The work carried out in this study was supported in part by the Royal Veterinary College through the student research projects fund, as well as the Biotechnology and Biological Sciences Research Council and the Department for International Development (grant number BB/H009337/2). This manuscript has been assigned the reference PPB_00795 by the RVC.

1.模板制备注:怀疑含有的DNA从该感染的鸡的七个艾美球虫物种之一衍生的任何基因组DNA模板可以被用作模板用于基于LAMP的艾美尔球虫种的识别。这里描述在例行验应收集肠组织样本进行现场诊断分析。 <ol><li>选择肠的部分（或部分），以进行测试。请参阅表1为指导，以取样点的选择和最有可能出现18和图1的取样点分布和位置的品种，特定范围的艾美尔球虫种。 注:这是至关重要，因为艾美特有尤其是主机站点。每个物种感染的鸡是由它的目标19肠道区域定义。这一决定可以由农庄以往的经验，兴趣在一个或Mo的影响再具体的艾美球虫或其它诊断 ​​指标6。 </li><li>消费5厘米或选择来测试使用艾美球虫无菌剪刀或手术刀肠部（S）的较长的长度。任选地，存储样品用于随后的分析中固定剂例如在RNAlater中18％或95％的乙醇。 注:如果在乙醇储存样品应当在无菌三 - 乙二胺四乙酸充分洗涤（TE）缓冲液在使用前。 </li><li>切取试样打开纵向，从自由使用无菌玻璃显微镜载玻片的任一边缘或乙醇/火焰灭菌剪刀片粘膜层除去大部分肠内容物（如果存在）和刮除细胞。任选的合并样本包括来自所有4的物种特异性肠位点在单管细胞。 </li><li>把刮下材料到含有100微升无菌TE缓冲液含1无菌1.5毫升螺杆顶部的离心管为0％（重量/体积）的Chelex 100树脂。 </li><li>用力摇动每个样品1分钟。确保螺杆顶部是牢牢封闭，然后孵育在沸水浴中10分钟。 </li><li>煮沸后允许每个样品在环境温度下冷却1-2分钟。 </li><li>离心机使用微量的最高速度（ 例如，〜万XG），持续1分钟各样品。 </li><li>收集2微升所得上清液为将要进行的模板中的每个灯检测。任选地，集中多个肠部位在一个单一的管，以提供一个多站点检测。 </li></ol> 2. 艾美 LAMP引物制备（预检测） <ol><li>准备艾美 LAMP底漆股票足够100测定: <ol><li>通过增加分子级水至100微米的浓度（所指定的制造商）重建每个冻干艾美 LAMP底漆。如果未指定，计算分子级所需的水全光照的体积克各引物的物理和分子量。 </li><li>待测定吸移管60微升分子级水到一个单独0.5毫升翻盖微量离心管中的每个的艾美球虫种。 </li><li>添加的引物FIP，BIP，F3，B3的LF和LB特异于靶艾美尔球虫种使用表2中所示的卷的水，形成了一系列的7种特异性引物混合物。 </li><li>简要涡混合底漆的解决方案，然后脉冲离心和冷冻备用。 </li></ol></li><li>制备LAMP反应mastermix每个艾美尔球虫种待测定。由样本数目乘以在表3所示的体积，并添加三至阳性对照，阴性对照和移液备用。吸取到0.5或1.5毫升翻盖的离心管。 </li></ol> 3. 艾美 LAMP检测<ol><li>吸取23微升艾美球虫专用的Bst DNA聚合酶/ LAMP mastermix成0。5毫升离心管中。 </li><li>添加2微升DNA模板（在第1制备的），使得最终反应的25μl的体积。 </li><li>添加2微升的艾美球虫种特异性基因组DNA，以一个反应（阳性对照）。添加2微升的分子级水到反应（阴性对照）。 注意:如果物种特异性的基因组DNA是不可用，可以使用先前的阳性LAMP或标准的PCR产物来代替。 </li><li>孵育在水浴或加热块在62℃下30分钟。任选地，脱激活的Bst DNA聚合酶，通过加热至80℃达 10分钟，如果反应是不会被立即读出。 </li></ol> 4. LAMP检测读出<ol><li>在温育结束时评估由眼睛在室内光各反应的颜色。阴性结果出现粉红色到紫色，阳性结果出现天蓝20。 </li><li>可选，确认LAMP检测结果在实验室及其他实验室使用的核酸染色（每5微升ORY通过混合5微升LAMP反应产物与在1×的Tris /硼酸盐/ EDTA（TBE）缓冲液用2％琼脂糖凝胶的琼脂糖凝胶电泳将1μl的DNA凝胶上样缓冲液，预染50毫升琼脂糖）。加入5微升1Kb的分子大小的DNA梯的车道1的凝胶，以允许片段大小的计算。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

