This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

以下数据收集中使用的所有的猪都在美国俄亥俄州立大学机构动物护理和使用委员会保护（动物使用的协议号2009A0134-R1）。 1.准备病毒传输介质和样品收集瓶<ol><li>加37个克脑心浸液的900毫升纯化水中，并用搅拌棒和磁力搅拌器充分混合，同时加热至70℃以完全溶解粉末。 </li><li>高压灭菌的肉汤在121℃下进行15分钟。酷板凳上的顶部，直至汤汁达到室温。冷藏于4℃过夜如果需要的话。 </li><li>溶解，通过用磁力搅拌棒，6.02克青霉素G钠盐和10g硫酸链霉素到50ml的纯净水中，在室温下混合。添加额外的纯化水，使总体积为100毫升。过滤通过0.22μm聚醚砜膜的溶液到无菌瓶。 </li><li>无菌添加过滤青霉素和链霉素溶液到冷却的脑心浸液肉汤调匀（搅拌棒或用手涡旋烧瓶）以使病毒运输介质。 </li><li>无菌测试病毒运输介质的pH;如果需要的话，用盐酸或氢氧化钠将pH调节至7.4±0.2。 </li><li>通过测试的A型流感病毒基质蛋白RRT-PCR检测，所描述的张某和哈蒙执行病毒传输介质的质量保证测试。22 </li><li>分配将5ml病毒运输介质的到无菌的高密度聚乙烯瓶用8ml容量。标签小瓶在现场使用。 </li><li>存储准备使用的小瓶中标准的冷冻箱在-20℃下直到样品收集。 </li></ol> 2.收集鼻湿巾从猪<ol><li>解冻小瓶样品采集前夕适当数量的;一小瓶将每头猪的采样是必要的。解冻在室温温度大约需要30-45分钟。保持解冻瓶清凉冰袋直至使用。 </li><li>唐适当的个人防护装备的情况所决定（ 如工作服，靴套，口罩，耳塞等 ）。 </li><li>进入动物区。如果需要，限制了猪的小区域，但也没有限制。不要吵醒休息的动物。 </li><li>穿上一双一次性检查手套。请注意不要触摸猪，人，或无生命的物体污染的手套。 </li><li> （在2×2英寸）从包装用戴着手套的手，除去无菌5.08厘米×5.08厘米棉花纱布垫。无菌纱布垫应尽可能使用。独立包装纱布垫比那些包裹着每个套餐二纱布垫使用起来更加方便。 </li><li>持棉花纱布垫用一只手指尖暴露尽可能多的垫尽可能进行采样。 </li><li>擦拭整个猪的口鼻部的纱布垫，以如果可能的话格外小心，输入外部鼻孔。收集可见鼻腔分泌物（约1毫升）使用相同的纱布垫。 </li><li>其自身使用相同的手，折叠纱布垫，以方便放置于含有病毒运输介质中的小瓶中。 </li><li>使用另一只手，除去从药瓶的盖和将折叠的纱布垫入小瓶中。重新盖上小瓶并摇动小瓶以确保纱布垫和病毒运输介质的混合。 </li><li>在适当的容器中取出手套和地点。更改每头猪的手套。 </li><li>验证小瓶标识符。记录猪识别号码，年龄，性别，临床症状，并认为必要的研究者其它注释。 </li><li>采集后立即冷却该样品。考虑冻结防止多个冻融循环之前使样品的等分试样。一旦收集后可能，将样品在干冰上运送到实验室。存放小瓶于-80°C，直到试验开始。 </li></ol>检测流感3.病毒核酸<ol><li>解冻在37℃的干燥珠浴样品5分钟，然后完成解冻置于工作台顶部，将样品在室温下20-30分钟。同时加入几个小瓶至珠会导致浴加热;应谨慎行事，以不过热的小瓶。 </li><li>取出100μl病毒传输介质的RNA提取。 <ol><li>使用高通量（96孔板格式）磁珠平台，用于从大量样本中提取RNA。31 </li></ol></li><li>使用实时逆转录聚合酶链反应检测的快速检测甲型流感病毒。31 </li></ol> 4.隔离流感从鼻湿巾病毒<ol><li>如第3.1节上述解冻的样本</li><li>治疗解冻样品硫酸庆大霉素（1000微克/毫升），两性霉素B（22.5微克/毫升），和硫酸卡那霉素（325微克/毫升）。 </li><li>接种入的Madin-Darby犬肾（MDCK）的上皮细胞如前所述。32 </li></ol>

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The authors have nothing to disclose.

