我们诚挚地感谢萨宾Reckinger优秀的技术援助和支持。

1.准备<ol><li> 样品采集 注意:可用于单个或合并肌肉样品的磁性搅拌器方法。 <ol><li>从适当的好发部位，并以适当量9收集样品。请参阅该国要求或信息和通信技术，世界动物卫生组织或ISO的建议。 <ol><li>对于欧盟，例如，采取从隔膜支柱1g的样品在过渡到国内的猪的强壮有力的一部分。确保所有的肌肉样本不含所有的脂肪和筋膜。 </li></ol></li><li>如果舌头测试，删除之前测试的表面层。 </li><li>请注意，如果样品被冻结，因为大多数旋毛虫不抗冻和死亡后解开，是消化不太耐倾向于坚持容器壁，从而降低检测灵敏度。 注意:如果冻结，加倍为L样本量东和增加沉降时间（见下文）。 </li></ol></li><li> 消化液的制备 <ol><li>添加25％的盐酸（16±0.5毫升），以2升46预热自来水 - 48℃到3升的玻璃烧杯中。过低的温度会导致消化不完全;过高的温度停用胃蛋白酶的酶学性质。 </li><li>放置一个搅拌棒的烧杯中，并搅拌一预热板溶液（46 - 48℃）。 </li><li>添加10±0.2克胃蛋白酶（1:10,000 NF，1:12500 BP公司，2000 FIP）到酸性溶液中。 注意:胃蛋白酶的质量是最重要的和酶的活性必须由生产者进行认证。对于实验室安全原因，并保持胃蛋白酶活性，增加消化液的组件的顺序必须坚持。 </li></ol></li><li> 肌肉样品制备 <ol><li>砍在搅拌机或粉碎机百克肌肉样本。为了方便BLEN丁，加2毫升预热水样品和以最大速度融合为2 - 3秒。 </li><li>第二混合前，打开搅拌机和混合碗缘缘的底部，并重复交融的顶级传送任何肌肉样本。继续以2连发混合 - 3秒，直到肉没有明显的碎片依然存在。 注:太少混合，可能会导致消化不完全，而过多的混合可以在样品6损坏旋毛虫存在。 </li></ol></li></ol> 2.程序<ol><li> 肌肉样本消化 <ol><li>传送1升消化流体成圆筒。转移绞肉放入烧杯中，并在消化流体用勺子或叉子传播。彻底用500mL的预热消化流体进3升烧杯中漂洗搅拌器盖子，叶片和搅拌器碗。 注:可导致SENS的损失不完全漂洗itivity。 </li><li>用铝箔覆盖烧杯，并保持44一恒定温度 - 46℃，用一温度计监测。 </li><li>炒出的消化液30分钟，无飞溅创建了深刻的漩涡，直到肉颗粒消失。根据测试肉（野生动物如肉）的类型，增加消化时间直至最多60分钟。 </li></ol></li><li> 过滤和消化流体的沉降 <ol><li>以确定未消化的组织的量，调整刻度为零，在使用前称量筛，并注意其重量。筛应清洁，没有任何组织的粒子，这可能阻碍旋毛虫到达沉降漏斗。检查分液漏斗垂直平整。 </li><li>消化后，小心地倒入通过180微米筛消化液到分离玻璃漏斗。分离玻璃漏斗的宽度不应bË长度大于55％，使良好的幼虫沉淀。请注意，以避免任何溢出。 </li><li>为了避免灵敏度降低，彻底冲洗玻璃烧杯中，并从一个挤压洗涤瓶用约200毫升自来水的筛和漂洗水转移到沉淀玻璃漏斗。小心仔细冲洗3升烧杯中的边缘。提起筛子，彻底筛下冲洗的面积。 </li></ol></li><li> 未消化的组织量的确定 注意:由于在该方法的执行错误，肌肉组织可以保持在筛上，以及筋膜和结缔组织这是难消化。未消化的组织的量在第一10分钟沉淀步骤（见2.5）来测定。这允许大约30分钟的筛干，残留水会影响未消化的组织所确定的权重。 <ol><li>广场上规模纸巾，并权衡与undiges筛特德组织。 </li><li>从未消化组织筛子过滤重量减去前筛的重量。如果起始样品重量的不超过5％保留在筛（ 即 5克/ 100克原料）在消化过程被认为是令人满意的。 </li><li>如果未消化的组织的量超过5％时，重复使用新鲜肌肉样品的测试。如果剩余的组织主要是由未消化的肌肉组织的和新鲜样品不可用，再次消化未消化遗体和除了原始样品检查。 </li></ol></li><li> 消化液的沉淀 <ol><li>保持过滤消化流体在分液漏斗中30分钟。如果不受干扰，在旋毛虫会沉淀在漏斗的底部。 </li><li>如果使用冷冻的样品，增加沉降时间为最多60分钟。 </li><li>一旦沉积完成后，完全打开活塞分液漏斗中，让至少40消化液毫升迅速跑出来，放入量筒（80毫升，如果肌肉样品已经先前冻结）。 </li><li>首先，让一半的流体的进入气缸，然后在相反的方向完全打开活塞，以允许10毫升消化流体的通过。最后，在另外10毫升相反的方向打开活塞。此过程可确保旋毛虫不会被困在水龙头。 注意:需要足够的速度，以避免残留在分液漏斗中的幼虫，降低灵敏度。 </li></ol></li><li> 气缸中的消化流体的沉降 <ol><li>离开气缸中的沉淀10分钟，以允许幼虫沉积物。通过用移液管抽吸，而不会干扰10mL的沉积物除去30毫升上清液。 </li><li>以减少样品中的组织的纤维的量，30毫升的自来水中添加的沉积物和离开另一个10米英寸重复，直到液体是明确的。 </li><li>除去上清液，转移至10毫升洗净泥沙，以网格化培养皿或幼虫计数盆地。冲洗用10mL自来水筒，然后将水添加到样品中。 注意:在样品中作为大量碎片能够防止旋毛虫 （ 图1）的标识，如果需要的话应进行进一步的洗涤步骤，。 </li></ol></li><li> 镜检 <ol><li>使用trichinoscope或立体显微镜具有可调强度的子级透射光源在一个15至20倍的放大倍率的洗涤步骤后，立即以检查样品。 </li><li>浇样品放入网格培养皿后，检查由电网盘/盆系统网格。如果没有发现犯罪嫌疑人的结构，轻轻移动在网格培养皿或幼虫计数盆地流体以循环的方式让任何旋毛虫拉rvae，这潜在地忽略，在培养皿的中心积聚。重复检查。 </li><li>如果发现可疑的结构，提高放大倍数40 - 100X。从盘/盆用移液器除去旋毛虫 ，并与90％的乙醇的管收集。 </li><li>完整的菜已经被审查后，重复上述步骤，从盘/盆的轻轻摇动开始。重复该过程至少三次或直到没有更多的幼虫被找到。 </li><li>计数幼虫的数目。关联到肌肉组织的量测试和本为每克（LPG）的幼虫。 </li><li>如果太多的幼虫存在于消化流体以确定幼虫数可靠地，返回包含幼虫量筒中的流体，从挤压洗瓶用自来水冲洗，并留下幼虫沉降至底部。 </li><li>一个10分钟沉降时间后，降低上清液到规定体积（ 例如10毫升）中。以确定确切体积，转移上清液到新的筒用吸管。 </li><li>通过剧烈摇晃液均匀暂停幼虫。在摇动运动，加入20微升悬浮幼虫12滴到培养皿。 </li><li>检查显微镜每下降。确定在240微升的幼虫数和删除的最高和最低计数。推断幼虫的数目，以上清液的总体积（ 例如 ，10毫升）。 注意:立体显微镜的质量是最重要的为旋毛虫的识别。 </li><li>当幼虫在管通过吸移管收集，仔细检查幼虫不粘附在移液管壁而被转移到管中。 注:从合并的样品阳性结果旋毛虫必须跟踪从池中回原籍的尸体。在这里，池大小应逐步减少直至正面胎体鉴定者tified。样本大小也可以在这个过程中增加以增加灵敏度。其中集体样品的检查产生一个正的或不确定的结果，另外20克样品从每个猪服用。从五头猪各20 g样品汇集并使用所描述的方法检测。以这种方式，从20组的五头猪样本将被检查。当一个合并样品中的五头猪检测旋毛虫 ，将20g样品从该组中的个别猪收集并直到检测原点的正胎体每个单独检查。 </li></ol></li></ol>

