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 border="0" cellpadding="0" cellspacing="0" width="830">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">LabGard Class II, Type A2 biological safety cabinet</td>
			<td style="width:192px;">Nuaire</td>
			<td style="width:140px;">ES AIR NU-543&#160;</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Razor blades</td>
			<td style="width:192px;">VWR</td>
			<td style="width:140px;">55411-050</td>
			<td>0.22 mm thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Microfuge Tubes (Microtubes 1.5 mL)</td>
			<td style="width:192px;">Axygen</td>
			<td style="width:140px;">311-08-051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">AquaGenomic&#160;</td>
			<td style="width:192px;">MultiTarget Pharmaceuticals</td>
			<td style="width:140px;">2030</td>
			<td>DNA extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Microtube Pestle</td>
			<td style="width:192px;">Diamed</td>
			<td style="width:140px;">DIATEC610-1551</td>
			<td>Designed for 1.5 ml tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Water bath - Isotemp 202</td>
			<td style="width:192px;">Fishse Scientific</td>
			<td style="width:140px;">15-462-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Vortex</td>
			<td style="width:192px;">Labnet</td>
			<td style="width:140px;">S0100 CAN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Spectrafuge 24D Digital Microcentrifuge</td>
			<td style="width:192px;">Labnet</td>
			<td style="width:140px;">C2400</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Isopropanol</td>
			<td style="width:192px;">Sigma-Aldrich</td>
			<td style="width:140px;">190764</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">BIS-TRIS</td>
			<td style="width:192px;">Sigma-Aldrich</td>
			<td style="width:140px;">B9754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Primer Synthesis</td>
			<td style="width:192px;">Sigma-Aldrich</td>
			<td style="width:140px;">OLIGO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">PCR Cabinet</td>
			<td style="width:192px;">Misonix</td>
			<td style="width:140px;">PCR6-482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">PCR tube with attached flat caps 0.2mL</td>
			<td style="width:192px;">VWR</td>
			<td style="width:140px;">20170-012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">GoTaq Green Master Mix 2x</td>
			<td style="width:192px;">Promega</td>
			<td style="width:140px;">M7123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Nuclease-free water</td>
			<td style="width:192px;">Promega</td>
			<td style="width:140px;">M7123</td>
			<td>The water comes with the GTG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Spectrafuge Mini</td>
			<td style="width:192px;">Labnet</td>
			<td style="width:140px;">C1301</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">SYBR Safe DNA Stain 10,000X Concentration</td>
			<td style="width:192px;">EDVOTEK</td>
			<td style="width:140px;">608</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Froggarose LE (Molecular Biology Grade Agarose)</td>
			<td style="width:192px;">FroggaBio</td>
			<td style="width:140px;">A87-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">GD 100 bp DNA Ladder</td>
			<td style="width:192px;">FroggaBio</td>
			<td style="width:140px;">DM001-R500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Electrophoresis power pack&#160;</td>
			<td style="width:192px;">VWR</td>
			<td style="width:140px;">VWR 105</td>
			<td>EC Apparatus Corporation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Fluor-S MultiImager&#160;</td>
			<td style="width:192px;">BioRad</td>
			<td style="width:140px;">170-7700 (Serial number 433B0154)</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:299px;">Quantity One 1-D Analysis Software (V. 4.5.2)</td>
			<td style="width:192px;">BioRad</td>
			<td style="width:140px;">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

17.8K 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

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

快速PCR热循环利用微型热对流

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

检测莱姆病螺旋体,螺旋体螺旋体, 在蜱中使用巢式 PCR

摘要

探索更多视频

此视频中的章节