/毫升) 补充 1 L 的无菌 LB 培养基. </li>
		<li> 使用一次性接种循环来接种3毫升含有卡那霉素和氯霉素的 LB 培养基, 以选择每个板块的一个菌落. </li>
		<li> 将 mini-cultures 放置在37和 #176 的恒温箱中; C 以 225 rpm 为 5-6 h 的恒定晃动. </li>
		<li> 将 mini-cultures 添加到 Autoinduction 的原始 1 l 媒体的其余部分, 以及用于诱导蛋白质表达的 Express 系统 1. </li>
		<li> 将养殖置于2升烧瓶中, 或更大, 在37和 #176 的孵化器中; C 以 200 rpm 为 20-24 h 的恒定晃动. </li>
		<li> 将 1 L 区域性拆分为 4 x 250 毫升离心管. </li>
		<li> 在室温下向下旋转含有 3750 x g 的培养物的管子20分钟. </li>
		<li> 丢弃上清液并在50毫升离心管中收集细菌颗粒, 然后在-80 和 #176 ° C 下冷冻, 直到进一步处理。由此产生的颗粒已重3至4克, 以获得良好的蛋白质产量. </li>
	</ol> </li> <li> 准备包含体 <ol> <li> 将冻结的颗粒 (3 到4克) 解冻到37和 #176; C 分钟. </li>
		<li> 在-80 和 #176 中再一次冻结10分钟的颗粒; C 和再次解冻。重复这一步骤, 冻结和解冻两次. </li>
		<li> 重1X 和 #160 中的细菌颗粒; 裂解试剂 (如 独来独往主组合) 由移彻底。使用5毫升的试剂为1克的细菌颗粒。确保完全质混合. </li>
		<li> 室温下在摇杆上孵育匀浆20分钟. </li>
		<li> 在室温下离心 1.6万 x g 的匀浆20分钟. </li>
		<li> 通过仔细地将清扔掉, 从而丢弃它. </li>
		<li> 质在前一步骤中使用的1X 裂解试剂体积相同的颗粒. </li>
		<li> 室温下在摇杆上孵育匀浆15分钟. </li>
		<li> 添加足够数量的 1:10 (0.1x) 裂解试剂, 以获得40毫升的总匀浆量。由倒装混合, 以确保质好. </li>
		<li> 离心匀浆在 7900 x g 为 15 min 在4和 #176; C. </li>
		<li> 通过仔细地倒掉上清. </li>
		<li> 重40毫升的0.1x 裂解试剂中的颗粒. </li>
		<li> 离心匀浆在 1.6万 x g 为 15 min 在4和 #176; C. </li>
		<li> 将上清液浇注后仔细地丢弃, 并将含有包含体的小球储存在-20 和 #176; C 直到进一步处理. </li>
	</ol> </li> <li> 蛋白质纯化 <ol> 在室温下解冻包含体. <li>
		<li> 溶解在14毫升8米胍盐酸 (38 克胍 0.1 m 那坡县 4 pH 8.0, 并填补与 H 2 O 到50毫升的悬浮的解冻包涵体; 不要调整最终解决方案的 ph 值) 溶解蛋白质. </li>
		<li> 确保通过移上下质好. </li>
		<li> 室温下在摇杆上孵育裂解物50分钟 </li>
		<li> 制备18克镍 NTA 树脂珠子 (18 克3至4克细菌颗粒); 用100毫升的水冲洗, 用真空和100毫升0.22 和 #181; m 瓶顶过滤器。
		<ol> <li> 将18克 NTA 树脂珠放入50毫升的管中, 并添加足够数量的变性缓冲 (100 毫米磷酸钠、10 mm 三、ph 8.0、6米胍、ph 8), 使最终容量达50毫升. </li>
			<li> 在室温下, 在一个摇杆上平衡50分钟的变性缓冲器中的珠子. </li>
