Name: 作者聚焦：用于环境分析和疾病监测的水 中的钩端螺旋体 DNA 检测
Uploaded: 2024-06-14T12:30:00
Description: The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by ...

我们感谢墨西哥国立自治大学兽医和动物技术学院微生物学和免疫学系的钩端螺旋体收藏。我们感谢对参考钩端螺旋体菌株的慷慨捐赠;Lebtospira fainei 血清型 Hurstbridge 菌株 BUT6 和 Leptospira biflexa 血清型 Patoc 菌株 Patoc I 给 Alejandro de la Peña Moctezuma 博士。我们感谢CIBAC协调员José Antonio Ocampo Cervantes博士和工作人员的后勤支持。EDT属于Metropolitan Autonomous University-Campus Cuajimalpa本科生的终端项目计划。我们承认用于创建图 1 和图 3 至 9 的 Biorender.com 软件。

1. 样品制备
<ol><li>通过在4°C下以8,000× g 离心10分钟，将每个水样品浓缩在1.5mL微量离心管中。 根据需要多次重复此步骤，将样品浓缩到 250 μL 的体积中。</li>
	<li>根据制造商的说明使用 DNA 提取试剂盒（参见 材料表）。</li>
	<li>根据将使用的斑点印迹探针进行特异性PCR（表1）。<ol><li>在PCR管中进行扩增，最终体积为25μL，含有1X缓冲液，2.5单位Taq聚合?...

用于检测水样中 钩端螺旋体 DNA 的斑点印迹检测

斑点印迹技术的关键步骤包括：（1）DNA固定化，（2）用非同源DNA阻断膜上的游离结合位点，（3）退火条件下探针和靶片段之间的互补性，（4）去除未杂交的探针，以及（5）检测报告分子41。
PCR-Dot-blot具有一定的局限性，例如该技术不提供有关杂交片段37大小的信息，它需要在与第二个或第三个探针重新杂交之前对单个膜进行去杂交，并?...

