Name: 一种多检测试验为疟疾蚊子发射 (视频) | JoVE Journal
Uploaded: 2015-02-28T14:00:00.000+00:00
Duration: 9 min
Description: 了解基于MALDI-TOF的SNP基因分型平台如何同时检测疟疾媒介蚊子的物种鉴定、杀虫剂抗性和寄生虫感染等多种关键特征，为疟疾防控策略提供重要的分子技术支持。

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我们感谢加州大学戴维斯分校的 Anthony Cornel 博士和 Laura Norris 博士以及格拉斯哥大学的 Katharina Kreppel 博士提供来自坦桑尼亚的蚊类标本。我们感谢女士&#160;我们感谢约翰斯·霍普金斯大学公共卫生学院的 Smita Das 和 Douglas Norris 博士分享来自赞比亚的蚊子样本。我们还感谢兽医遗传学实验室的 Lee V. Millon 先生在检测设计方面的培训。本研究得到了美国国立卫生研究院基金 R01AI 078183 和 R21AI062929 的资助。

1. PCR 扩增
<ol>
	<li>将所有PCR引物（参见补充表S1）混合于一个1.5 ml微量离心管中。每个SNP包含两条引物（正向和反向）。确保每条PCR引物的储备浓度为100 μM。配制足够用于500–1,000次反应的引物混合液，以减少移液误差和实验台操作时间（参见补充表S2）。如需要，可将混合液分装为每管100–200次反应的量，并于-20°C保存。</li>
	<li>按照试剂生产商提供的说明书配制PCR预混液（见表1）。配制96次反应的总体积需额外增加20%的余量。轻轻涡旋混匀含有PCR预混液的离心管，并在2,000 × g条件下离心30秒&#160;。</li>
	<li>使用排液器将3 μl&#160;PCR预混液加入非裙边96孔板的每一孔中。使用多通道移液器可显著节省此步骤的时间。</li>
	<li>向每孔中加入2 μl相应的蚊虫基因组DNA。 
	注：从蚊虫组织（头部/胸部、腹部或整体）提取基因组DNA的方法详见其他文献29–32。应选择合适的DNA提取方案，以区分血液中含Plasmodium（腹部）的个体与具有感染性的蚊虫（Plasmodium位于头部/胸部）。我们通常使用从蚊虫身体部位（头部/胸部或腹部）提取的DNA，因此DNA浓度范围为0.05–2 ng/μl。尽管总DNA投入量低于生产商推荐值（5–10 ng/反应），但在未进行全基因组扩增的情况下，我们通常仍能从98%的DNA样本中获得可靠的SNP分型结果。</li>
	<li>密封反应板，轻柔混匀或短暂涡旋，并在370 × g条件下离心1分钟&#160;。</li>
	<li>采用以下热循环程序进行PCR扩增：(1) 94°C 预变性2分钟，(2) 94°C 变性30秒，(3) 56°C 退火30秒，(4) 72°C 延伸1分钟，(5) 重复步骤(2)–(4)共45个循环，(6) 72°C 最终延伸1分钟，随后4°C保存。</li>
</ol>
2. SAP 治疗
<ol>
	<li>在96孔板中配制SAP酶溶液（见表2）。每块板处理96个样本，96次反应的总体积需额外增加20%的余量。</li>
	<li>轻轻涡旋SAP酶溶液管，然后在2,000 × g条件下离心30秒&#160;。向每个孔中加入2 μl SAP酶溶液。此步骤可使用多通道移液器以节省时间。</li>
	<li>密封反应板，轻轻混匀或短暂涡旋，并在370 × g条件下离心1分钟&#160;。按以下条件孵育反应板：（1）37°C孵育40分钟，（2）85°C孵育5分钟&#160;以灭活酶，（3）4°C保存。此步骤完成后，将反应板储存于-20°C，待后续使用。储存的反应板需在两周内完成后续处理。</li>
</ol>
3. SNP 扩增
<ol>
	<li>若样本板在SAP处理后处于冷冻状态，请先使其升至室温后再进行后续操作。</li>
	<li>制备延伸引物混合液（补充表S2）。每个SNP对应一条延伸引物。每条延伸引物的储备浓度应保持为500 μM。可选：按每管100-200次反应量分装，并于-20°C保存。</li>