		</ol> </li> <li> 在 1.6万 x g 离心包含体裂解物5分钟以去除不溶性碎片. </li>
		<li> 将上清液加入平衡珠并丢弃颗粒. </li>
		<li> 在室温下将混合料放在摇杆上40分钟, 以使蛋白质与镍 NTA 树脂结合. 
		注: 镍螯合珠被用来纯化 PrP 的镍亲和性。PrP 的多组氨酸 N 端区使与镍 (II) 的亲和力, 这使得蛋白质绑定到镍离子没有任何他的标签. </li>
		<li> 用清水冲洗 FPLC, 清除两条线 (A 和 B). </li>
		<li> 运行变性缓冲器 (100 毫米磷酸钠, 10 毫米三, ph 8.0, 6 米胍, ph 8) 通过两行 (A 和 B), 以使系统成为质数 (该列尚未附加) </li>
		<li> 将树脂加载到柱上。确保尽量减少气泡. </li>
		<li> 将该列附加到 FPLC, 并从100% 变性缓冲 a 和0% 的复性缓冲 B (100 mm 磷酸钠、10 mm 三、pH 8.0) 开始, 到0% 个变性缓冲和100% 复用缓冲的线性渐变。这种渐进的转变是运行在0.75 毫升/分钟的流量为240分钟, 并需要适当的折叠 PrP. 
		注: 在色谱纯化过程中, 变性 PrP 首先在树脂上缓慢折叠, 应用复性缓冲的线性梯度代替变性缓冲液。此步骤还删除了 chaotropic 胍-HCl. </li>
		<li> 确保100% 复用缓冲缓冲区继续流经该列, 在0.75 毫升/分钟的额外30分钟. </li>
		<li> 冲洗线与水, 然后洗脱缓冲 (100 毫米磷酸钠, 10 毫米三, 500 毫米咪唑, pH 5.8) 绕过该列。这将清除 AKTA 系统中已被洗脱缓冲器取代的变性缓冲. </li>
		<li> 洗蛋白在2毫升/分钟运行线性梯度40分钟从100% 复性缓冲 B 和0% 洗脱缓冲 a 在第一到0% 复性缓冲和100% 洗脱缓冲. 
		注: 在洗脱缓冲液中高浓度的咪唑会与 PrP 和 #39 相结合, 对镍 (II) 具有约束力。主要蛋白质峰值应开始洗约1/3 的方式通过梯度。
		<ol> <li> 观察色谱为 OD 280 nm 增加. </li>
		</ol> </li> <li> 只收集在大峰值的中心包含蛋白质的管. 
		注: 2 毫升的洗分数分别收集到15毫升管中. </li>
		<li> 用大约1/3 卷 (1 毫升) 的透析缓冲液 (10 毫米磷酸钠, pH 5.8) 立即将管内所含的蛋白质稀释. </li>
		<li> 立即将管子放在冰上. </li>
		<li> 将管中包含的洗蛋白池. </li>
		<li> 将蛋白质放入透析盒 (分子量切断 10 kDa). </li>
		<li> 将磁带放在 pre-chilled 透析缓冲液 (3.6 升) 中, 2 小时在4和 #176; C, 然后在一夜之间将磁带放入新的透析缓冲液 (3.6 l) 中。使当然, 透析缓冲是在不断搅拌. </li>
		<li> 通过0.2 和 #181 透析后过滤蛋白; acrodisc 注射器过滤器. </li>
		<li> 测量蛋白质浓度, 使用一个评估蛋白检测试剂盒. </li>
		<li> 如果需要, 集中或稀释, 将蛋白质浓度调整到0.3 毫克/毫升. 
		注: 通过 SDS 页和马斯亮蓝染色确认纯度. </li>
		<li> 在离心管中等分1毫升的蛋白质, 并立即存储在-80 和 #176; C 直到进一步使用, 如 RT QuIC 协议所述. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。

