作者希望感谢卡米哈里斯的入门设计, 阿什利 McKibbon 和杰西卡托马斯-Ebbett 的拍摄, 和约翰布雷克利, 亚历山德拉福利-伊, 克里斯兰利, 朱莉刘易斯, 凯西太, 阿什利 McKibbon, 克里斯 Zinck, 和罗西的狗出现在视频.a.m.k. 得到加拿大莱姆疾病研究基金会的支持, 项目资金由加拿大自然科学和工程研究理事会 (NSERC) 提供。m.k.b. W. 收到新不伦瑞克省创新基金会 (NBIF) 的薪资资助。

1. 从蜱的 DNA 分离
<ol><li>通过野外采集或兽医和公众的被动监视来获取蜱虫。蜱应死于冷冻, 放置在一个密封的袋子, 并在室温下邮寄。<ol>
<li>使用提交表单, 收集有关该主机的相遇日期和地理位置、蜱类附着状态、寄主种类和最近旅行历史的信息。</li>
		<li>由于巢式 PCR 是固有的易受污染, 确保实验室工作空间的设置, 以尽量减少交叉暴露的样本。这涉及到在独立的专用空间中执行协议的不同元素, 它们相互隔离, 完全清洁和灭菌, 并确保所有仪器都没有污染物。</li>
		<li>在收到标本, 照片的蜱和确定的物种, 发展阶段, 性别, 和充血的状态比较的标识键32 。</li>
		<li>在无菌条件下, 用无菌刀片或手术刀对蜱, 并将两个蜱片放到单独的离心管中。</li>
	</ol>
</li>
	<li>提取总 DNA, 使用任何产生 PCR 相容模板的隔离程序;该协议演示了一种简单的基于螯合的方法。<ol>
<li>首先添加一个适当的体积 (通常介于 50-200 µL) 的裂解螯合试剂到蜱片段。具体数量将取决于标本的大小和充血状态;指南应由制造商提供。质用微杵。</li>
		<li>在60° c 的水浴中孵育样品45分钟, 并短暂的涡旋。</li>
		<li>离心机样品4分钟在 16276 x g (13300 rpm) 在桌面离心。</li>
		<li>将上清液转移到一个新的, 标记微含有50µL 异丙醇, 由倒置混合, 重新离心和在 (1.2.3)。</li>
		<li>醒酒上清液, 用50µL 70% 乙醇冲洗 DNA 颗粒。</li>
		<li>用吸管除去过量的乙醇, 并允许颗粒在室温下风干15分钟。</li>
		<li>为了重 DNA, 添加50µL 1 毫米的 pH 值 7.0, 并在水浴中孵育样品1小时, 在60° c。DNA 现在可以储存在-20 ° c, 用于未来的分子分析。</li>
	</ol>
</li>
</ol>2.螺旋体 OspA和松弛的嵌套 PCR 检测。
注: 一般 PCR 原理和实践的概述由洛伦兹提供, 201233。
<ol><li>合成或获得螺旋体基因特异的外、内寡核苷酸引物。请参见表 1以了解底漆集、它们各自的增大小和熔化温度。</li>
</ol><table border="1"><tbody>
<tr>
<td>底漆名称</td>
			<td>基因靶</td>
			<td colspan="2">序列 (5 "-3")</td>
			<td>增尺寸</td>
			<td>退火温度</td>
		</tr>
<tr>
<td>松弛出 Fw</td>
			<td>松弛</td>
			<td colspan="2">gcatcactttcagggtctca</td>
			<td rowspan="2">503 bp</td>
			<td rowspan="2">c</td>
		</tr>