	<li>按表3所述配制延伸反应混合液。96次反应的总体积包含20%的过量。</li>
	<li>轻轻涡旋混匀延伸反应混合液管，于2,000 × g离心30秒&#160;。向每个孔中加入2 μl延伸反应混合液。重复移液器可显著节省此步骤的时间。</li>
	<li>密封反应板，轻轻混匀或短暂涡旋，并于370 × g离心1分钟&#160;。按图1所示程序进行热循环。此后，可直接进行质谱分析，或将反应板在-20°C保存最多2周。</li>
</ol>
4. 优化质谱分析的单核苷酸多态性延伸产物的处理
<ol>
	<li>如果样本板在SNP延伸后已冷冻，请在继续操作前使其升至室温（RT）完全解冻。</li>
	<li>使用长柄勺从容器中转移15 mg树脂至凹孔板中。用刮刀将树脂分散至凹孔板的孔内。横向来回刮动刮刀，使树脂均匀分布，确保每个孔中均有树脂。</li>
	<li>用刮刀将凹孔板表面多余的树脂刮除。让树脂在凹孔板中静置至少20分钟。在静置期间，向样本板的每个孔中加入41 μl HPLC级水。</li>
	<li>将样本板轻轻倒置，放置在凹孔板上。确保样本板紧贴凹孔板上的小圆柱定位柱，以使样本板的孔与凹孔板的孔精确对齐。</li>
	<li>将样本板和凹孔板一起握紧，轻轻翻转，使树脂从凹孔板落入样本板的孔中。轻敲凹孔板，确保所有树脂均完全落入样本板的孔内。以3,200 × g离心30秒。</li>
	<li>将样本板置于旋转器上，在室温（RT）下旋转5分钟&#160;，旋转器必须围绕样本板的长轴完成360°旋转。</li>
	<li>以3,200 × g离心样本板5分钟。此步骤完成后，可将样本板在-20°C保存最多2周，之后再进行下一步操作。</li>
</ol>
5. 基质辅助激光解吸电离飞行时间质谱
<ol>
	<li>根据制造商说明，将条件化的 SNP 延伸产物转移至生物芯片（如 SpectroCHIP）。此步骤我们使用 MassArray 纳米点样仪。</li>
	<li>转移完成后，在检测数据库中定义检测和板的设置方式。为检测和板命名时，应确保每块板具有唯一的标识符，以关联到特定的检测设计（补充表 S3）。</li>
	<li>在检测和板定义完成后，使用质谱仪获取质谱数据。</li>
</ol>
6. 数据分析
<ol>
	<li>使用实时 PCR 分析数据解读软件（如 Typer Analyzer 或 TyperViewer（Sequenom））进行数据分析。获取包含样品布局、每个孔的质谱图、标记信息（包括名称、对应 SNP 每个变异体的预期质量）以及与每项反应相关的任何错误或警告信息的 .xml 格式数据。结果质谱图参见图 2。</li>
	<li>通过设定检测阈值（信号强度或信噪比）以及每种基因型允许的容差/方差，进行 SNP 基因型聚类分析。图 3 展示了一个 04707-KDR#2 SNP 基因型聚类的示例。</li>
	<li>将获得的 SNP 基因型分型结果导出至电子表格程序中。</li>
</ol>

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作者无任何利益冲突可披露。

多重等位基因特异性扩增（MDA）包含五个主要步骤：PCR扩增、虾碱性磷酸酶（SAP）反应、SNP延伸、延伸产物纯化以及基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱分析33-37。第一步PCR扩增用于扩增每个SNP位点侧翼的DNA序列，以确保在SNP延伸步骤中有足够的模板DNA。SAP反应可中和未使用的dNTPs，防止其干扰后续的SNP延伸步骤。SNP延伸步骤针对每个SNP位点使用一条特异性延伸引物以及质量修饰的终止核苷酸。3’端修饰的核苷酸可阻止后续核苷酸继续掺入延伸引物中，因此通过测定单个碱基的质量即可确定相应SNP的基因型。
确保本方法成功最关键的一步是拥有目标生物体中已知多态性的一个优质数据集。我们采用了特征明确的单核苷酸多态性（SNP）用于物种鉴定（N&#62;900）10,12,38-40 和 kdr 基因（N&#62;1,000）15,18。恶性疟原虫（Plasmodium）的物种特异性SNP是通过比对来自非洲的123个P. falciparum</...