<ol>
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作者宣称，他们没有竞争的经济利益。

虽然磁力搅拌器方法可以容易地进行，而不是严格遵守的技术细节导致灵敏度不足，从而危及消费者的健康。关键控制点已经凸显在上面的文字。此外，使用适当的设备是为测试的成功结果至关重要。所有船只应作出的涂有硅，以减少对表面幼虫附着玻璃和枪头。 消化方法的灵敏度取决于在肌肉幼虫密度和肌肉样品的测试的量。为3的幼虫密度- 5液化石油气肌肉组织的，100％的灵敏度被报道，但低于1 LPG的灵敏度下降至40％，13。取决于所测试的宿主动物种类，试验的灵敏度可通过增加样品的使用量得到改善。第i在野生动物nfection负担通常较低，因此大尺寸的样品使用14。好发部位也宿主动物的不同而不同。这里，一些肌肉组织的下消化率如舌需要被考虑在内，这也可导致更大的样本在尺寸15。 此外，要明白，磁力搅拌器方法不包括内部控制是重要的。因此，从采样质量保证管理的文档是必不可少的，以获得准确的和精确的结果。实验室性能检测旋毛虫肌肉样品中的质量应在能力测试每个分析师经常参与进行监测。 该方法的进一步限制是由显微镜肌幼虫的最终识别阶段是高度主观和HEAvily依赖于幼虫形态的研究分析师的知识。 绕过这个问题的一个有趣的替代方法是磁力搅拌器方法/过滤器上分离之后幼虫检测通过胶乳凝集试验。不过，根据欧盟法规此方法仅视为等同于家猪的而不是动物，它们在感染风险较高的肉类的测试，如野猪4。用单克隆抗体幼虫抗原的识别使得测试更加客观，但否认实验室确定每克肉16的幼虫数的可能性。 磁力搅拌器方法的进一步变化包括机械辅助合并样本消化法/沉淀技术，机械辅助合并样本挖estion方法/过滤器上分离的技术，并为最多至35g技术的合并样品的自动消化方法。这些技术都是基于肉和旋毛虫的可视化及数量的人工消化，但在用于过滤和沉降步骤的设备不同。 分离出的幼虫可以进一步确定在通过分子测试（ 如多重PCR）17种的水平。根据欧盟法律，所有的阳性样品必须转发国家参考实验室或EURLP为旋毛虫决心。