<tr>
<td>松弛的 Rv</td>
			<td>松弛</td>
			<td colspan="2">tggggaacttgattagcctg</td>
		</tr>
<tr>
<td>在 Fw 松弛</td>
			<td>松弛</td>
			<td colspan="2">ctttaagagttcatgttggag</td>
			<td rowspan="2">447 bp</td>
			<td rowspan="2">°</td>
		</tr>
<tr>
<td>Rv 松弛</td>
			<td>松弛</td>
			<td colspan="2">tcattgccattgcagattgt</td>
		</tr>
<tr>
<td>OspA 出 Fw</td>
			<td>ospa</td>
			<td colspan="2">cttgaagttttcaaagaagat</td>
			<td rowspan="2">487 bp</td>
			<td rowspan="2">c</td>
		</tr>
<tr>
<td>OspA 出 Rv</td>
			<td>ospa</td>
			<td colspan="2">caactgctgacccctctaat</td>
		</tr>
<tr>
<td>OspA 在 Fw</td>
			<td>ospa</td>
			<td colspan="2">acaagagcagacggaaccag</td>
			<td rowspan="2">350 bp</td>
			<td rowspan="2">°</td>
		</tr>
<tr>
<td>OspA 在 Rv</td>
			<td>ospa</td>
			<td colspan="2">ttggtgccatttgagtcgta</td>
		</tr>
</tbody></table>表 1: 螺旋体 burgdorferiFlaB 和 OspA nPCR 的内、外底漆。 在实践中,松弛底漆检测B. 螺旋体和其他密切相关的螺旋体基因, 而OspA引物捕获仅b. 螺旋体两个位点的放大将表明b。螺旋体.s。s
<ol start="2"><li>用 UV 光和70% 乙醇对 PCR 机柜进行预灭菌。 注意: 为了尽量减少潜在的样品污染, 这个工作区应该有别于蜱解剖, DNA 提取, 凝胶电泳的位置。<ol>
<li>对于初始 PCR 运行, 使用外部引物与在上面步骤1.0 中恢复的模板 DNA 结合, 并按照表 2中的描述设置反应混合物。同时, 执行由验证的螺旋体DNA 组成的正控制运行, 负控制运行包括无模板反应来检测试剂和气溶胶污染。在末端添加 DNA 以尽量减少试剂的潜在污染。确保在任何时候, 当试剂在 DNA 添加后和其他管子打开之前没有被加入和关闭, 每管都是闭合的。</li>
		<li>程序热循环如下:95 ° c 为 5 min;40循环95° c 为十五年代, 退火温度为三十年代, 72 ° c 为四十五年代;72° c 为 5 min;并保持在4° c。</li>
	</ol>
</li>
	<li>执行第二轮 pcr 类似的第一反应, 除了使用内引物与2µL 的第一个 pcr 产品 (产生于 2.2.2)。 注意: 在表 2中再次提供了反应卷。必须小心, 以避免交叉污染的增, 确保模板 DNA 的最后添加, 只有一个与一个样本对应的管道是开放的一次。 
	 