Anopheles arabiensis，Anopheles coluzzii 和 Anopheles gambiae 是非洲疟疾传播的主要媒介1这三种物种在形态上无法区分2 只能通过分子检测方法加以区分3-9此外，还有许多常规的下游检测方法可用于辅助流行病学和群体遗传学研究。这些方法包括：（1）用于物种形成岛的基因分型检测10-12，（2）一种基因分型检测方法，可识别1014位点的非同义SNPth 氨基酸密码子位置 para 基因（the 击倒抗性，或 kdr, SNP13-18，（3）寄生虫检测 PCR19-23，以及（4）对蚊虫中肠内宿主特异性DNA进行筛查24,25.
我们开发了一种多检测分析法（MDA），将所有这些检测方法整合到一个单一的多重反应中，旨在分析非洲疟疾媒介中具有流行病学重要意义的特征。MDA 分析包含多个用于检测（1）物种的标记冈比亚按蚊、埃及伊蚊、科卢齐按蚊 或其他（三种均无），（2）杀虫剂抗性表现于 kdr SNPs（包括L1014F）和 L1014S）， (3) 两种主要疟原虫的存在, 恶性疟原虫 和 P. vivax，以及（4）来自一种鸟类和六种哺乳动物宿主的血源。
传统上，这些检测需在多个独立的聚合酶链式反应中进行。当使用常规PCR平台完成所有这些检测时，需要进行8-10个PCR反应以及相应的凝胶电泳步骤。每个PCR反应从准备到结果记录需耗时4-5小时，而本文介绍的多重检测扩增（MDA）方法总共仅需约5小时，相当于仅劳动力成本即可节省90%。本文所述的MDA方法每个样本的基因分型成本为5美元，可检测全部33个单核苷酸多态性（SNP）。这明显低于基于单个琼脂糖凝胶的检测方法（每个样本约1.50美元）。若要通过单独的琼脂糖凝胶检测覆盖MDA所包含的全部特征，则至少需要8-10个独立检测，每个样本成本达12-15美元。此外，MDA通过为每种寄生虫或宿主来源的检测使用至少三个标记，显著降低了产生假阳性或假阴性的可能性。
我们所使用的平台不仅限于疟疾媒介的研究，还可广泛应用于医学、兽医学和基础生物学等多个领域26-28。对于涉及大量样本（数量级达数百个）的深入关联研究或群体遗传学研究，需要采用经济高效的检测方法，以同时筛查多个标记位点。大多数需要使用两种或更多独立PCR检测的研究，均可采用多重置换扩增（MDA）方法，以更低的成本更快地获得结果。

<table><tbody><tr><td>MassARRAY Analyzer Compact</td><td>Sequenom</td><td>MT9</td><td>用于基因组学应用中分析核酸的 MALDI-TOF 质谱仪</td></tr><tr><td>MassARRAY Nanodispenser</td><td>Sequenom</td><td>RS1000</td><td>将完成的 iPLEX 反应产物转移至 SpectroCHIP</td></tr><tr><td>iPLEX Gold Genotyping Reagent Set</td><td>Sequenom</td><td>10158</td><td>用于 iPLEX 检测的试剂，包括 SAP 试剂盒</td></tr></tbody></table>