属旋毛虫寄生线虫可在全球范围内的食肉动物和杂食的哺乳动物，鸟类和爬行动物的横纹肌进行检测。这些人畜共患线虫可以从猪，马及人的时候生的或半生肉和肉类衍生产品，以及野生动物摄入。此人畜共患可以是在人类特征在于特异病征性体征和症状， 例如腹泻，发烧，眶周水肿和肌痛和可能的并发症，如心肌炎，血栓栓塞性疾病和脑炎1一种严重的疾病。 旋毛虫是野生和家养动物旋毛虫感染的最普遍的病原体，导致大部分人类感染全世界。在欧洲，北非和西非，和西亚， 旋毛虫britovi是人类感染的另一个来源。此外，另外十个类群较少通常报告为致病<html>人类疾病和剂在世界的不同地区被发现，通常在野生动物2。除了野生动物如野猪，熊，海象和獾，猪代表了人类感染的全球最重要的来源。 为防止旋毛虫病的最好方法就是从吃生肉产品不要和煮任何肉类，尤其是野味到安全温度（&gt; 65℃1分钟， 即从粉红肉的颜色变成棕色的核心肉产品）3。欧盟和美国农业部已指定冷冻和烹调时间和温度，可以用来杀死旋毛虫幼虫肉4,5猪肉制品。此外，对于旋毛虫在肉及肉制品灭活建议由国际委员会发表的旋毛虫病"外部参照"&gt; 6。在欧洲，除了引进的商业猪群那里旋毛虫感染的风险可以忽略不计控制住房条件，人类旋毛虫病的预防是基于易感动物4肌肉样品的实验室检查。 对于食品安全而言，消化试验是在肉3,7直接检测旋毛虫的唯一可靠方法。即使有消化测定的几个变型中，磁力搅拌器的方法是国际公认的参考方法3，4，7。该测定是基于在横纹肌组织的检测旋毛虫 。肌肉样品的酶消化和随后的过滤和沉淀步骤， 三棱后chinella幼虫通过显微镜鉴定。根据贸易义务和国家立法，也有磁力搅拌器方法的一般协议的小变化，众说纷纭。在这里，技术细节是根据欧盟要求的4，ISO规格8，信息和通信技术指导6，和欧盟参考实验室的寄生虫（EURLP）的经验和德国的参考实验室旋毛虫 。

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	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Knife,Scissors, Tweezers&#160;</td>
			<td style="width:356px;"></td>
			<td style="width:80px;"></td>
			<td style="width:363px;">Cutting samples and removing non-digestible tissue</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Blender&#160;</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">With a sharp chopping blade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Magnetic stirrer</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">With thermostatically controlled heating plate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Stirring Rod</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Teflon-coated, approximately 5 cm long</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:220px;">Conical glass separation funnel</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity of at least 2 litres, preferably fitted with Teflon safety plugs. The width should not be larger than 55% of the length to allow a good larva sedimentation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Stands, rings and clamps</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Sieve</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Mesh size 180 microns, external diameter 11 cm, with stainless steel mesh</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Funnel</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Internal diameter not less than 12 cm, to support the sieves</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Glass beaker</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity 3 litres</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Glass measuring cylinder</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity 50 to 100 ml, or centrifuge tube</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:220px;">Stereo-microscope</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">With a substage transmitted light source of adjustable intensity or trichinoscope with a horizontal table</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:220px;">Petri dishes</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">9 cm diameter, marked on their undersides into 10 &#215; 10 mm square examination areas using a pointed instrument or a larval counting basin for the tricinoscope</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Aluminium foil</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Alternatively a lid to cover the beakers</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">25 % hydrochloric acid</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:220px;">Pepsin</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Strength: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (F&#233;d&#233;ration internationale de pharmacie), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Ethanol</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">70-90% ethyl alcohol</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Tap water</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Heated to 46-48 &#176;C before use</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Balance</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Accurate to at least 0.1 g</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Metal tray</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity 10 to 15 litres, to collect the remaining digestive juice</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Pipettes and pipette holder</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Different sizes (1, 10 and 25 ml)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:220px;">Thermometer&#160;</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Accurate to 0.5 &#176;C, temperature range at least from 20 &#176; C to 70 &#176; C</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Siphon&#160;</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">For tap water</td>
		</tr>
	</tbody>
</table>

magnetic stirrer method for the detection of trichinella larvae in muscle samples

旋毛虫病是人类一种使人衰弱的疾病，被感染了旋毛虫属的线虫幼虫动物的生的或未煮熟的肉类消费量造成的。全球人类感染的最重要来源是野味及猪肉或猪肉制品。在许多国家，人类旋毛虫病的预防是基于肌肉样本的易感动物尸体的人工消化的方式识别受感染的动物。 有基于肉的消化，但磁力搅拌器方法被认为是金标准的几种方法。此方法允许肌肉样品和随后的过滤和沉降步骤的酶消化后的检测旋毛虫通过显微镜。虽然这种方法不需要特殊的和昂贵的设备，就不能使用的内部控制。因此，严格的质量管理应applie整个试验D。目前的工作的目的是要提供详细的操作说明和方法来分析的基础上，欧盟参考实验室寄生虫和旋毛虫德国国家参考实验室的经验的关键控制点。

