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...

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>Materials</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Commercial buffers and solutions</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Standards and commercial kits</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment setup</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sofware</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 (<strong class="...

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

Research

JoVE Journal

Immunology and Infection

13.5K 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

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 ...

科研

免疫与感染

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

用于狂犬病诊断的泛源病毒实时RT-PCR

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Although stool culture for Campylobacter is imprecise, it is still considered the gold standard for identification. Methods to determine the limit of detection and survival in transport media of C. jejuni in human stool are described and compared with a new immunoassay with better accuracy.

人类粪便标本中弯曲杆菌Jejuni在运输介质中确定检测和生存极限的培养方法

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician ...

Fecal (micro) RNA Isolation

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs from fecal samples of animal and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The advantages of this optimized protocol in the miRNA isolation are capabilities of isolating highly purified RNA products with additional washing steps described, increased quantity of RNA obtained with an improved method in the resuspension of sample, and important tips of decontamination. One limitation is the inability to process and purify larger sample of more than 200 mg as these sample sizes would cause a difficulty in the clear formation of the interphase. Consequently, the large sample size may contaminate the aqueous phase to be extracted as described in the protocol with organic matters that affect the quality of RNA isolated in the end. However, RNA isolates from a sample of up to 200 mg are sufficient for most of downstream analyses.

Fecal micro RNA Isolation

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

粪便（微量）RNA分离

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...