The Eimeria species-specific LAMP assays described in this paper offer a new diagnostic tool kit in support of effective control of coccidia and the disease coccidiosis. The outcomes of eimerian infection can include severe economic loss as well as seriously compromised bird welfare and increased susceptibility to colonisation by zoonotic pathogens21. Opportunities to monitor flocks for the occurrence of some, or all Eimeria species can provide early warning of a breakdown in anticoccidial control efficacy. Key advantages of LAMP include robust target specificity, resulting from the requirement for six different DNA sequence targets, as well as high sensitivity, boosted by the inclusion of loop primers13, although the qualitative, not quantitative nature of LAMP may be considered a limitation. It is not currently possible to discriminate low level parasite escape from routine chemoprophylaxis or live vaccine replication from unchecked eimerian replication. Nonetheless, the technical ease of the protocol and definitive readout offers considerable improvement over the existing specialist and frequently subjective pathology- and morphology-led approaches6,7. Each assay may be completed at a cost of ~&#163;0.75 per sample, independent of labour and equipment set up expenses. Thus, LAMP assays are also more cost effective than other molecular diagnostics such as PCR, since they require an isothermal incubation with no need for specialist equipment. 
For many years access to Eimeria genomic DNA as template has limited the development and application of molecular field diagnostics. The oocyst is the most readily accessible phase of the eimerian lifecycle, but routine DNA extraction requires laboratory facilities22. Other, more labile intestinal lifecycle stages require purification prior to DNA preparation to prevent PCR inhibition and a consequent loss of sensitivity23,24. The ability to extract eimerian DNA of a quality suitable for LAMP using equipment no more specialised than a microcentrifuge and a water bath, supplemented by inhibitor adsorption using chelex resin, now promotes the wider use of molecular biology in eimerian diagnostics. Intriguingly, the reported detection of quantitative PCR-measurable Eimeria DNA in intestinal tissue 20 days after the initiation of parasite infection, 11 days after the last detectable oocyst output, raises the suggestion that LAMP may be used to detect resolved parasite exposure as well as ongoing infection, even after any visible lesions may have been resolved25. 
The relatively low cost and low technical requirements of LAMP Eimeria diagnostics can promote their application in the developing world where other more established approaches may not be available or appropriate. For this to be applicable each assay must be capable of detecting all strains which may be circulating within each region. While understanding of the genetic diversity prevailing among Eimeria species is limited26, the use of target sequences previously validated for use in quantitative PCR with strains from Africa, Asia, Europe and South America provides some evidence of conservation, supporting the utility of these LAMP assays around the world10.