人类钩端螺旋体病主要源于环境来源1,2。钩端螺旋体存在于湖泊、河流和溪流中，是钩端螺旋体病在野生动物以及最终可能接触这些水体的家畜和生产动物中传播的一个指标1,3,4。此外，钩端螺旋体已在非自然资源中被发现，包括污水、死水和自来水 5,6。
钩端螺旋体是一种全球分布的细菌7,8，环境在其保存和传播中的作用已得到充分认识。钩端螺旋体可以在可变 pH 值和矿物质9 下的饮用水中生存，并且可以在自然水体中生存1。它也可以在蒸馏水中长期存活10，在恒定的 pH 值 （7.8） 下，它可以存活长达 152 天11。此外，钩端螺旋体可能在细菌联合体中相互作用，以在恶劣的条件下生存12,13。它可能是淡水中生物膜的一部分，含有偶氮螺菌和鞘氨醇单胞菌，甚至能够生长和承受超过 49 °C的温度 14,15。它还可以在涝渍的土壤中繁殖，并保持活力长达 379 天16，保持其引起疾病的能力长达一年17,18。然而，人们对水体内的生态学及其在水体中的分布知之甚少。
自发现以来，钩端螺旋体属的研究基于血清学测试。直到本世纪，分子技术才在这种螺旋体的研究中变得更加普遍。点印迹法很少用于使用 （1） 基于 16S rRNA 和简单序列重复序列 （ISSR） 19,20 的同位素探针进行鉴定，（2） 作为应用于尿液的人钩端螺旋体病的基于纳米金的免疫测定21，或 （3） 作为基于抗体的牛尿液样本测定22.该技术被废弃了，因为它最初是基于同位素探针的。然而，它是一种众所周知的技术，与PCR相结合，可以产生更好的结果，并且由于使用了非同位素探针，它被认为是安全的。PCR在钩端螺旋体DNA的富集中起着至关重要的作用，它通过扩增可能在样品中微量发现的特定DNA片段。在每个PCR循环中，反应中靶向DNA片段的量增加一倍。在反应结束时，扩增子已乘以超过一百万倍23。通过PCR扩增的产物在琼脂糖电泳中通常不可见，通过在斑点印迹24,25,26中使用DIG标记的探针进行特异性杂交而变得可见。
斑点印迹技术简单、稳健，适用于大量样品，使资源有限的实验室能够使用。它已被用于各种细菌研究，包括 （1） 口腔细菌27，（2） 其他样品类型，如食物和粪便28，以及 （3） 不可培养细菌的鉴定29，通常与其他分子技术一致。斑点印迹技术的优点包括：（1）该膜具有高结合载量，能够结合超过200μg/cm 2 的核酸和高达400μg/cm2的核酸;（2） 斑点印迹结果无需特殊设备即可目视解读，（3） 它们可以在室温 （RT） 下方便地储存多年。
钩端螺旋体属已分为致病性、中间性和腐生分支30,31。这些分支之间的区别可以基于特定的基因来实现，例如 lipL41、lipL32 和 16S rRNA。LipL32 存在于致病分支中，在各种血清学和分子工具中表现出高敏感性，而在腐生菌属21 中不存在。管家基因 lipL41 以其稳定的表达而闻名，并在分子技术中使用32，而 16S rRNA 基因用于分类。
一旦通过离心浓缩了大量的水，这种方法就可以应用于水。它允许评估水体内的各个点和深度，以检测钩端螺旋体DNA的存在及其所属的分支。该工具对于生态学和一般筛查目的都很有价值，也可用于检测水中可能存在的其他不可培养细菌。
此外，PCR和斑点印迹检测对于各种实验室来说在技术和经济上都是负担得起的，即使是那些缺乏复杂或昂贵设备的实验室也是如此。本研究旨在应用基于地高辛的斑点印迹法鉴定从自然水体收集的水样中的三个 钩端螺旋体 分支。
菌株 本研究纳入 12 个 钩端螺旋体 血清型（Autumnalis、Bataviae、Bratislava、Canicola、Celledoni、Grippothyphosa、Hardjoprajitno、Icterohaemorrhagiae、Pomona、Pyrogenes、Tarassovi 和 Wolffi）。这些血清型是墨西哥国立自治大学兽医和动物技术学院微生物学和免疫学系收集的一部分，它们目前用于微凝集试验 （MAT）。
所有 钩端螺旋体 血清型均在 EMJH 中培养，并使用商业 DNA 提取试剂盒提取其 DNA（参见 材料表）。将 12 个血清型的基因组 DNA 混合物用作 钩端螺旋体 致病分支的阳性对照。作为 钩端螺旋体 中间分支的阳性对照，纳入了 Fainei钩端螺旋 体血清型Hurstbridge菌株BUT6的基因组DNA，作为钩 端螺旋 体腐生菌分支的阳性对照，还包括 双曲钩端螺旋体 血清型Patoc菌株Patoc I的基因组DNA。
阴性对照包括空质粒、来自非亲缘细菌（解脲支原体、金黄色葡萄球菌、流产布鲁氏菌、鼠伤寒沙门氏菌、博伊氏志贺氏菌、肺炎克雷伯菌、鲍曼不动杆菌 和 大肠杆菌）的 DNA 和 PCR 级水，用作非模板对照。
水样 使用分层随机抽样方法从Cuemanco生物和水产养殖研究中心（CIBAC）（19° 16' 54“ N 99° 6' 11” W）收集了12个试验抓取样本。这些样品是在三个深度获得的：浅表、10 和 30 厘米（图 1A、B）。取水程序没有影响任何濒危或受保护的物种。每个样品都收集在无菌 15 mL 微量离心管中。为了收集样品，将每个试管轻轻浸入水中，在选定的深度填充，然后密封。将样品保持在22°C，并迅速运送到实验室进行处理。
通过在无菌 1.5 mL 微量离心管中在室温下以 8000 x g 离心 20 分钟浓缩每个样品。重复此步骤，直到所有样品浓缩到一个试管中，然后用于DNA提取（图1C）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65435/65435fig01v2.jpg"> 图1：通过离心浓缩水样。 （A） 水样池塘，以及 （B） 自然溪流。（C） 基于离心法的水样处理，根据需要重复多次（n）。 <a href="https://www.jove.com/files/ftp_upload/65435/65435fig01largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
DNA提取 根据制造商的说明，使用商业基因组 DNA 试剂盒分离总 DNA（参见 材料表）。将DNA提取物在20μL洗脱缓冲液中洗脱，通过UV分光光度计在260-280nm处测定DNA浓度，并在4°C下储存直至使用。
PCR扩增 PCR 靶标是 16个 S rRNA、lipL41 和 lipL32 基因，它们识别钩端螺旋体属的 DNA，并允许区分三个分支：致病性、腐生性和中间体。引物和探针的设计均基于 Ahmed 等人、Azali 等人、Bourhy 等人、Weiss 等人和 Branger 等人之前的工作33,34,35,36,37。每种探针、引物和扩增片段的序列见表1，其与参考序列的比对见补充文件1、补充文件2、补充文件3、补充文件4和补充文件5。PCR试剂和热循环条件在协议部分进行了描述。