<html>消化后，将感染性肌幼虫的形状可以变化，使得识别更加困难。典型形式包括紧密缠绕的幼虫，轻轻地盘绕幼虫或C形或完全解开幼虫（ 图2B - 2C）。每克发现幼虫的数目可以显着变化，并且可以从一个幼虫到几百幼虫范围。 感染性肌幼虫的形态示于图2A。 旋毛虫是0.7至1.5毫米长，宽度大约0.3mm。食管狭窄，有一个略显圆润结束。角质层是光滑的。在体腔内，该stichosome的前半部分，一个结构构成的细长管45至55的大细胞（stichocytes）一排包围的，可以观察到。直肠也是圆的，没有任何突出物或附属物10,11。 t"的&gt;其他的结构，除了旋毛虫 ，可以肌肉消化后发现的一些例子示于图3A。 - 3F野生动物常常感染其他寄生虫或可用于测试的肌肉样品中由动画的剜过程被污染或无生命的结构/生物。幼虫的长度，前部和后部端部的外观和的stichosome帮助的发生，以不同的线虫属12区分。 <img alt="图1" src="/files/ftp_upload/55354/55354fig1.jpg" /> 图1: 旋毛虫后镜检（A）不足和（B）有足够的漂洗步骤。刻度条表示100μm左右。 <a href="http://ecsource.jove.com/files/ftp_upload/55354/55354fig1large.jpg" target="_blank">请点击此处查看大图已经</a>这个数字rsion。 <img alt="图2" src="/files/ftp_upload/55354/55354fig2.jpg" /> 图2: 感染形态旋毛虫显示幼虫（A）的直肠，Stichosome和食道 。消化显示（B）后肌幼虫可能的形状紧密缠绕，轻轻地盘绕动的幼虫和（C）解开幼虫。刻度条表示100μm左右。 <a href="http://ecsource.jove.com/files/ftp_upload/55354/55354fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55354/55354fig3.jpg" /> 图3: 肌肉的消化样品中的磁力搅拌器方法后偶然发现的例子。 （A）的鬃毛般的头发野猪发现蚯蚓; （B）从野猪Alaria翅 ; （C）从野猪的肌纤维; （D）Metastrongylus藻。从野猪; 弓首 SP:（E）植物纤维野猪（F）中。幼虫从野猪。刻度条表示100μm左右。 <a href="http://ecsource.jove.com/files/ftp_upload/55354/55354fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples at JoVE.com

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

旋毛虫病的人在许多国家预防全球是基于消化，其中的磁力搅拌器方法被认为是黄金标准的方法，从易感动物肌肉样品的实验室检查。

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

magnetic-stirrer-method-for-detection-trichinella-larvae-muscle

Research

JoVE Journal

Immunology and Infection

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

28.5K 次观看.  Federal Institute for Risk Assessment. 旋毛虫病的人在许多国家预防全球是基于消化，其中的磁力搅拌器方法被认为是黄金标准的方法，从易感动物肌肉样品的实验室检查。 

视频: Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to the best of these authors' knowledge, no method has been developed to allow for the in situ visualization of the 
bacteria in histological sections.
While bacterial localization has been determined in vivo in previous studies using plate counts 3, radiometric labeled 4, 
or bioluminescent bacteria 5, most of these studies have only been performed at the gross organ level, with one exception 6. This limitation 
is of particular concern because E. ictaluri has a complex infection cycle 1,7, and it has a variety of virulence factors 8,9. 
The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi 10, which is in the same taxonomic family.
Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.11.
Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the 
sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC 
labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium. 
This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.

使用荧光免疫化学检测爱德华ictaluri</em斑点叉尾鮰>（一毛虫）样品

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

人乳样品靶向16S 测序方法的研究

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

对于检测磁力搅拌器方法

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