全球鸡肉产量增加了10倍，在过去50年发展中世界的托管见证了发达国家扩大近四倍<a href="http://www.faostat.org">（www.faostat.org） 。</a>由于鸡的生产对世界粮食安全的相关性已变得如此得具有病原体可在鸡引起严重的疾病的配置文件。一个典型的例子是艾美尔球虫种，无处不在的原生动物寄生虫这会导致肠道疾病球虫病1。无论鸡饲养的一种或多种艾美球虫物种很可能是常见2-4。在发达国家艾美耳球虫主要是由化学预防控制，使用航天飞机或轮换计划，以减少阻力5的影响。活疫苗还用在系统中的鸟值足以证明费用（ 例如，种畜，层和一些肉鸡5）。作为一名RESULT这些措施的临床球虫病常常很好的控制，虽然亚临床感染是常见的5。在发展中国家，疫苗接种是罕见的，药物的应用程序频繁少消息灵通。作为结果的亚临床和临床球虫病是更常见的，施加一个显著经济影响3。 eimerian感染的诊断已传统上依赖于病变得分验尸，尽管使用最广泛的评分系统连作者指出对于有些种类"看来值得怀疑是否这样一个程序应在任何但中度严重的感染可以尝试"6。补充证据可通过显微镜检测粪便或枯枝落叶样品中的环保性卵囊生命周期阶段的聚集，但重叠的形态可以混淆所有，但专家6,7。利用聚合酶链式反应的分子的替代（PCR），随机amplificatioÑ ​​多态性DNA的PCR（RAPD-PCR）和定量PCR技术，已可用于长达20年8-10，但到目前为止，他们都未能普及。相对费用和专科实验室设备或处理的要求限制了它们的摄取，尽管往往主观，技术要求高的老pathology-的性质和显微镜为基础的方法10,11。这样的限制可能被夸大许多世界上较贫穷的地区，如东南亚，那里的球虫病对贫困的影响可能更大比例12。在回答有新的直接和敏感的，但成本效益， 艾美尔球虫种特异的诊断试验的明确要求。 环介导等温扩增（LAMP）是容易制备的DNA聚合酶驱动的技术，它是能够扩增大量的DNA组成。最重要的是，LAMP使用的Bst.DNA聚合物A DNA本身代替的Taq DNA聚合酶在PCR中常用的，这有利于DNA扩增在单一恒定温度下，而不用于热循环13,14的要求。灯泡可以是适合于应用，即使在最起码的实验室或在外地。通过对许多PCR抑制剂，高灵敏度和特异性的相对阻力Characterised，LAMP法检测已被开发用于广泛的病原体，包括传染性法氏囊病病毒， 产气荚膜梭菌和隐孢子虫 15-17。在回答具体到每个感染鸡的七个艾美球虫的LAMP检测的面板也已开发出18个新的具有成本效益的艾美尔球虫种特定的诊断要求。应用新的检测包括监测寄生虫的发生，尤其是价值的特定物种的协会，如巨型艾美耳球虫和毒害艾美耳球虫经济PE差rformance 3,4。其它应用包括评估一个农场的抗球虫策略，亚临床感染或临床疾病和风险由艾美球虫所构成的一个农场的评估诊断的功效。