收集使用聚酯嘴鼻拭子样品的猪在开展IAV监控已经证明是有益的;然而，使用鼻拭子过程的阻碍，由于所需使用圈套克制的监测工作。鼻湿巾代表目前的猪采样技术的细化，旨在样品采集过程中减少压力对人和猪。而该方法已被开发用于和验证展猪设置，它可以很容易地应用于其它情形宰前检测IAV的猪是必要的（ 即商业猪，活的动物中的市场，生物医学研究等 ） <p class="jove_content"...

<html> A型流感病毒（IAV）引起的呼吸系统疾病在许多物种，包括家禽，猪和人类。由于分段IAV基因组快速病毒进化的重配可发生新IAV株频频出现。猪是可以作为从多个宿主物种重配IAVs的混合容器中的物质。1目前IAV三个主要亚型间的北美猪（H1N1，H1N2，H3N2）通常循环，但来自人多IAV引入有导致大量IAV多样性的亚型内。IAVs感染猪2快速发展自1998年以来，当含有来自人类，禽流感和猪的病毒基因片段一个三重配IAV 3成为普遍在美国猪群中已可见一斑。4，内部基因从三重配IAV段保持IAVs目前感染猪中非常普遍。5 “&gt;在世界范围内，IAV是显著造成呼吸系统疾病的猪其典型的临床症状包括发烧，厌食，嗜睡，咳嗽，呼吸困难，打喷嚏，流鼻涕和体重不增。IAV可以是特别昂贵的母猪养殖场，其中生殖失败是由于IAV引起发热和弱仔猪已被记录在案，6,7在美国，IAV是常用的商业猪群和广泛的抗原和基因组多样性与持续发展之间的IAV感染猪检测阻碍了该控制病毒8-11 关于大流行IAV株来自重配导致猪分别实现在2009年时包含从三重配的北美猪血统和欧亚禽样猪的血统基因片段猪谱系IAV引起了世界范围内大流行的出现公共健康问题在人类12大流行性流感病毒（A型（H1N1）pdm09）具有自重配地方性猪IAV株13,14和一些新的重配株已传回人类。15重配事件和新IAV株可能引起大流行的出现，使循环IAV病毒在猪势在必行的主动监测的频率，特别是在猪的人机界面。 猪 - 人接口是IAV的双向种间传播的重要。人到猪的商业化养猪生产产生的传输是负责大量当前存在于猪群IAV多样性。农产品展销会是在美国人民和猪的comingling最大的设置和已知位点IAV的动物传播。15-21在2012年，一个变种H3N2 IAV爆发期间，案件93％的受访出席农业博览会在未来的日子发病前15基因组分析21展猪感染IAV从展猪病毒分离株与人分离株相比，证实人畜共患传输。常常不显示疾病，21-23指示需要直接诊断测试的临床症状。 对明显的病猪采样本身并不能成功地识别IAV流行的猪，不能依赖于识别IAV新毒株出现的猪之一。主动监测为检测IAV的猪，并评估其对两个猪和公共卫生威胁新兴菌株的绝对必要的。大多数IAV侦察活动是自愿的，因此需要最低限度的破坏性的方法。为IAV感染猪三大生前样本收集程序是：鼻拭子，唾液，鼻腔擦拭。对于个别采样猪检测IAV名单合成纤维插入当前建议拭子插入鼻孔的首选方法收集鼻分泌物和上皮细胞。24,25由于猪可能尽量避免此过程中，一组训练有素的人员必须手动或使用圈套根据动物的大小抑制猪26的限制处理是费力的人员，和紧张的猪。此外，展览猪经常参与多个比赛在一个公平的这样增加的压力对比赛的动物的感受可以让车主耐监测工作。 随着IAV检测的概率感染牛群从80-100％的IAV，口服液体已经成为一种流行的替代鼻拭子在猪群体的分子检测IAV的。27,28此外，口服液体可以提供更广泛的窗口IAV检测不是遵循最初的感染鼻拭子。然而，从唾液IAV隔离一直是个问题，只有50％的病毒分离的尝试导致IAV恢复。29 代替期间在猪IAV监视鼻拭子的使用鼻腔擦拭物克服了上述限制。鼻腔擦拭物不需要使用限制圈套的，可以在不强调过程的动物或证人来执行。最少的技术培训是需要收集鼻腔擦拭，这使得非兽医专业人士，包括猪的业主，收集监测样本。鼻腔擦拭物以前相比鼻拭子对甲病毒30和对于这种非侵入性采样的方法的详细的协议下面描述的检测和流感的隔离。