QuIC 以前被用来检测 CWD 朊的尿液和粪便提取物的口腔感染白尾鹿和骡鹿38。本手稿中所示的系统是一种适应的 RT-QuIC 法。另外的步骤被纳入 "经典" RT QuIC 测定, 以提高检测和灵敏度的 CWD 朊在粪便材料的感染动物。
在粪便提取物中, 检测灵敏度低, 使我们对 QuIC 协议进行了改进。为了实现敏感的体外检测粪便中的朊, 为了去除干扰朊病毒转化和/或传播的成分, 提高检测的灵敏度, 在样品制备中添加了关键步骤。在 QuIC 和 NaPTA/sarkosyl 治疗方案中纳入基质置换对检测前临床和 CWD 感染的麋鹿的播种活性和提高粪便提取物中朊病毒转化率的检测范围至关重要.
NaPTA 沉淀是一种常见的技术, 通常伴随着 sarkosyl 提取分离和浓缩 PrPSc到一个可检测的水平。NaPTA 是已知的最好沉淀 prpSc在 prpC 43 , 而 sarkosyl 是一种洗涤剂已知, 以促进朊病毒转换在低浓度的细胞自由系统44。与此相一致, 使用 NaPTA 和 sarkosyl 的组合可能会生成更高的纤维聚合体的收率, 如45、46、47之前所示。这一方法已被纳入前 QuIC 检测, 以检测外周 CWD 朊成功的标本, 如纯化唾液39和全血40。我们的研究提供了第一个证据, 将 NaPTA/sarkosyl 纯化纳入粪便样品制备的协议, 使 CWD 朊病毒检测的 RT-QuIC。此外, 通过这种方法, 我们能够减少 rPrP 基质在 CWD 阴性粪匀中的自发转化, QuIC 试验。
提出了一种潜在的机制来解释基底置换的效果30。在第一轮反应中 (在添加新鲜基质之前), 只有少量的种子加入到 RT-QuIC 反应中。虽然只有一部分的 rPrP 被纳入到种子的反应, 其余的基板可以通过与反应板孔的墙壁互动, 以形成 non-amyloid 聚合体, 或被改变为不太容易被转换的形式, 见ded RT-QuIC 产品而不是原始种子。因此, rPrP 的加入率是缓慢的, 而纤维的形成在滞后阶段是不可探测的。由于在滞后阶段生长的种子 RT QuIC 产品最终被拉长, 以达到 "快速组装阶段", 朊可以通过震动或横向加入较小的集料迅速放大。在这一阶段, 添加到反应中的新鲜基质容易地融入到种子产品中, 而不是形成不的聚合体。在我们的协议中加入基板替换步骤是一种提高灵敏度的修改;这是很有用的检测朊病毒种子的样本与一个非常低数额的 PrPSc从前临床动物和/或含有抑制化合物。
使用 RT-QuIC 法而不是 PMCA, 这是第一个体外蛋白折叠扩增试验所描述的21, 有几个优点。在 PMCA 中, 管在 sonicator 内的水浴中孵育和声;与位于中心48中的管道相比, 位于 sonicator 外围的管道显示的放大效果较差。QuIC 系统的颤抖似乎更容易控制。使用96井格式是 RT QuIC 的真正优势, 但是, 由 Moudjo et al.开发的 mb PMCA49,50显示了类似的优点, 但在 PMCA 中仍然较少使用。
作为基质, RT-QuIC 使用重组 rPrP 进行转化;相比之下, PMCA 在大多数情况下使用正常的脑匀浆。此外, 在 RT-QuIC 中, 种子和基底之间不需要序列同源51-53。
因子的存在是必要的朊病毒复制在 PMCA 和结果的产品是传染性的。然而, 在 RT-QuIC 检测, 放大 PrP 是不传染的。在体外放大试验中, 可能是由于 PrP 的自发转换而引起的假阳性反应的出现, 这是一个反复出现的问题.因此, 本研究采用的分析和条件, 以尽量减少此类干扰, 最大限度地提高种子 rPrP 转换和自发转换之间的差异。
总之, NaPTA/sarkosyl 治疗可以去除粪便中的测定抑制剂, 减少自发转化。有趣的是, 这种治疗并没有妨碍 precipitatedPrPSc的播种活动。此外, 当基体置换同时采用时, 检测灵敏度明显提高。