通过在TAE（40mM Tris碱，20mM乙酸和1mM EDTA;pH 8.3）中，在60 V下电泳分离45 min，使用乙锭溴化物检测45分钟来观察扩增产物，如补充图1所示。根据致病性钩端螺旋体的基因组大小（4， 691， 184 bp）38，腐生钩端螺旋体的基因组大小（3， 956， 088 bp）39，从每个血清型获得的基因组DNA的浓度范围为6 x 106至1 x 104个基因组当量拷贝（GEq）， Fainei serovar Hurstbridge菌株BUT6的基因组大小（4,267,324 bp），种质数为AKWZ00000000.2。
在每个实验中，使用来自每个致病性血清型的 DNA 评估探针的灵敏度，即 L. biflexa 血清型 Patoc 菌株 Patoc I 和 L. fainei 血清型 Hurstbridge 菌株 BUT6。为了评估PCR和斑点印迹杂交测定的特异性，包括来自非亲缘细菌的DNA。
表1：PCR引物和探针，用于扩增产物，用于鉴定钩端螺旋体的致病性、腐生菌和中间分支。<a href="https://www.jove.com/files/ftp_upload/65435/TABLE_1_%207th_PR_%20EMCC.xlsx" target="_blank">请按此下载此表格。</a>
斑点印迹杂交试验 该技术被称为点印迹，因为放置 DNA 样品的孔具有点状，当它们被吸入通过真空吸力固定到位时，它们会获得这种形状。该技术由 Kafatos 等人开发[40]。该技术允许对每个PCR阳性样品中的 钩端螺旋体 进行半定量。该方案包括在室温下用NaOH 0.4M变性，将具有30ng至0.05ng的 钩端螺旋 体DNA样品，对应于6×106 至1×104 钩端螺旋体，用96孔斑点印迹装置吸迹到尼龙膜上。固定后，DNA 通过暴露于 120 mJ 紫外线与膜结合。每个 DNA 探针通过 3' 端的末端转移酶催化步骤与地高辛-11 dUTP 偶联（地高辛是从 洋地黄获得的植物类固醇，用作报告基因41）。在将标记的 DNA 探针 （50 pmol） 在特定温度下严格杂交到靶 DNA 上后，通过化学发光反应与抗地高辛碱性磷酸酶抗体与其底物 CSPD 共价偶联来观察 DNA 杂交体。通过暴露于X射线胶片来捕获发光（图2）。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65435/65435fig02v2.jpg"> 图 2：PCR-dot-blot 检测的程序步骤。 <a href="https://www.jove.com/files/ftp_upload/65435/65435fig02largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purelink DNA extraction kit</td>
			<td>Invitrogen</td>
			<td>K182002</td>
			<td></td>
		</tr>
		<tr>
			<td>Gotaq Flexi DNA Polimerase (End-Point PCR Taq polymerase kit)</td>
			<td>Promega</td>
			<td>M3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman filter paper, grade 1,</td>
			<td>Merk</td>
			<td>WHA1001325</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Membranes, positively charged Roll 30cm x 3 m</td>
			<td>Roche</td>
			<td>11417240001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-AP, Fab fragments Sheep Polyclonal Primary-antibody</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium Base EMJH</td>
			<td>Difco</td>
			<td>S1368JAA</td>
			<td></td>
		</tr>
		<tr>
			<td>Leptospira Enrichment EMJH</td>
			<td>Difco</td>
			<td>BD 279510</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking Reagent</td>
			<td>Roche</td>
			<td>11096176001</td>
			<td></td>
		</tr>
		<tr>
			<td>CSPD ready to use Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2&#8242;-(5&#8242;-chloro) tricyclo [3.3.1.13,7] decan}8-4-yl) phenyl phosphate</td>
			<td>Merk</td>
			<td>11755633001</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acid from herring sperm</td>
			<td>Sigma Aldrich</td>
			<td>D3159</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer Carestream</td>
			<td>Carestream Health Inc</td>
			<td>GBX5158621</td>
			<td></td>
		</tr>
		<tr>
			<td>Digoxigenin-11-ddUTP</td>
			<td>Roche</td>
			<td>11363905910</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, Disodium Salt (Dihydrate)</td>
			<td>Promega</td>
			<td>H5032</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Sigma Aldrich</td>
			<td>F8016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixer Carestream</td>