	<table border="1"><tbody>
<tr>
<td>组件</td>
				<td>PCR 1 容量</td>
				<td>PCR 2 容量</td>
			</tr>
<tr>
<td>Taq 聚合酶主混合2X</td>
				<td>12.5 µL</td>
				<td>12.5 µL</td>
			</tr>
<tr>
<td>无核酸水</td>
				<td>8.5 µL</td>
				<td>8.5 µL</td>
			</tr>
<tr>
<td>10µM 正向和反向引物</td>
				<td>1.0 µL 外底漆</td>
				<td>1.0 µL 内底漆</td>
			</tr>
<tr>
<td>DNA 模板</td>
				<td>2.0 µL, 从样品提取 (1.2.7)</td>
				<td>2.0 µL, 从1轮 PCR (2.2.2)</td>
			</tr>
</tbody></table>
表 2: 第一和第二放大的 PCR 反应混合物。 
	 
	<ol>
<li>程序的热循环, 但调整退火温度, 以适应内引物 (参见表 1)。</li>
	</ol>
</li>
</ol>3. 琼脂糖凝胶电泳及成像
注: 有关用电泳分离 DNA 的基本说明, 请参阅 Lee et al., 201234。
<ol><li>准备一个1.2% 琼脂糖凝胶使用 20X SB 缓冲器 (0.2 米氢氧化钠, 0.8 米硼酸 pH 8), 稀释到 1X, 并添加约5µL 的 DNA 染色, 在浇注前, 如果前染色的凝胶是理想的。</li>
	<li>从每个实验和对照样品中的第二 (内) PCR 反应中装载10µL 的产品, 同时还有 100 bp 梯 (5 µL)。<ol>
<li>Electrophorese 为1小时, 在 107 v (5 伏/厘米)。</li>
	</ol>
</li>
	<li>使用 transilluminator 和相关的照相机和软件查看和记录凝胶。</li>
</ol>