朊病毒疾病或传染性海绵状脑 (东京) 是神经退行性疾病, 包括克雅氏病 (CJD) 在人类, 牛海绵状脑病 (疯牛病) 在奶牛, 病在绵羊和山羊, 和慢性浪费疾病 (CWD) 在 cervids 1,2。TSEs 的特点是有明显的海绵状的外观和神经元在大脑中的损失。根据 "仅蛋白质" 假说, 朊主要由 prpsc ("sc" 为病) 3, 错误型的主机编码的细胞朊蛋白, prpC。prpSc将 prpC转换为在ᵦ中丰富的构象4,5,6 , 它可以作为种子来绑定和转换其他 prpc分子。新生成的 PrPSc分子被合并成一个生长的聚合物7,8 , 它会分解成更小的寡聚物, 从而导致传染性核的数量增加。PrPSc易于聚合, 并部分抗蛋白酶9、10。
CWD 影响野生和养殖的麋鹿 (鹿黄花), 骡鹿 (Odocoileus 野驴), 白尾鹿 (WTD;Odocoileus 鹑)、驼鹿 (驼鹿驼鹿) 和驯鹿 (驯鹿 tarandus tarandus) 11,12,13。它被认为是最具传染性的朊病毒疾病与横向传播所青睐的 cervid 相互作用和环境持续性的传染性14,15。不同于其他朊病毒的疾病, PrPSc的积累和传染性被限制在大脑, 在 CWD 这些也发现在外围组织和体液, 如唾液, 尿液和粪便16,17,18。
免疫组织化学被认为是 CWD 诊断的金标准, 以检测 PrPSc分布和海绵状病变1920。ELISA 和更罕见的情况下, 西方印迹也用于 CWD 诊断。因此, 目前的朊病毒疾病诊断主要是基于检测朊在死后组织。CWD 的宰前诊断可通过扁桃体或直肠肛粘膜相关淋巴组织 (RAMALT) 活检获得;然而, 这个过程是侵入性的, 需要捕获的动物。因此, 使用容易接近的标本, 如尿和粪便, 将是一种切实可行的方法, CWD 朊病毒检测。然而, 那些排泄物港口相对地低浓度的朊低于目前诊断方法的检测限度。因此, 需要一个更敏感和高吞吐量的诊断工具。体外转换系统, 如蛋白质折叠循环放大试验 (PMCA) 21, 淀粉样播种试验, 和 real-time 颤抖诱导转换 (RT QuIC) 化验22,23,24是非常强大的工具, 可以利用 prpsc的蔓延能力模仿体外朊病毒转化过程, 从而放大少量 prp 的存在sc至可检测级别25 ,26。然而, RT-QuIC 方法利用了在β板二级结构中富集的转化产物可以具体地结合 thioflavin t (Th t) 的事实。因此, 重组 PrP (rPrP) 在种子转化后生长成淀粉样纤维, 并通过测量荧光的时间 t 表示为相对荧光单位 (RFU), 从而可以实时检测。一旦被监测, RFU 可用于评估相对播种活动, 以及定量参数, 如滞后阶段。滞后阶段表示到达阈值所需的时间 (h), 在此期间, 反应的早期阶段的 rPrP 转换低于 Th t 荧光的检测限度。明显滞后阶段的结束, 伴随着足够的淀粉样核的形成 (成核/伸长), 发生时, T 荧光超过阈值水平, 并成为积极的。淀粉样纤维的生长可以实时检测, 样品中含有的初始 PrPSc或播种活动通过分割产生更多的种子来放大。这些种子反过来诱发淀粉样纤维生长的快速指数阶段。
由于这种检测方法能够检测出 prpsc 24的低至 1 fg, 因此高灵敏度可以通过检测 prpsc在各种外围组织、排泄物或其他种类的标本, 窝藏低水平的传染性。RT-QuIC 肯定提供比其他化验的优势, 其重现性, 实用性, 快速 (少于50小时) 和低成本相比, 生物鉴定。避免了 PMCA 中使用的超声等技术复杂性;此外, 它是在胶带密封的微, 最大限度地减少了每井的气溶胶污染的风险。井格式可以在同一实验中对多达96样本进行分析。为了应对 rPrP 在体外转换分析中出现的误报和自发转换的复发问题, QuIC 中的阈值 (截止) 的实现非常有用。事实上, 根据负控制的结果 (负样本的平均 RFU +5 SD 27), 建立一个基线, 从中可以进行正负样本之间的区分。对每个样本使用四复制可以帮助定义一个样本为正数, 当至少50% 的复制显示一个正信号时,即跨越截断28。种子和基质之间的同源性在 RT-QuIC 中不需要, 如在以前的研究中, 仓鼠 rPrP 被发现是一个比较敏感的基板与人类 PrP 的同源基底相比,vCJD种子和绵羊病种子反应29. 仓鼠-绵羊嵌合 rPrP 也被认为是一个比人类 rPrP 更适合的基质, 以检测人类变体 CJD 朊30。因此, 在这种方法中使用不同种类的 rPrP 基质是非常普遍的。这种检测方法已成功应用于几种朊病毒疾病, 如零星 CJD 31,32,33, genetic 朊病毒疾病 34,疯牛病35,36,37, 病23,36, 和 CWD 38,39,40,41, 42。研究使用经加工的脑脊液, 全血, 唾液, 尿作为种子在 RT QuIC 都成功地检测 PrPSc 38,39,40,41,42. 为了培养可能含有淀粉样蛋白形成抑制剂的血浆样品的检测能力, Orrú et al。(2011) 通过梳理 PrPSc沉淀 (IP) 步骤和 RT QuIC (称为 "增强 QuIC") 检测 (eQuIC), 制定了消除淀粉样蛋白形成的潜在抑制剂的策略。此外, 为了提高灵敏度, 在反应时间为 24 h 后采用了衬底置换步骤。最终, 低至 1 ag PrPSc可通过 eQuIC 30检测到。
为了净化粪便提取物, 清除粪便中可能的化验抑制剂, 从麋鹿在实验性口腔感染的前、临床分期采集的粪便样品均在含有洗涤剂和蛋白酶抑制剂的缓冲液中匀浆。粪便提取物进一步提交到不同的方法, 以集中 PrPSc在样品中利用蛋白质沉淀通过磷钨酸钠 (NaPTA) 沉淀。NaPTA 沉淀法, 首先由萨法尔et al.描述43, 用于将 PrPSc集中在测试示例中。NaPTA 与样品的孵化导致 prpSc而不是 prpC的优先沉淀。然而, 分子机制还不清楚。这一步骤还有助于遏制和防止 rPrP 的自发转化, 在某些情况下, 这种转换是被观察到的。最后, 以小鼠 rPrP (aa 23-231) 为基质, 对粪便萃取物进行了优化的 RT QuIC 测试, 并在协议中包括基片置换, 以提高检测的灵敏度。
研究结果表明, 这种改进的方法可以检测到极低浓度的 CWD 朊, 增加了粪便样品中的检测和特异性的灵敏度, 而不需要 NaPTA 沉淀和基质置换。这种方法可能适用于其他组织和体液, 可以很好地用于野生和圈养 cervids 的 CWD 监测。

