Summary

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

Published: February 20, 2015
doi:

Summary

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.

Abstract

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’s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Introduction

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(www.faostat.org) 。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。

Protocol

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。 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。 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。 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。 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。 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。 煮沸后允许每个样品在环境温度下冷却1-2分钟。 离心机使用微量的最高速度( 例如,〜万XG),持续1分钟各样品。 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。 2. 艾美 LAMP引物制备(预检测) 准备艾美 LAMP底漆股票足够100测定: 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。 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。 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。 简要涡混合底漆的解决方案,然后脉冲离心和冷冻备用。 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。 3. 艾美 LAMP检测吸取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。 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。 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。 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。 4. LAMP检测读出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。 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。

Representative Results

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”/"> 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)。 请点击此处查看该图的放大版本。 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。 请点击此处查看该图的放大版本。 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 。 请点击此处查看该图的放大版本。 示例站点 艾美尔球虫种测定法(最有可能的) 十二指肠(D) 堆型艾美球虫,大肠杆菌雷竹 空肠/回肠*(J / I) E.最大值,E.球虫 盲肠(C) E.毒害艾美耳球虫,球虫 回肠末端(TI) E. BRUNETTI,缓症E. </tR> 合并的样品(P)的 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 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。 入门* 股票浓度(μM) 体积(微升) 水 – 60 正向内引物(FIP) 100 40 落后的内蒙古小学滨海(BIP) 100 40 正向外引物(F3) 100 10 落后的外引物(B3) 100 10 正向循环(LF) 100 20 环向后(LB) 100 20 总 200 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。 股票浓度ñ 最终的反应浓度ñ 每个反应体积(微升) DDW – – 10.1 ThermoPol缓冲区 10× 1个 2.5 硫酸镁 100毫米 2毫米 0.5 引物混合物* 表2 2.5 的dNTP 25毫米 400林M 0.4 甜菜碱 5米 1M的五 Hydroxynaphthol蓝色 3毫米 120μM 1 BST DNA聚合酶 8000 U / ml的 8ü 1 总 23 表3.准备一个LAM的P反应mastermix。* 艾美耳球虫种属特异性。

Discussion

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 ~£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.

Declarações

The authors have nothing to disclose.

Acknowledgements

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.