			<td>Carestream Health Inc</td>
			<td>GBX 5158605</td>
			<td></td>
		</tr>
		<tr>
			<td>Lauryl sulfate Sodium Salt (Sodium dodecyl sulfate; SDS) C12H2504SNa</td>
			<td>Sigma Aldrich</td>
			<td>L5750</td>
			<td></td>
		</tr>
		<tr>
			<td>N- Lauroylsarcosine sodium salt CH3(CH2)10CON(CH3) CH2COONa</td>
			<td>Sigma Aldrich</td>
			<td>L-9150</td>
			<td>It is an anionic surfactant</td>
		</tr>
		<tr>
			<td>Polivinylpyrrolidone (PVP-40)</td>
			<td>Sigma Aldrich</td>
			<td>PVP40</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol Sorbitan monolaurate (Tween 20)</td>
			<td>Sigma Aldrich</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma Aldrich</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Terminal transferase, recombinant</td>
			<td>Roche</td>
			<td>3289869103</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride (Tris HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSPE 20X</td>
			<td>Sigma-Aldrich</td>
			<td>S2015-1L</td>
			<td>It can be Home-made following Supplementary File 6</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Sigma-Aldrich</td>
			<td>On demand</td>
			<td>Follow table 1</td>
		</tr>
		<tr>
			<td>Probes</td>
			<td>Sigma-Aldrich</td>
			<td>On demand</td>
			<td>Follow table 1</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop&#8482; One Spectrophotometer</td>
			<td>Thermo-Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated microcentrifuge Sigma 1-14K, suitable for centrifugation of 1.5 ml microcentrifuge tubes at 14,000 rpm</td>
			<td>Sigma-Aldrich</td>
			<td>1-14K</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfected adjustable pipettes, range 2-20 &#181;l, 20-200 &#181;l</td>
			<td>Gilson</td>
			<td>SKU:F167360</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1.5 ml microcentrifuge tubes (autoclaved)</td>
			<td>Axygen</td>
			<td>MCT-150-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 600 &#181;l microcentrifuge tubes (autoclaved)</td>
			<td>Axygen</td>
			<td>3208</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipette tips 1-10 &#181;l</td>
			<td>Axygen</td>
			<td>T-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipette tips 1-200 &#181;l</td>
			<td>Axygen</td>
			<td>TR-222-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Dot-Blot apparatus Bio-Dot</td>
			<td>BIORAD</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable Hergom Suction</td>
			<td>Hergom</td>
			<td>7E-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Light Box (Visible-light PH90-115V)</td>
			<td>Hoefer</td>
			<td>PH90-115V</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Hoefer</td>
			<td>UVC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Hybaid PCR Express Thermocycler</td>
			<td>Hybaid</td>
			<td>HBPX110</td>
			<td></td>
		</tr>
		<tr>
			<td>Radiographic cassette with IP Plate14 X 17</td>
			<td>Fuji</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