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acrodisc seringe filters</td><td>PALL</td><td>4652</td><td></td></tr><tr><td>amicon Ultra-15 Centrifugal filter Unit</td><td>Millipore</td><td>UCF901024</td><td></td></tr><tr><td>BD 10 ml seringe</td><td>VWR</td><td>CA75846-842</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td></td></tr><tr><td>Corning bottle-top vacuum filters</td><td>Sigma-Aldrich</td><td>431118</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>gentleMACS M Tube</td><td>Miltenyi Biotec</td><td>130-093-236</td><td></td></tr><tr><td>Guanidine hydrochloride</td><td>Sigma-Aldrich</td><td>G4505</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>I9516</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Luria-Bertani (LB) broth</td><td>ThermoFisher Scientific</td><td>12780029</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>N2 supplement (100X)</td><td>ThermoFisher Scientific</td><td>15502048</td><td></td></tr><tr><td>N-lauroylsarcosine sodium salt (sarkosyl)</td><td>Sigma-Aldrich</td><td>ML9150</td><td></td></tr><tr><td>Nanosep centrifugal devices with omega membrane 100K</td><td>PALL</td><td>OD100C34</td><td></td></tr><tr><td>Nunc sealing tapes</td><td>ThermoFisher Scientific</td><td>232702</td><td></td></tr><tr><td>Parafilm M</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>Protease inhibitor tablet</td><td>Roche</td><td>4693159001</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Calbiochem</td><td>7910-OP</td><td></td></tr><tr><td>sodium phosphate</td><td>Sigma-Aldrich</td><td>342483</td><td></td></tr><tr><td>Sodium phosphate dibasic anhydrous</td><td>Sigma-Aldrich</td><td>S9763</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>Sodium phosphotungstate hydrate (NaPTA)</td><td>Sigma-Aldrich</td><td>496626</td><td></td></tr><tr><td>Thioflavin T</td><td>Sigma-Aldrich</td><td>T3516</td><td></td></tr><tr><td>Tris-Hydroxy-Methyl-Amino-Methan (Tris)</td><td>Sigma-Aldrich</td><td>T6066</td><td></td></tr><tr><td>Triton-100</td><td>Calbiochem</td><td>9410-OP</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P7949</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Commercial buffers and solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BugBuster Master Mix</td><td>Nogagen</td><td>71456-4</td><td></td></tr><tr><td>Ni-NTA superflow</td><td>Qiagen</td><td>1018401</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS) pH 7.4 (1X)</td><td>Life Technoligies</td><td>P5493</td><td></td></tr><tr><td>UltraPure Distilled Water</td><td>Invitrogen</td><td>10977015</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Standards and commercial kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Express Autoinduction System 1</td><td>Novagen</td><td>71300-4</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23227</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment setup&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AKTA protein purification systems FPLC</td><td>GE Healthcare Life Sciences</td><td></td><td></td></tr><tr><td>Beckman Avanti J-25 Centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Beckman rotor JA-25.50</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Beckman rotor JA-10</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>FLUOstar Omega microplate reader</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Sofware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MARS Data Analysis</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>GraphPad Prism6</td><td>GraphPad software</td><td></td><td></td></tr></tbody></table>