作者没有什么可透露的。

经过几十年的使用, PCR 技术一直证明它们的价值, 在检测螺旋体从节肢动物和哺乳动物标本, 无论是螺旋体来自环境或临床来源。PCR 提供了许多改进前的现有方法的监测。值得注意的是, 它不依赖于抗体试剂的开发和性能, 而是可以简单地通过修改底漆序列来检测新的感兴趣的目标。PCR 也可以独立地或通过复用反应对多个基因座进行平行的评估。它可以应用于不同的标本输入, 包括新鲜和存档蜱22, 动物或人体体液, 和切除组织25。PCR 也产生增, 可以进一步处理, 例如通过限制酶消化, 探针杂交, 或测序, 以提供更多的洞察微生物的身份21。
作为一种临床工具, PCR 在概念上比标准的血清学诊断更可取, 因为它提供了细菌存在的直接指示, 而不是依赖于宿主免疫应答作为继发性措施。然而, 莱姆疏是与一个相对适中的微生物负担 (< 50 生物体/毫升的尿或血浆) 和瞬态 spirochetemia, 这可能导致假阴性结果25。该技术在临床上的敏感性因组织类型、感染阶段和样本状况的不同而有很大差异, 在现有的血液、脑脊液和活检组织的研究中, 在12.5% 和62% 之间下降了36。如果 PCR 是在细菌培养之后进行的, 那么分子敏感性限制就不是一个问题, 然而从临床标本中恢复可行的螺旋体也同样具有挑战性。只有最近才对协议进行了优化, 以获得更高的收益率35。同时, 受感染的成人蜱的肠道平均可以从2000到 5万螺旋体37,38,39, 这是在 nPCR 的检测范围内稳固。因此, 该议定书非常适合于监测工作。
尽管它有许多优点, nPCR 确实带来了某些挑战, 因此, 这种技术准确报告蜱感染的能力取决于基因的战略选择和增, 细致的实验工作流程, 以恢复质量的 DNA从标本, 同时尽量减少交叉暴露样品, 并使用适当的控制, 可以报告污染物在实验室环境和试剂。在进行任何试验之前, 应明确界定调查的范围和意图, 以便能够选择适当的基因所在地和其中的区域。如果目标是为莱姆导致的螺旋体螺旋体广义复数提供一个无偏的屏幕, 则应创建底漆, 以检测具有相似亲和力和放大效率的所有相关菌株, 而不捕获无关有机体25。新的引物可以设计和评估在硅片使用的软件工具, 如底漆爆破40, 虽然它们也应该在实验中对标准参照分离器进行验证, 然后再应用于 uncharacterized 样品。DNA 提取过程的目的是从混合环境样品 (蜱匀浆) 中回收完整的微生物模板。在进行 PCR 之前的一个可选步骤是评估提取的 DNA 的完整性。此外, 还可以设置平行反应, 以在蜱中的一个看家基因为目标。后者的方法也可以表明在反应混合物中存在抑制剂, 提供了更多的信心, 消极的螺旋体反应是由于缺乏有机体, 而不是存在的抑制性污染物。
嵌套 PCR 方法的灵敏度增加, 是以产生假阳性结果的潜在污染为代价的。外源模板可以被引入到样品的蜱解剖和 DNA 恢复, 或在建立的过程中, 外部和内部放大反应。因此, 遵循最佳的 PCR 方法以避免模板或增交叉污染是特别重要的。这些措施包括使用独立的气流, 在 PCR 和生物安全柜中适当地进行隔离, 对表面和试剂进行彻底的化学和物理清洗和消毒, 并谨慎处理 DNA41. 无模板控制对于识别库存试剂的污染也是至关重要的。nPCR 的一个特殊的脆弱性是在将第一反应的产物转移到第二个 PCR 容器时发生的增处理。由于目标 DNA 在阳性样本中已经经历了指数的富集, 这一步尤其容易交叉污染, 因此开发了单管 nPCR 协议来规避这一限制。在这种方法中, 这两套引物的一个给定的基因被加在一起的反应管, 仍然密封的两个回合的 PCR31。外部和内部底漆对必须热力不同, 这样, 外部夫妇放大模板在高退火温度是禁止内引物。在第二轮, 退火温度降低, 以适应内部对。这不仅绕过了一个潜在的混淆步骤, 它还允许使用附加的防污染措施31,42。然而, 这种修改可能会牺牲一些化验灵敏度。无论采用何种方法, 增加在样本上独立进行的技术复制的数量将有助于识别杂散污染。
自从引入 nPCR 以来, 分子技术一直在不断发展, 而这些新的方法比最初的设计更有选择的优势, 尽管财政支出增加了。顾名思义, 定量 pcr (qPCR 或实时 (RT) pcr) 允许在样本中列举病原体, 而传统的技术在性质上是定性的43。据报告, qPCR 的灵敏度与 nPCR38相似, 但它可能会根据底漆特性的不同而波动28。使用分子信标 (MB) 代替常规 TaqMan 探针的 qPCR 的变体在临床标本中的螺旋体检测中也显示了希望44、45、46。由于其独特的二级结构, 分子信标产生较低的背景荧光和更高的合法信号, 从而提供了优越的灵敏度47。临床前评估建议在一个和十螺旋体44,45之间有一个潜在的检测阈值。此外, 该技术已成功应用于多重反应, 同时检测哺乳动物寄主基因44和其他蜱源性致病菌45, 这是不容易实现与 nPCR。其他设计修改包括液滴数字 PCR (ddPCR) 技术, 它可以定量的 DNA 不使用标准曲线39,48。此技术对螺旋体的较低检测限制同样是大约十螺旋体/示例39。与 nPCR 相比, 这些方法还具有降低潜在污染的优点, 因为它们只需要一个单一的放大协议。
如果监测的目的是为了描述蜱的不同细菌含量, 16S rRNA 基因筛选是一个有吸引力的选项49。虽然这种方法是昂贵的, 需要专门的 DNA 器在所有分子生物学实验室没有发现, 并要求更复杂的生物信息学为基础的解释, 它可以捕捉到更准确的微生物广泛的光谱表示载体的病原体负荷。
虽然 qPCR 及其衍生物是定量应用的选择方法, 而基因屏幕提供了蜱菌群的广泛的库存, 目标监视工作往往涉及二进制报告的存在或缺乏一个或几个病原体在一个载体。在这种情况下, 这些新的、更复杂的做法可能会给这一进程带来不必要的复杂性和财政负担。由于这些原因, nPCR 已经经受住了时间的考验, 成为蜱测试的关键技术。