polymerase chain reaction and dot-blot hybridization for leptospira detection in water samples

斑点印迹是一种简单、快速、灵敏且用途广泛的技术，能够在存在载体 DNA 的情况下鉴定探针杂交特异性靶向的最小量 DNA。它基于将已知量的 DNA 转移到惰性固体支持物（例如尼龙膜）上，利用斑点印迹装置，无需电泳分离。尼龙膜具有核酸结合载量高（400 μg/cm2）、强度高、带正电荷或带中性电荷等优点。使用的探针是高度特异性的 ssDNA 片段，由 18 至 20 个碱基组成，用地高辛 （DIG） 长标记。探针将与 钩端螺旋体 DNA 结合。一旦探针与目标 DNA 杂交，它就会被抗地高辛抗体检测到，从而可以通过 X 射线胶片中显示的发射物轻松检测。带有发射的点将对应于感兴趣的 DNA 片段。该方法采用探针的非同位素标记，其半衰期可能非常长。这种标准免疫标记物的缺点是灵敏度低于同位素探针。然而，通过偶联聚合酶链反应 （PCR） 和斑点印迹测定可以缓解这种情况。这种方法可以富集靶序列并进行检测。此外，与知名标准品的系列稀释液相比，它可以用作定量应用。本文介绍了一种斑点印迹法检测水样中三个主要分支钩 端螺旋体 的应用方法。一旦通过离心浓缩了大量的水，就可以将这种方法应用于大量的水，以提供钩端螺旋体DNA存在的证据。对于一般筛查目的，这是一种有价值且令人满意的工具，可用于水中可能存在的其他不可培养细菌，从而增强对生态系统的理解。