<table border="0" cellpadding="0" cellspacing="0" width="1051">
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			<td height="21" style="height:21px;width:216px;">BBL Brain Heart Infusion</td>
			<td style="width:360px;">Becton, Dickinson and Company</td>
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			<td style="width:356px;"></td>
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			<td height="21" style="height:21px;width:216px;">Penicillin G Sodium Salt</td>
			<td style="width:360px;">MP Biomedicals, LLC</td>
			<td style="width:119px;">021194537</td>
			<td>1500 u/mg</td>
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			<td height="21" style="height:21px;width:216px;">Streptomycin Sulfate</td>
			<td style="width:360px;">AMRESCO LLC</td>
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			<td style="width:360px;">Mediatech, Inc.</td>
			<td style="width:119px;">30-005-CR</td>
			<td>50 mg/ml</td>
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			<td style="width:360px;">Fisher</td>
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			<td>250 ug/ml</td>
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			<td>HDPE with white PP closure</td>
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			<td>5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in)&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Nitrile Exam Gloves</td>
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nasal wipes for influenza a virus detection and isolation from swine

在监测猪流感A型病毒对人类和动物的健康至关重要，因为A型流感病毒迅速演变的猪群和新品种不断涌现。猪能够被感染甲型流感病毒的使得它们的出现和维持的新的流感病毒株重要主机多样谱系。抽样猪在不同的设置，如商业猪场，农产品集贸市场和活禽交易市场是非常重要的，提供当前流行IAV株的全面视图。目前的黄金标准宰前抽样技术（鼻拭子的，即集合）是劳动密集型的，因为它需要猪的物理限制。鼻抹布擦涉及在尽量减少猪的口鼻部一块布来节制的动物。鼻腔擦拭过程简单进行，并不需要专业的兽医或动物处理人员培训。 W¯¯往往微不足道略高于鼻拭子不太敏感，病毒检测和​​隔离率足以使鼻湿巾抽检个别猪在低应力抽样方法都需要一个可行的选择。该诉讼协议概述了需要收集一个可行鼻的步骤从个体猪擦拭。

成功使用这种方法产生RRT-PCR结果是，伴随着RNA提取和RRT-PCR过程中使用的内部控制，显示样本不含有PCR抑制剂从任何环境碎片捡起取样过程。样品在接种后，分离病毒井应不受样品可见环境碎屑。有与PCR通常产生更高IAV阳性率较病毒分离因为PCR检测病毒核酸，不一定可行病毒的理解RRT-PCR结果和病毒分离结果之间吻合。 结果表明，鼻抹布是一个有用的替代鼻拭子，这对于IAV目?...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K 次观看.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

鼻湿巾从猪流感病毒检测和​​隔离

鼻湿巾从猪流感病毒检测和隔离