Lyme 疾病 (LD) 是最普遍的传染媒介传染在北半球, 并且它的发病率继续增加1。这种衰弱的疾病是由 spirocheteal 的病原体的莱姆疏 (LB) 复合体 (通常称为螺旋体螺旋体狭义广义, 或广义), 其中历史上包括主要的北美洲病原体, B。螺旋体严格意义上的 (s), 除了b. afzelii和b. garinii,在欧洲和亚洲广泛存在, 并且是新兴临床相关性的物种2,3。这些细菌通过叮咬被传染的蜱滴答声2被传染给人.虽然主要北美洲传染媒介是i. scapularis和i. pacificus, 这个属中的多个物种已被发现窝藏和传播细菌4。在人类中, B. 螺旋体可导致多症状影响皮肤、关节、心脏、神经系统、内分泌腺体、胃肠道和内脏器官5、6、7, 8、9、10。疾病控制和预防中心目前每年估计超过30万新的人类病例在美国11,12。虽然在早期诊断和治疗疾病时, 预后往往是有利的, 但研究表明, 在10% 和60% 的患者中, 接受推荐的抗生素方案的病人在治疗后继续出现症状停止, 在一种现象被称为后处理 Lyme 疾病综合征 (PTLDS)13,14,15。此外, 临床干预的延迟可能是由于缺乏对蜱叮咬的认识、对最初疾病的非特定表述, 以及在感染开始时使用的传统血清学诊断方法的低灵敏度造成的。如果不能迅速而适当地治疗, 就能使症状进展在日益衰弱的并发症中表现出来16,17。因此 Lyme 疾病预防是风险管理的基石。对付这一日益严重的威胁的战略包括强有力的监测措施, 以表明蜱虫病的流行程度, 并确定关注的地理区域。
本文展示了嵌套聚合酶链反应 (nPCR) 作为一种分子筛查工具的效用, 用以识别感染蜱。为了提高特异性, 两个螺旋体基因用于平行放大。鞭毛 B (松弛)对鞭毛18的主要灯丝蛋白进行编码, 而该基因位于单个线性染色体上, 而外表面蛋白 a (OspA) 的脂蛋白产物介导蜱中肠殖民化, 并且是质粒编码的19,20。该工作流包括蜱收集, DNA 提取, 然后第一轮 PCR 检测螺旋体特定的基因座。随后的 PCR 将第一反应的产物作为一个新的模板来生成更小的、内部的放大片段。当从两个螺旋体基因的内增可以通过琼脂糖凝胶电泳检测到螺旋体螺旋体时, 蜱被认为是阳性的。
nPCR 技术, 和松弛和OspA基因目标, 已被广泛用于生态监测和临床检测的莱姆螺旋体自二十世纪九十年代代初2122,23 ,24,25。在分子协议的发展之前, 蜱被评估通过服从肠道内容对反螺旋体免疫荧光 (如果) 染色, 微生物培养或它的组合26。这些方法受到固有的限制, 包括螺旋体27的缓慢增长和吹毛求疵的性质、抗体性能问题, 以及如果处理21的活蜱的要求。随后采用 PCR 技术对感染病媒进行快速、灵敏、特异的鉴定。它提供了明显的改进传统方法, 包括独立于文化的直接应用到不同的标本类型, 如死和存档蜱, 否则将不适合测试21,22.嵌套实验设计通过在连续两轮放大2528中使用两组不同的基因特异引物, 进一步提高了经典 PCR 的特异性和灵敏度。
实验成功的关键在于战略基因的选择和增的设计。为了准确估计螺旋体螺旋体的存在, 引物应在螺旋体属中检测到与复发发热螺旋体的相关病原体, 而不进行交叉反应。在松弛的情况下, 这种特异性是通过针对基因的可变内部区域来实现的, 而不是由不同细菌 (图 1)24,29所共享的相对保守的侧翼序列。,30。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56471/56471fig1.jpg"> 图 1: nPCR 的概念, 如所示使用螺旋体松弛基因作为目标.基因的5和 3 ' 总站是共同的对有机体在螺旋体螺旋体广义之外, 使这些区域不适合对 Lyme 导致的病原体的具体评估。采用不太保守的内部序列作为引物靶, 进行两轮 PCR 检测最终的内部增。<a href="//cloudflare.jove.com/files/ftp_upload/56471/56471fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在测试他们的歧视性能力, 内部的松弛底漆评估了80不同的螺旋体隔离, 并发现只识别那些与莱姆病相关的24。nPCR 的较低检测限度已记录在六24和十31细菌从纯文化, 虽然灵敏度可以进一步改善南部印迹的增, 并杂交 radiolabelled 或化学发光探针。耦合技术将检测阈值减少到单个螺旋体31。通过直接比较, 发现常规的 unprobed 单圆 PCR 报告至少存在 104螺旋体31。然而, 应该指出的是, 在处理复杂的环境和临床样本时, 由于不相关的 DNA 和潜在的抑制物质的存在, 化验灵敏度会降低。这些挑战在很大程度上可以通过使用 nPCR 来规避。
尽管分子技术在过去的几十年中取得了进展, nPCR 仍然是现代监测工作的主要技术。如果在这个协议的设计和执行中被注意到, 它是一种强大的、适应性强且相对简单的方法来捕获病原体在蜱媒中的存在。