Leptospirosis in humans mainly originates from environmental sources1,2. The presence of Leptospira in lakes, rivers, and streams is an indicator of leptospirosis transmission among wildlife, and domestic and production animals that may eventually come into contact with these bodies of water1,3,4. Furthermore, Leptospira has been identified in non-natural sources, including sewage, stagnant and tap water5,6.
Leptospira is a worldwide distributed bacteria7,8, and the role of the environment in its preservation and transmission has been well recognized. Leptospira can survive in drinking water under variable pH and minerals9, and in natural bodies of water1. It can also survive for long periods in distilled water10, and under constant pH (7.8), it may survive up to 152 days11. Moreover, Leptospira may interact in bacterial consortiums to survive harsh conditions12,13.&#160;It may be part of biofilms in freshwater with Azospirillum and Sphingomonas and is even capable of growing and enduring temperatures exceeding 49 &#176;C14,15. It can also multiply in waterlogged soil and remain viable for up to 379 days16, preserving its ability to cause the disease for as long as a year17,18. However, little is known about the ecology within water bodies and how it is distributed within them.
Since its discovery, the study of the genus Leptospira was based on serological tests. It was not until the current century that molecular techniques became more prevalent in the study of this spirochaete. The dot-blot has been scarcely used for its identification using (1) an isotopic probe based on the 16S rRNA and on an inter-simple sequence repeat (ISSR)19,20, (2) as a nanogold based immunoassay for human leptospirosis applied to urine21, or (3) as an antibody-based assay for bovine urine samples22. The technique fell in disuse because it was originally based on isotopic probes. However, it is a well-known technique that, coupled with PCR, yields enhanced results, and it is considered safe due to the use of non-isotopic probes. PCR plays a crucial role in the enrichment of the Leptospira DNA by amplifying a specific DNA fragment that may be found in trace amounts in a sample. During each PCR cycle, the amount of the targeted DNA fragment is doubled in the reaction. At the end of the reaction, the amplicon has been multiplied by a factor of more than a million23. The product amplified by PCR, often not visible in agarose electrophoresis, becomes visible through specific hybridization with a DIG-labeled probe in the dot-blot24,25,26.
The dot-blot technique is simple, robust, and suitable for numerous samples, making it accessible to laboratories with limited resources. It has been employed in a variety of bacteria studies, including (1) oral bacteria27, (2) other sample types such as food and feces28, and (3) the identification of unculturable bacteria29, often in agreement with other molecular techniques. Among the advantages offered by the dot-blot technique are: (1) The membrane has a high binding capacity, capable of binding over 200 &#956;g/cm2 of nucleic acids and up to 400 &#956;g/cm2; (2) Dot-blot results can be visually interpreted without requiring special equipment, and (3) they can be conveniently stored for years at room temperature (RT).
The genus Leptospira has been classified into pathogenic, intermediate, and saprophytic clades30,31. The distinction among these clades can be achieved based on specific genes such as lipL41, lipL32, and the 16S&#160;rRNA. LipL32 is present in the pathogenic clades and exhibits high sensitivity in various serological and molecular tools, whereas it is absent in saprophyte species21. The housekeeping gene lipL41 is&#160;known for its stable expression and used in molecular techniques32, while the 16S&#160;rRNA gene is utilized for their classification.
This methodology can be applied to large volumes of water once they have been concentrated by centrifugation. It allows the assessment of various points and depths within a water body to detect the presence of leptospiral DNA and the clade to which it belongs. This tool is valuable for both ecological and general screening purposes and can also be employed to detect other non-culturable bacteria that may be present in water.
Additionally, PCR and dot-blot assays are technically and economically affordable to a wide range of laboratories, even those lacking sophisticated or expensive equipment. This study aims to apply the digoxigenin-based dot-blot for the identification of the three Leptospira clades in water samples collected from natural bodies of water.
Bacterial strains 
Twelve Leptospira serovars (Autumnalis, Bataviae, Bratislava, Canicola, Celledoni, Grippothyphosa, Hardjoprajitno, Icterohaemorrhagiae, Pomona, Pyrogenes, Tarassovi, and Wolffi) were included in this study. These serovars are part of the collection at the Department of Microbiology and Immunology, Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico,&#160;and they are currently used in the microagglutination test (MAT).
All Leptospira serovars were cultured in EMJH, and their DNA was extracted using a commercial DNA extraction kit (see Table of Materials). A genomic DNA mix of the twelve serovars was used as a positive control for the Leptospira pathogenic clade. As a positive control of the Leptospira intermediate clade, genomic DNA from Leptospira fainei serovar Hurstbridge strain BUT6 was included, and as a positive control for the Leptospira saprophyte clade, genomic DNA of Leptospira biflexa serovar Patoc strain Patoc I, was also included.
Negative controls consisted of an empty plasmid, DNA from non-related bacteria (Ureaplasma urealyticum, Staphylococcus aureus, Brucella abortus, Salmonella typhimurium, Shigella boydii, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli), and PCR-grade water, which served as non-template control.
Water samples 
Twelve trial grab samples were collected using a stratified-haphazard sampling method from the Cuemanco Biological and Aquaculture Research Center (CIBAC) (19&#176; 16&#39; 54&#34; N 99&#176; 6&#39; 11&#34; W). These samples were obtained at three depths: superficial, 10, and 30 cm (Figure 1A, B). The water collection procedures did not impact any endangered or protected species. Each sample was collected in a sterile 15 mL microcentrifuge tube. To collect the sample, each tube was gently submerged in the water, filled at the selected depth, and then sealed. The samples were maintained at 22 &#176;C and promptly transported to the laboratory for processing.
Each sample was concentrated by centrifugation in sterile 1.5 mL microcentrifuge tubes at 8000 x g for 20 min at room temperature. This step was repeated until all the samples were concentrated into one tube, which was then used for DNA extraction (Figure 1C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65435/65435fig01v2.jpg" /> 
Figure 1: Concentration of water samples by centrifugation. (A) Water sampling ponds, and (B) Natural streams. (C) Centrifugation-based water sample processing in repeated steps as many times as needed (n). <a href="https://www.jove.com/files/ftp_upload/65435/65435fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
DNA extraction 
Total DNA was isolated using a commercial Genomic DNA kit according to the manufacturer&#39;s instructions (see Table of Materials). DNA extractions were eluted in 20 &#181;L of elution buffer, and DNA concentration was determined by a UV Spectrophotometer at 260-280 nm, and stored at 4 &#176;C until use.
PCR amplification 
The PCR targets were the 16S rRNA, lipL41, and lipL32 genes, which identify DNA from the genus Leptospira and allow the distinction among the three clades: pathogenic, saprophytic, and intermediate. Both primers and probe designs were based on the previous works by Ahmed et al., Azali et al., Bourhy et al., Weiss et al., and Branger et al.33,34,35,36,37. The sequence of each probe, primer, and amplified fragment are described in Table 1, and their alignment with reference sequences are provided in Supplementary File 1, Supplementary File 2, Supplementary File 3, Supplementary File 4, and Supplementary File 5. The PCR reagents and thermocycling conditions are described in the protocol section.
Amplification products were visualized by electrophoretic separation on a 1% agarose gel in TAE (40 mM Tris base, 20 mM Acetic acid, and 1 mM EDTA; pH 8.3), at 60 V for 45 min with ethidium-bromide detection, as shown in Supplementary Figure 1. Genomic DNA obtained from each serovar was used with concentrations ranging from 6 x 106 to 1 x 104 genomic equivalent copies (GEq) in each PCR reaction, based on the genome size of L. interrogans (4, 691, 184 bp)38 for pathogenic Leptospira, the genome size of L. biflexa (3, 956, 088 bp)39 for saprophytic Leptospira, and the genome size of L. fainei serovar Hurstbridge strain BUT6 (4, 267, 324 bp) with accession number AKWZ00000000.2.
The sensitivity of the probes was assessed with DNA from each pathogenic serovar, L. biflexa serovar Patoc strain Patoc I, and L. fainei serovar Hurstbridge strain BUT6 in each experiment. To assess the specificity of the PCR and dot-blot hybridization assay, DNA from non-related bacteria was included.
Table 1: PCR primers and probes to amplify products for identifying the pathogenic, saprophyte, and intermediate clades of Leptospira. <a href="https://www.jove.com/files/ftp_upload/65435/TABLE_1_ 7th_PR_ EMCC.xlsx" target="_blank">Please click here to download this Table.</a>
Dot-blot hybridization assay 
The technique is called dot-blot because the holes in which the DNA sample is placed have a dot shape, and when they are sucked to be fixed in place by vacuum suction, they acquire this shape. This technique was developed by Kafatos et al.40. The technique allows the semi-quantification of Leptospira in each PCR-positive sample. The protocol consists of a denaturation with NaOH 0.4 M at room temperature, samples with Leptospira DNA from 30 ng to 0.05 ng, corresponding to 6 x 106 to 1 x 104 leptospires, are blotted onto a nylon membrane with a 96-well dot-blot apparatus. After immobilization, the DNA is bound to the membrane by exposure to 120 mJ UV light. Each DNA probe is conjugated with digoxigenin-11 dUTP by a terminal transferase catalysis step at the 3&#39; end (Digoxigenin is a plant steroid obtained from Digitalis purpurea, used&#160;as a reporter41). Following the stringent hybridization of the labeled DNA probe (50 pmol) at the specific temperature onto the target DNA, the DNA hybrids are visualized by the chemiluminescence reaction with the anti-digoxigenin alkaline phosphatase antibody covalently conjugated with its substrate CSPD. The luminescence is captured by exposure to an X-ray film (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65435/65435fig02v2.jpg" /> 
Figure 2: Steps of the procedure for the PCR-dot-blot assay. <a href="https://www.jove.com/files/ftp_upload/65435/65435fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