<table><tbody><tr><td>RNAlater</td><td>Ambion</td><td>AM7024</td><td></td></tr><tr><td>Ethanol</td><td>VWR Chemicals</td><td>20821.321</td><td>Caution, highly flammable</td></tr><tr><td>100 x Tris-EDTA (TE) buffer concentrate</td><td>Sigma-Aldrich</td><td>T9285</td><td></td></tr><tr><td>Chelex 100 resin</td><td>Bio-Rad</td><td>142-1253</td><td></td></tr><tr><td>Molecular grade water</td><td>Invitrogen</td><td>10977035</td><td></td></tr><tr><td>E. acervulina F3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CCTAACATTTCGCTTCACGGAC</td></tr><tr><td>E. acervulina B3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*ATGAGCAAGTGGAACACCTTG</td></tr><tr><td>E. acervulina FIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*AGAGCACAGTGGCAGTGC-AGCAGACAGCATGGCTTACCT</td></tr><tr><td>E. acervulina BIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GAAGACCCTCTGAAGAACGGA-CCTTCTCACCGCTTACCGG</td></tr><tr><td>E. acervulina LB</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*TAAGGTTACACCCGTGGAGG</td></tr><tr><td>E. acervulina LF</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GCCATGCACAAAGCGACTT</td></tr><tr><td>E. brunetti F3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GGCCATCAAGTTCCATGAGC</td></tr><tr><td>E. brunetti B3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*TCAACCTCCTGAGTGTGGTT</td></tr><tr><td>E. brunetti FIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GAAAATGCCTTCGTAGCTGCT-GCTGGGTACGGAGCGTCTT</td></tr><tr><td>E. brunetti BIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*TACTTCCTAGGATCCATCCTCGC-AGTTTCGCTGCCGCCTC</td></tr><tr><td>E. brunetti LB</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GAAACGCTCGAACATGGC</td></tr><tr><td>E. brunetti LF</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CTTCTCCACAGACCCAGAGGT</td></tr><tr><td>E. maxima F3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*ACTACGGAAAAGTGCGTAGCT</td></tr><tr><td>E. maxima B3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CCTTCCTCCCTTCTGAAAACTG</td></tr><tr><td>E. maxima FIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GAGTCACTGCTGATGTACCAAA&lt;br/&gt; AG-GAACTATGCCGCTTTCCCCTG</td></tr><tr><td>E. maxima BIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*AGAATGCGGATTTGTTAGCAGC-AGCAAGTCCAAGGTGTGTGTA</td></tr><tr><td>E. maxima LB</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CAAGCCTACGCGGACATC</td></tr><tr><td>E. maxima LF</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*TTATGCAGCTGGGTCAACG</td></tr><tr><td>E. mitis F3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*ACGATAGCCAAGACACGTAAGG</td></tr><tr><td>E. mitis B3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CCCCGTGATAAGAGTAGGAACA</td></tr><tr><td>E. mitis FIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CGCGGGTCGTGAGATTTAAATT&lt;br/&gt; AT-GGAAGATCAGGACGGGCACT</td></tr><tr><td>E. mitis BIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GTTTCAGTTGATGAACAAGCGA&lt;br/&gt; GA-TGCGCCTCTAGAATCAAGACG</td></tr><tr><td>E. mitis LB</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*TCCATGCATCCCCTTGTT</td></tr><tr><td>E. mitis LF</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CGTGGGCACAGATTGATTC</td></tr><tr><td>E. necatrix F3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*TGGCTTTCCCGCGTACC</td></tr><tr><td>E. necatrix B3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CGGCCCAACACAAAGACTG</td></tr><tr><td>E. necatrix FIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CGCTTGAGTTTTAAGCTATGCA&lt;br/&gt; CA-GACCCAAGCAGCTCACCAA</td></tr><tr><td>E. necatrix BIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CGCCATGCCATTCAATGAACG-*GAGGCATACCGGCGTTGTC</td></tr><tr><td>E. necatrix LB</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GTCTGTAACTTGGGACGTTGT</td></tr><tr><td>E. necatrix LF</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GAACAGCCGGAGCCTCTC</td></tr><tr><td>E. praecox F3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GCCCTTGTATGTTGCTGTTTCT</td></tr><tr><td>E. praecox B3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GCGCACGAATCTGAATCACAC</td></tr><tr><td>E. praecox FIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*ATCTCCTCAAAGACTTTCGCGT&lt;br/&gt; A-GCGCTTGGCTATATCCATAGG</td></tr><tr><td>E. praecox BIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GCTCTCGTGGCATACTTGC-GCCAGGAGCCACTGATTGT</td></tr><tr><td>E. praecox LB</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GAATAGCATTGCCAGGTGG</td></tr><tr><td>E. praecox LF</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GTCCACTGTCATTAATATTGC&lt;br/&gt; TGC</td></tr><tr><td>E. tenella F3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GCTTGTGAAGGTCAGCGTG</td></tr><tr><td>E. tenella B3</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GCTGAGTCCATACGTACTTCCT</td></tr><tr><td>E. tenella FIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GCCACTGCTATGGAAAGTCAC&lt;br/&gt; AC-CATAACTGGCATGCAGGGGT</td></tr><tr><td>E. tenella BIP</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*GTTTGGCCCGAAAGTTGTGAA&lt;br/&gt; GA-CGTCAGAAATTGCTGCCCAAT</td></tr><tr><td>E. tenella LB</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CGCATGTGCAGTTGAAGACA</td></tr><tr><td>E. tenella LF</td><td>Sigma-Aldrich</td><td>VC00021</td><td>*CCAAATGTATCTGCTAGTTATA&lt;br/&gt; TTAACAAG</td></tr><tr><td>10 x ThermoPol reaction buffer</td><td>New England Biolabs</td><td>B9004S</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>dNTPs</td><td>Promega</td><td>U1330</td><td></td></tr><tr><td>Betaine solution (5 M)</td><td>Sigma-Aldrich</td><td>B0300</td><td></td></tr><tr><td>Bst polymerase</td><td>New England Biolabs</td><td>M0275S</td><td></td></tr><tr><td>Hydroxynaphthol blue</td><td>Sigma-Aldrich</td><td>33936</td><td>Dissolved in molecular grade water.</td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>10 x Tris/Borate/EDTA (TBE) buffer</td><td>Invitrogen</td><td>AM9863</td><td></td></tr><tr><td>Blue/Orange DNA loading dye (x6)</td><td>Promega</td><td>G1881</td><td></td></tr><tr><td>GeneRuler 1Kb DNA ladder</td><td>Thermo Scientific</td><td>SM0313</td><td></td></tr><tr><td>SafeView nucleic acid stain</td><td>NBS Biologicals</td><td>NBS-SV</td><td></td></tr></tbody></table>

loop-mediated isothermal amplification (lamp) assays for the species-specific detection of eimeria that infect chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