<table><tbody><tr><td>LabGard Class II, Type A2 biological safety cabinet</td><td>Nuaire</td><td>ES AIR NU-543&amp;nbsp;</td><td></td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td>0.22 mm thickness</td></tr><tr><td>Microfuge Tubes (Microtubes 1.5 mL)</td><td>Axygen</td><td>311-08-051</td><td></td></tr><tr><td>AquaGenomic&amp;nbsp;</td><td>MultiTarget Pharmaceuticals</td><td>2030</td><td>DNA extraction reagent</td></tr><tr><td>Microtube Pestle</td><td>Diamed</td><td>DIATEC610-1551</td><td>Designed for 1.5 ml tubes</td></tr><tr><td>Water bath - Isotemp 202</td><td>Fishse Scientific</td><td>15-462-2</td><td></td></tr><tr><td>Vortex</td><td>Labnet</td><td>S0100 CAN</td><td></td></tr><tr><td>Spectrafuge 24D Digital Microcentrifuge</td><td>Labnet</td><td>C2400</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>190764</td><td></td></tr><tr><td>BIS-TRIS</td><td>Sigma-Aldrich</td><td>B9754</td><td></td></tr><tr><td>Primer Synthesis</td><td>Sigma-Aldrich</td><td>OLIGO</td><td></td></tr><tr><td>PCR Cabinet</td><td>Misonix</td><td>PCR6-482</td><td></td></tr><tr><td>PCR tube with attached flat caps 0.2mL</td><td>VWR</td><td>20170-012</td><td></td></tr><tr><td>GoTaq Green Master Mix 2x</td><td>Promega</td><td>M7123</td><td></td></tr><tr><td>Nuclease-free water</td><td>Promega</td><td>M7123</td><td>The water comes with the GTG</td></tr><tr><td>Spectrafuge Mini</td><td>Labnet</td><td>C1301</td><td></td></tr><tr><td>SYBR Safe DNA Stain 10,000X Concentration</td><td>EDVOTEK</td><td>608</td><td></td></tr><tr><td>Froggarose LE (Molecular Biology Grade Agarose)</td><td>FroggaBio</td><td>A87-500G</td><td></td></tr><tr><td>GD 100 bp DNA Ladder</td><td>FroggaBio</td><td>DM001-R500&amp;nbsp;</td><td></td></tr><tr><td>Electrophoresis power pack&amp;nbsp;</td><td>VWR</td><td>VWR 105</td><td>EC Apparatus Corporation</td></tr><tr><td>Fluor-S MultiImager&amp;nbsp;</td><td>BioRad</td><td>170-7700 (Serial number 433B0154)</td><td></td></tr><tr><td>Quantity One 1-D Analysis Software (V. 4.5.2)</td><td>BioRad</td><td>1709600</td><td></td></tr></tbody></table>

detecting the lyme disease spirochete, borrelia burgdorferi, in ticks using nested pcr