Materials

Name Company Catalogue number Comments
RNAlater Ambion AM7024
Ethanol VWR Chemicals 20821.321 Caution, highly flammable
100 x Tris-EDTA (TE) buffer concentrate Sigma-Aldrich T9285
Chelex 100 resin Bio-Rad 142-1253
Molecular grade water Invitrogen 10977035
E. acervulina F3 Sigma-Aldrich VC00021 *CCTAACATTTCGCTTCACGGAC
E. acervulina B3 Sigma-Aldrich VC00021 *ATGAGCAAGTGGAACACCTTG
E. acervulina FIP Sigma-Aldrich VC00021 *AGAGCACAGTGGCAGTGC-AGCAGACAGCATGGCTTACCT
E. acervulina BIP Sigma-Aldrich VC00021 *GAAGACCCTCTGAAGAACGGA-CCTTCTCACCGCTTACCGG
E. acervulina LB Sigma-Aldrich VC00021 *TAAGGTTACACCCGTGGAGG
E. acervulina LF Sigma-Aldrich VC00021 *GCCATGCACAAAGCGACTT
E. brunetti F3 Sigma-Aldrich VC00021 *GGCCATCAAGTTCCATGAGC
E. brunetti B3 Sigma-Aldrich VC00021 *TCAACCTCCTGAGTGTGGTT
E. brunetti FIP Sigma-Aldrich VC00021 *GAAAATGCCTTCGTAGCTGCT-GCTGGGTACGGAGCGTCTT
E. brunetti BIP Sigma-Aldrich VC00021 *TACTTCCTAGGATCCATCCTCGC-AGTTTCGCTGCCGCCTC
E. brunetti LB Sigma-Aldrich VC00021 *GAAACGCTCGAACATGGC
E. brunetti LF Sigma-Aldrich VC00021 *CTTCTCCACAGACCCAGAGGT
E. maxima F3 Sigma-Aldrich VC00021 *ACTACGGAAAAGTGCGTAGCT
E. maxima B3 Sigma-Aldrich VC00021 *CCTTCCTCCCTTCTGAAAACTG
E. maxima FIP Sigma-Aldrich VC00021 *GAGTCACTGCTGATGTACCAAA
AG-GAACTATGCCGCTTTCCCCTG
E. maxima BIP Sigma-Aldrich VC00021 *AGAATGCGGATTTGTTAGCAGC-AGCAAGTCCAAGGTGTGTGTA
E. maxima LB Sigma-Aldrich VC00021 *CAAGCCTACGCGGACATC
E. maxima LF Sigma-Aldrich VC00021 *TTATGCAGCTGGGTCAACG
E. mitis F3 Sigma-Aldrich VC00021 *ACGATAGCCAAGACACGTAAGG
E. mitis B3 Sigma-Aldrich VC00021 *CCCCGTGATAAGAGTAGGAACA
E. mitis FIP Sigma-Aldrich VC00021 *CGCGGGTCGTGAGATTTAAATT
AT-GGAAGATCAGGACGGGCACT
E. mitis BIP Sigma-Aldrich VC00021 *GTTTCAGTTGATGAACAAGCGA
GA-TGCGCCTCTAGAATCAAGACG
E. mitis LB Sigma-Aldrich VC00021 *TCCATGCATCCCCTTGTT
E. mitis LF Sigma-Aldrich VC00021 *CGTGGGCACAGATTGATTC
E. necatrix F3 Sigma-Aldrich VC00021 *TGGCTTTCCCGCGTACC
E. necatrix B3 Sigma-Aldrich VC00021 *CGGCCCAACACAAAGACTG
E. necatrix FIP Sigma-Aldrich VC00021 *CGCTTGAGTTTTAAGCTATGCA
CA-GACCCAAGCAGCTCACCAA
E. necatrix BIP Sigma-Aldrich VC00021 *CGCCATGCCATTCAATGAACG-*GAGGCATACCGGCGTTGTC
E. necatrix LB Sigma-Aldrich VC00021 *GTCTGTAACTTGGGACGTTGT
E. necatrix LF Sigma-Aldrich VC00021 *GAACAGCCGGAGCCTCTC
E. praecox F3 Sigma-Aldrich VC00021 *GCCCTTGTATGTTGCTGTTTCT
E. praecox B3 Sigma-Aldrich VC00021 *GCGCACGAATCTGAATCACAC
E. praecox FIP Sigma-Aldrich VC00021 *ATCTCCTCAAAGACTTTCGCGT
A-GCGCTTGGCTATATCCATAGG
E. praecox BIP Sigma-Aldrich VC00021 *GCTCTCGTGGCATACTTGC-GCCAGGAGCCACTGATTGT
E. praecox LB Sigma-Aldrich VC00021 *GAATAGCATTGCCAGGTGG
E. praecox LF Sigma-Aldrich VC00021 *GTCCACTGTCATTAATATTGC
TGC
E. tenella F3 Sigma-Aldrich VC00021 *GCTTGTGAAGGTCAGCGTG
E. tenella B3 Sigma-Aldrich VC00021 *GCTGAGTCCATACGTACTTCCT
E. tenella FIP Sigma-Aldrich VC00021 *GCCACTGCTATGGAAAGTCAC
AC-CATAACTGGCATGCAGGGGT
E. tenella BIP Sigma-Aldrich VC00021 *GTTTGGCCCGAAAGTTGTGAA
GA-CGTCAGAAATTGCTGCCCAAT
E. tenella LB Sigma-Aldrich VC00021 *CGCATGTGCAGTTGAAGACA
E. tenella LF Sigma-Aldrich VC00021 *CCAAATGTATCTGCTAGTTATA
TTAACAAG
10 x ThermoPol reaction buffer New England Biolabs B9004S
MgSO4 Sigma-Aldrich M7506
dNTPs Promega U1330
Betaine solution (5 M) Sigma-Aldrich B0300
Bst polymerase New England Biolabs M0275S
Hydroxynaphthol blue Sigma-Aldrich 33936 Dissolved in molecular grade water.
UltraPure agarose Invitrogen 16500-500
10 x Tris/Borate/EDTA (TBE) buffer Invitrogen AM9863
Blue/Orange DNA loading dye (x6) Promega G1881
GeneRuler 1Kb DNA ladder Thermo Scientific SM0313
SafeView nucleic acid stain NBS Biologicals NBS-SV

Referências

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Barkway, C. P., Pocock, R. L., Vrba, V., Blake, D. P. Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens. J. Vis. Exp. (96), e52552, doi:10.3791/52552 (2015).

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