作者聚焦：用于环境分析和疾病监测的水 中的钩端螺旋体 DNA 检测

聚合酶链反应和斑点-印迹杂交用于水样中 钩端螺旋体 检测

This protocol is designed to identify the presence of Leptospira DNA in water samples. The dot-blot technique is utilized for this purpose, which enables accurate detection and analysis of the DNA. The dot-blot is an easy and versatile non-isotopic technique and may help to answer key questions about the environmental distribution of Leptospira.This method has the potential to assess huge water bodies and aid in disease surveillance.

为了评估该技术的有效性，使用了来自每种 钩端螺旋体 血清型的纯培养物的基因组 DNA 以及分支特异性探针。每个血清型的每次 PCR 反应用 100 ng 基因组 DNA 制备膜，然后用非相关细菌的 8 个基因组 DNA 和 临时钩端螺旋体 血清型的可变浓度基因组 DNA 制备膜。每种测定包括阳性、阴性和非模板对照。这些不相关的基因组DNA对斑点印迹探针没有显示出亲和力。膜分布和斑点印迹膜如 <stro...

在这项研究中，设计了一种斑点印迹应用来检测水样中三个主要分支的 钩端螺旋体 。该方法允许鉴定地高辛标记探针特异性靶向的最小 DNA 量，很容易被抗地高辛抗体检测到。这种方法是用于筛选目的的有价值和令人满意的工具。

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by ...

该协议旨在确定水样中钩端螺旋体DNA的存在。斑点印迹技术用于此目的，可以准确检测和分析 DNA。斑点印迹是一种简单且通用的非同位素技术，可能有助于回答有关钩端螺旋体环境分布的关键问题。这种方法有可能评估巨大的水体并帮助进行疾病监测。

polymerase-chain-reaction-dot-blot-hybridization-for-leptospira

Research

JoVE Journal

Biology

Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 &#181;g/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.

In this study, a dot-blot application was designed to detect Leptospira from the three main clades in water samples. This method allows for the identification of minimal DNA quantities specifically targeted by a digoxigenin-labeled probe, easily detected by an anti-digoxigenin antibody. This approach is a valuable and satisfactory tool for screening purposes.

作者聚焦：用于环境分析和疾病监测的水 中的钩端螺旋体 DNA 检测

作者聚焦：用于环境分析和疾病监测的水中的钩端螺旋体 DNA 检测