莱姆病是一个严重的传染媒介感染, 是由螺旋体螺旋体严格广义家庭螺旋体, 这是通过叮咬感染的蜱蜱传给人类。北美洲的主要病因病原体为螺旋体螺旋体。随着地理风险区域的扩大, 谨慎的做法是, 支持能够测量蜱感染率的强健的监视计划, 并将调查结果传达给临床医生、兽医和公众。嵌套聚合酶链反应 (nPCR) 的分子技术早已用于这一目的, 它仍然是检测蜱和野生动物中的螺旋体的中心、廉价和健壮的方法。
本文说明了 nPCR 在 DNA 提取物中的应用, 以识别感染的标本。两个独立的b. 螺旋体目标, 编码鞭毛 b (松弛) 和外表面蛋白 A (OspA) 的基因, 已广泛使用此技术。该协议涉及蜱收集, DNA 提取, 然后第一轮 PCR 检测每一个螺旋体特定的基因座。随后的聚合酶链反应 (PCR) 使用第一反应的产品作为一个新的模板, 以产生更小的, 内部放大片段。嵌套方法提高了常规 PCR 的特异性和灵敏度。当从两个螺旋体基因的内增可以通过琼脂糖凝胶电泳检测时, 病原体的蜱被认为是阳性的。

nPCR 是一个优雅的方法, 以提高特异性和灵敏度的病原体检测, 特别是当复杂的环境样本正在调查。如图 1所示, 针对螺旋体松弛轨迹的战略区域 (和OspA, 而不是图) 的两轮 PCR, 可以通过生成短的内层来报告螺旋体螺旋体细菌的存在。增.当通过凝胶电泳解决时, 每个刻度的松弛和OspA反应产物可以直观地得分, 并与对照组进行比较。在图 2中, 在每个凝胶的顶部都提供了唯一的标本识别符, 而 no 模板和气溶胶控制分别在最左、右的两个梯子旁边表示。
在选择实验样品中, 健壮的增带明显可见, 它们很容易与剩余的引物和残留的 DNA 区分开来 (图 2)。根据所阐述的实验设计原则, 当并行负控制显示不放大时,螺旋体的刻度被视为正值, 而内部增则由松弛和OspA底漆产生。在这种情况下, 标本 T907, T604, 和 T606 符合监视标准的 Lyme 病原体 (图 2)。相比之下, T923 只对OspA是积极的, 这一结果有多种可能的解释: A) 对螺旋体而言, 原点的刻度是负数, 但外部或内部OspA PCR 制剂被地污染模板 DNA (注意, 通过负控制排除了试剂的系统污染), B) 蜱携带莱姆螺旋体, 但低模板量或实验错误阻止放大松弛, 或 C) 有机体存在它包含了保守的OspA序列, 但缺少鞭毛或在松弛的目标区域中没有足够的标识来退火到底漆。实际上, OspA序列的相似性在各种生物体中已经被注意到, 包括植物和动物28。相反的情况, 放大的更保守的鞭毛基因, 但不是OspA基因, 可以表明一个相关的螺旋体物种。实际上, 该协议比松弛引物产生更多的OspA单阳性结果, 这表明方案 "A" 是最不可能的解释。然而, 如果没有对有争议的标本进行进一步的实验分析, 就不可能确定单一阳性结果的来源。增加在模棱两可的样品上进行的技术复制的数量可能有助于调和它们的真实状态, 因为任何被地引入到先前反应中的污染物都不会出现在存档的 DNA 中。然而, 如果随后与这些引物的反应未能提供确凿的结果, 其他的螺旋体位点可以通过 PCR 进行研究。从这些反应产生的增也可以测序和比较的分离, 以帮助分配的身份, 并估计应变发散程度。Sapi 和同事使用人体临床样本35提供了此工作流的示例。
考虑到所有因素, 所列的议定书和解释标准分别估计为假阳性和假阴性率为0.17% 和0.0063%。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56471/56471fig2.jpg"> 图 2: 检测螺旋体螺旋体在单个刻度中按 nPCR 的松弛和OspA.分配给每个刻度的代码都表示在凝胶顶部, 并且标识垂直对齐以报告每个标本中的OspA和松弛的检测。在图左边的 B 的车道代表无模板控制, 而 A (右手车道) 是气溶胶控制捕获实验室环境的污染。<a href="//cloudflare.jove.com/files/ftp_upload/56471/56471fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR at JoVE.com