real-time quaking-induced conversion assay for detection of cwd prions in fecal material

RT QuIC 技术是一种敏感的体外无细胞朊病毒扩增法, 主要基于重组朊蛋白 (PrP) 基质的种子折叠和聚集, 以朊病毒种子为模板进行转化。RT QuIC 是一种新的高通量技术, 类似于 real-time 聚合酶链反应 (PCR)。淀粉样纤维生长的检测是基于染料 Thioflavin T, 这荧光在特定的相互作用与ᵦ片丰富的蛋白质。因此, 可以实时检测淀粉样蛋白的形成。我们试图建立一个可靠的非侵入性筛选试验, 以检测粪便提取物中的慢性 CWD 朊。在这里, 我们已经特别适应了 RT QuIC 技术, 以揭示 PrPSc播种活动的粪便 CWD 感染 cervids。最初, 我们准备的粪便提取物的播种活性在 RT-QuIC 中相对较低, 可能是由于粪便中的潜在化验抑制剂。为了提高粪便提取物的播种活性和去除电位测定抑制剂, 我们将粪便样品在含有洗涤剂和蛋白酶抑制剂的缓冲液中均匀化。我们还提交了样品到不同的方法, 以集中 PrPSc的基础上的蛋白质沉淀使用磷钨酸钠, 和离心力。最后, 通过优化的 RT QuIC 对粪便萃取物进行了测试, 其中包括在协议中对基体进行置换, 以提高检测的灵敏度。因此, 我们建立了一个 CWD 朊病毒播种活性的敏感检测方法, 在临床前 cervids 的粪便 QuIC, 这是一种实用的非侵入性 CWD 诊断的工具。