实验验证在验证过程中各艾美尔球虫种特异性LAMP方法是使用表示该感染的鸡，以及鸡基因组DNA作为主机控制所有七个艾美尔球虫种纯的DNA样品的一组测试。琼脂糖凝胶电泳来解析每个测定并证明绝对物种特异性无主机交叉反应18。接着，使用纯化的禽艾美球虫基因组DNA揭示基因组1 10之间，并拷贝18的检测灵敏度极限的10倍连续稀释系列制备。没有上限与取得了积极成果，其中包括最高浓度（100,000基因组拷贝）18确定。 与现场采样应用收集艾美球虫测试样品可能被从发现死鸡衍生，扑杀为p的结果OOR健康或宰杀前哨健康监测，表明的1至3个可能的一个样本大小时的程序的一部分。测试从美国肉鸡养殖场作为监测计划的一部分，收集了三只小鸟取得了三组肠道样品。目标应用的种类特异性的LAMP测定的，优先的优选的肠部位的每个的艾美球虫种（ 表1），允许视觉识别eimerian感染的所有鸟类使用hydroxynaphthol蓝作为指示剂（ 图2A）进行测试。使用hydroxynaphthol蓝色时实现具有负LAMP反应的颜色范围可以从紫罗兰到粉红色，但是总是由一个肯定的结果所取得的蓝色鲜明。确认通过琼脂糖凝胶电泳提供类似的结果（ 图2B）。在现场应用中，用户可以选择适用全屏对所有7种，或只针对那些优先物种重要的或已知的可循环在农场或周围区域。 的PCR技术为基础的方法未能成为确立为诊断艾美球虫的发生强调在任何新的测试为了简单的要求。而LAMP提供简单的准备和处理比PCR，要求测试每只鸡多发性肠部位依然令人沮丧。生产每只鸡的单个合并的DNA样品，然后可以与一个或多个灯测定测试的，很可能是更有吸引力。处理每只鸡1合并样本，表示从各表1中的所有7 LAMP检测所述和之前的DNA制剂，以汇集，用于测试的特定的肠部位收集的材料提供的相同的结果时，每个肠位点分别进行处理（ 图2与图3相比）。 <img alt="图1" src="/files/ftp_upload/52552/52552fig1.jpg"/&gt; 图1.肠道采样点为LAMP检测艾美球虫寄生虫感染的鸡。每个艾美球虫有针对性的肠道地区是由彩色线条突出，采样由虚线黑线之间的数字表示的首选网站（E.堆型艾美 :黄/ 1，E. BRUNETTI:粉红色/ 2，E最大值 :蓝/ 3， 缓症E.:橙/ 4，E.球虫 :红/ 5，E.雷竹 :绿色/ 6和球虫 :灰/ 7）。 <a href="https://www.jove.com/files/ftp_upload/52552/52552fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52552/52552fig2.jpg" /> 图2. LAMP eimerian感染的诊断来回回米三个商业肉鸡。LAMP反应，用（A）解析hydroxynaphthol蓝色，其中一个天蓝反应是积极的和紫粉色的反应是负面的，而（B）的琼脂糖凝胶电泳。取样的肠部位表1中所示为每个寄生虫物种。 A = E.堆型艾美 ，B = E. BRUNETTI，马= E.千里马 ，MI = E.缓症 ，N = E.毒害艾美耳球虫 ，P = E.雷竹和T = E.球虫 。 1巷包含GeneRuler 1Kb的DNA阶梯。 <a href="https://www.jove.com/files/ftp_upload/52552/52552fig2large.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/52552/52552fig3.jpg" /> 使用汇集图3. LAMP eimerian感染的诊断样品从三个不同的商业肉鸡。LAMP REAC使用hydroxynaphthol蓝色，其中一个天蓝反应是积极的，紫粉色的反应是消极的系统蒸发散解决。 A = E.堆型艾美 ，B = E. BRUNETTI，马= E.千里马 ，MI = E.缓症 ，N = E.毒害艾美耳球虫 ，P = E.雷竹和T = E.球虫 。 <a href="https://www.jove.com/files/ftp_upload/52552/52552fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 示例站点 </td><td> 艾美尔球虫种测定法（最有可能的） </td></tr><tr><td>十二指肠（D） </td><td> 堆型艾美球虫，大肠杆菌雷竹 </td></tr><tr><td>空肠/回肠*（J / I） </td><td> E.最大值，E.球虫 </td></tr><tr><td>盲肠（C） </td><td> E.毒害艾美耳球虫，球虫 </td></tr><tr><td>回肠末端（TI） </td><td> E. BRUNETTI，缓症E. </td></tR&gt; <tr><td>合并的样品（P）的</td><td> 堆型艾美球虫，大肠杆菌BRUNETTI，巨型艾美球虫，大肠杆菌和缓，E.毒害艾美耳球虫，大肠杆菌雷竹，球虫 </td></tr></tbody></table>要抽样表1肠区域专用的选择候补的艾美球虫种测定。地区的选择而变化为如图1各艾美尔球虫种。合并的样品包括来自所有四个特定网站然后将其合并为DNA制备收集的材料。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 入门* </td><td> 股票浓度（μM） </td><td> 体积（微升） </td></tr><tr><td>水</td><td> - </td><td> 60 </td></tr><tr><td>正向内引物（FIP） </td><td> 100 </td><td> 40 </td></tr><tr><td>落后的内蒙古小学滨海（BIP） </td><td> 100 </td><td> 40 </td></tr><tr><td>正向外引物（F3） </td><td> 100 </td><td> 10 </td></tr><tr><td>落后的外引物（B3） </td><td> 100 </td><td> 10 </td></tr><tr><td>正向循环（LF） </td><td> 100 </td><td> 20 </td></tr><tr><td>环向后（LB） </td><td> 100 </td><td> 20 </td></tr><tr><td> 总 </td><td></td><td> 200 </td></tr></tbody></table> 表2.