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

巢式 PCR 是一种灵敏的、特异的、直接的技术, 可以应用于蜱 DNA 提取物来探测莱姆病的致病剂螺旋体螺旋体。最初的 PCR 实验使用基因特异的引物产生长的增, 然后成为模板为随后反应使用内部引物。

检测莱姆病螺旋体,螺旋体螺旋体, 在蜱中使用巢式 PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...

detecting-lyme-disease-spirochete-borrelia-burgdorferi-ticks-using

Research

JoVE Journal

Biology

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

18.0K 次观看.  Mount Allison University. 巢式 PCR 是一种灵敏的、特异的、直接的技术, 可以应用于蜱 DNA 提取物来探测莱姆病的致病剂螺旋体螺旋体。最初的 PCR 实验使用基因特异的引物产生长的增, 然后成为模板为随后反应使用内部引物。

视频: Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

科研

免疫与感染

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

遗传学

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between vertebrates and ticks and dramatically remodels its transcriptional profile to survive in disparate environments during infection. A focus of B. burgdorferi studies is to clearly understand how the bacteria responds to its environment through transcriptional changes. In vitro transcription assays allow for the basic mechanisms of transcriptional regulation to be biochemically dissected. Here, we present a detailed protocol describing B. burgdorferi RNA polymerase purification and storage, sigma factor purification, DNA template generation, and in vitro transcription assays. The protocol describes the use of RNA polymerase purified from B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC is a previously published strain harboring a 10XHis-tag on the rpoC gene encoding the largest subunit of the RNA polymerase. In vitro transcription assays consist of the RNA polymerase purified from strain 5A4-RpoC, a recombinant version of the housekeeping sigma factor RpoD, and a PCR-generated double-stranded DNA template. While the protein purification techniques and approaches to assembling in vitro transcription assays are conceptually well understood and relatively common, handling considerations for RNA polymerases often differ from organism to organism. The protocol presented here is designed for enzymatic studies on the B. burgdorferi RNA polymerase. The method can be adapted to test the role of transcription factors, promoters, and post-translational modifications on the activity of the RNA polymerase.

Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays

In vitro transcription assays can decipher the mechanisms of transcriptional regulation in Borreliella burgdorferi. This protocol describes the steps to purify B. burgdorferi RNA polymerase and perform in vitro transcription reactions. Experimental approaches using in vitro transcription assays require reliable purification and storage of active RNA polymerase.

伯氏疏螺旋体（Borrelia）伯氏疏螺旋体外转录测定的基本成分

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between ...

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

噬菌体介导的莱姆病螺旋体伯氏疏螺旋体的遗传操作

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

血清中 疏螺旋体特异性 IgG 和 IgM 的多重免疫检测

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

作者聚焦:扩大用于莱姆疏螺旋体病诊断和病原体研究的多重免疫检测的范围 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

在多重血清学检测中同时检测不同抗体类别

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

下一代 16S rRNA 扩增子测序的蜱菌群鉴定

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

检测莱姆病螺旋体,螺旋体螺旋体, 在蜱中使用巢式 PCR

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此视频中的章节

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

全挂载原位杂交法可视化蜱内脏微生物群

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

伯氏疏螺旋体（Borrelia）伯氏疏螺旋体外转录测定的基本成分

噬菌体介导的莱姆病螺旋体伯氏疏螺旋体的遗传操作

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别

下一代 16S rRNA 扩增子测序的蜱菌群鉴定