10% (CWD) 制备的粪提取物能够 QuIC 反应, 但检测灵敏度较低, 为27。使用一个特定的缓冲区, 粪便均匀化是一个关键步骤, 以避免高背景荧光在 RT-QuIC 反应与使用鼠标 rPrP 衬底, 而不是鹿 rPrP, 允许获得更具体的结果27。NaPTA 降水的增加减少了 rPrP 在 RT-QuIC (图 1) 中的自发转换, 而不抑制反应的播种活动的放大 (图 2)。虽然在 NaPTA 治疗中观察到了更好的放大效果, 但 CWD 阳性粪便样本的荧光信号仍然很低 (图 1)。此外, 一些样品的朊病毒放大率在低荧光水平时达到了恒定的高度。这表明了反应的饱和度, 这可能是由于 rPrP 的退化/变性或 off-pathway 聚合的形成而消耗了 rPrP 池30。为了进一步提高朊的放大度, 提高检测的灵敏度, 在该协议中加入了基片置换步骤。在 rt-QuIC 协议 (图 2) 中引入基板替换, 提高了灵敏度 (77%; 14 出18样本中的 rt-QuIC 是阳性) 和特异性 (100%; 在 rt-QuIC 中没有任何负控制) 检测 (参见表1从程et al.27). 最后, 所有这些步骤 (图 3) 都导致优化了一个可靠和敏感的协议, 用于 RT QuIC 检测 CWD 朊, 使用含有低传染性的标本。
在 RT-QuIC 反应的前25小时, 通过补充含有新鲜基质和新鲜荧光染料的反应缓冲剂, 引入基体置换。通过在协议中引入基片置换步骤, 将 RT-QuIC 反应时间从50小时延长到75小时。如图 2所示, 随着基板置换的合并, 观察到朊病毒放大的显著改善。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56373/56373fig1.jpg"> 
图 1: 用纯化的 CWD-阴性粪便匀浆对 RT-QuIC 种子中小鼠 rPrP 基质的自发转化率降低.感染麋鹿 (C181-006) 或骡鹿的粪便匀在粪便提取液中匀浆, 最终浓度为 10% (w/五)。为净化, 这些粪便匀处理和10次集中 NaPTA 沉淀。固化粪匀 (a), NaPTA 纯化和浓缩的形式 (b) 被稀释如所示, 用于种子份 RT QuIC 反应与小鼠 rPrP 作为基板。y-axes 显示相对 T 荧光单位, 轴描述反应时间。在 NaPTA 纯化粪便样品 (b) 中发现了自发转化的减少。来自程et al.的数据27.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56373/56373fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56373/56373fig2.jpg"> 
图2。用基质置换法改进粪便中 CWD 朊的检测。C051-05、C052-05) 口腔感染 CWD 朊的粪便匀经 NaPTA 沉淀纯化浓缩。NaPTA 处理的样品被稀释在 2 x 10-1到 2 x 10-3之间, 用于种子份 RT-QuIC 反应与小鼠 rPrP 基质。RT-QuIC 检测是在50小时的正常期间内进行的, 而不是基板替换 (a), 或者是在 75 h (b) 的延长潜伏期内合并基底置换。对于后者, 在第一 25 h QuIC 反应后引入基片置换。90% 的反应体积被删除, 取而代之的是新鲜制备的 RT-QuIC 反应混合物, 含有 rPrP 基板和 Th T。通过在协议中引入基片置换步骤, 将 QuIC 反应时间从50小时延长到75小时. 从程et al.中使用的数据27.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56373/56373fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56373/56373fig3.jpg"> 
图 3: 描述 PrP 的流程图Sc利用 RT QuIC 分析法从 CWD 感染 cervids 的粪便提取和种子活性及朊病毒的扩增.从 CWD 感染动物的粪便样品在粪便萃取缓冲液中均质, 提交到 NaPTA 沉淀法, 并经 RT QuIC 测定。后者是通过引入基板更换步骤来执行的。NaPTA 和基质置换步骤的加入导致了自发转化的减少和种子活性和朊病毒放大的强烈改善。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56373/56373fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material at JoVE.com

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

在这里, 我们提出一个协议来描述一个简单, 快速和有效的朊病毒放大技术, real-time 颤抖诱导转换 (RT-QuIC) 方法。

实时颤振诱发的粪便中 CWD 朊的转化检测

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

real-time-quaking-induced-conversion-assay-for-detection-cwd-prions

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.6K 次观看.  University of Calgary. 在这里, 我们提出一个协议来描述一个简单, 快速和有效的朊病毒放大技术, real-time 颤抖诱导转换 (RT-QuIC) 方法。

视频: Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

科研

免疫与感染

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

朊病毒蛋白质错误折叠循环扩增

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

医学

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

实时定量聚合酶链反应检测感染细胞培养中的登革热病毒 rna

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

狂犬病病毒在各种牛脑结构中的量化

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

生物学

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

一个定量聚合酶重组酶扩增法的发展与内部阳性对照

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

朊病毒安全实验室滑动测试

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

实时颤振诱发的粪便中 CWD 朊的转化检测

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