准备一个LAMP引预混料，准备底漆预混料LAMP所需的组件和比例。显示容积为100 LAMP反应。 *所示的材料和底漆身份Barkway 等人，（2011）18。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 股票浓度ñ </td><td> 最终的反应浓度ñ </td><td> 每个反应体积（微升） </td></tr><tr><td> DDW </td><td> - </td><td> - </td><td> 10.1 </td></tr><tr><td> ThermoPol缓冲区</td><td> 10× </td><td> 1个</td><td> 2.5 </td></tr><tr><td> 硫酸镁 </td><td> 100毫米</td><td> 2毫米</td><td> 0.5 </td></tr><tr><td>引物混合物* </td><td colspan="2">表2 </td><td> 2.5 </td></tr><tr><td>的dNTP </td><td> 25毫米</td><td> 400林M </td><td> 0.4 </td></tr><tr><td>甜菜碱</td><td> 5米</td><td> 1M的</td><td>五</td></tr><tr><td> Hydroxynaphthol蓝色</td><td> 3毫米</td><td> 120μM </td><td> 1 </td></tr><tr><td> BST DNA聚合酶</td><td> 8000 U / ml的</td><td> 8ü </td><td> 1 </td></tr><tr><td> 总 </td><td></td><td></td><td> 23 </td></tr></tbody></table> 表3.准备一个LAM的P反应mastermix。* 艾美耳球虫种属特异性。

Watch this Scientific Journal Video about Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens at JoVE.com

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

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Immunology and Infection

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

26.9K 次观看.  Royal Veterinary College, London. Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

视频: Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

科研

生物化学

Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease 1, 2. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth 3. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing 3, 4. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation 3, 5-7, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1 8. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8&gamma;&delta;+, CD8&alpha; lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.

寄生在鸡模型诱导自身免疫性基因驱动恰加斯心脏疾病

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases ...

免疫与感染

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

环境科学

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

生物工程

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

环介导等温扩增（LAMP）法进行的物种特异性检测

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此视频中的章节

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

寄生在鸡模型诱导自身免疫性基因驱动恰加斯心脏疾病

循环介导等温扩增 (灯管) 法快速识别烟粉虱

冻干环介导等温扩增试剂的检测中的发展

毛细管阵列中多核酸的视觉检测

在动物标本呼吸道病原体的高通量检测通过纳米PCR

反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体