一种用于检测传播疟疾蚊虫的多指标检测方法

A Multi-detection Assay for Malaria Transmitting Mosquitoes

的 Anopheles gambiae 复合种包含非洲主要的疟疾传播蚊虫。由于这些蚊种具有重要的医学意义，通常需通过分子检测方法对多个性状进行表征，以辅助流行病学研究。这些性状包括物种鉴定、杀虫剂抗性、寄生虫感染状态以及宿主偏好。由于该复合种的种群 Anopheles gambiae 复合体在形态上难以区分，传统上使用聚合酶链式反应（PCR）来鉴定物种。一旦确定物种，通常会进行多项后续检测以进一步阐明其特征。例如，para基因中被称为KDR的突变可导致对DDT和拟除虫菊酯类杀虫剂的抗性。此外，酶联免疫吸附试验（ELISAs）或 疟原虫 寄生虫DNA检测PCR实验用于检测蚊虫组织中存在的寄生虫。最后，结合PCR与限制性内切酶消化的方法可用于阐明寄生虫的宿主偏好性例如，通过筛选蚊子血餐中宿主特异性的DNA来区分人血与动物血。我们开发了一种多重检测分析法（MDA），将上述所有检测方法整合到一个单一的多重反应中，可同时对96或384个样本进行33个SNP位点的基因分型。由于该MDA包含了多个物种特异性标记， 疟原虫 检测和宿主血液识别，与以往仅使用单一标记物检测每种性状的方法相比，本方法极大降低了假阳性或假阴性结果的产生概率。该检测方法稳定且操作简便，能够以更低的成本和远少于现有方法的时间，高效检测这些关键的蚊虫性状。

物种鉴定：
以下5个SNP共同用于鉴定三种物种（A. arabiensis、A. coluzzii 和 A. gambiae）（表4）。如果样本不属于这三种物种之一，则三个SNP位点（01073-213、04679-157 和 10313-052）无法扩增。
通过 kdr 基因型推断杀虫剂抗性：
1014th 密码子的 用于 电压门控钠通道对应于 kdr. 染色体2上的两个SNPnd 和 3rd 1014 的底部th 密码子决定三种可能的氨基酸。各种基因型的可能组合列于 表5该SNP适用于三种非洲疟疾传播媒介物种： A. arabiensis, A. coluzzii 和 <e...

传播疟疾的蚊子具有一些重要的流行病学特征，这些特征只能通过分子技术进行检测。我们利用基于 MALDI-TOF 的 SNP 基因分型平台，开发了一种可同时检测疟疾媒介多种关键性状（种类、杀虫剂抗性、寄生虫感染和宿主选择）的检测方法。

冈比亚按蚊复合体包含非洲主要的疟疾传播蚊子。由于这些物种具有重要的医学意义，……

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一种多检测试验为疟疾蚊子发射

一旦某个基因被鉴定为可能具有抗疟疾特性，就必须评估其在蚊子体内抑制疟原虫感染的作用。本方案展示了如何检测疟原虫在蚊子体内的感染程度。文中演示了配子体培养的制备、通过膜饲法使蚊子吸食含人类血液的样本，以及检测蚊子中肠内病毒滴度的技术。...

恶性疟原虫蚊虫感染方案及感染表型测定

通过将两种基因型不同的寄生虫喂给蚊子，可实现啮齿类疟原虫的遗传杂交。在允许蚊子叮咬感染小鼠后，从小鼠血液中分离并克隆重组子代。本视频展示了如何对Plasmodium...

啮齿类疟原虫遗传杂交实验方案

本视频展示了一种通过血液饲喂来评估具有新型作用机制的测试化合物对雌性蚊虫致死率和生殖力影响的实验方法。该技术有助于筛选新型杀虫剂，以杀灭抗药性蚊虫种群并控制疾病传播。...

蚊媒喂食实验：一种评估测试化合物对成年雌性蚊虫死亡率和繁殖力影响的方法

尽管高分辨率熔解分析能够区分异质群体中的单核苷酸多态性，但突变等位基因的扩增偏好性可增强其检测样本中相对低比例等位基因的能力。本方案描述了提高高分辨率熔解分析灵敏度的改进方法。...

提高疟疾高分辨率熔解单核苷酸多态性分型灵敏度的方法

由于啮齿类疟原虫在生命周期和生物学特性上与人类疟原虫具有显著相似性，啮齿类疟疾模型已成为疟疾研究不可或缺的工具。本文中，我们对野生型和转基因啮齿类疟原虫进行表型分析时所使用的一些最重要技术进行了标准化。...

啮齿类疟原虫无性与有性血液阶段及蚊期阶段的表型分析

目前用于检测寨卡病毒、基孔肯雅病毒和登革病毒的多重诊断方法需要复杂的样品制备过程和昂贵的仪器设备，难以在资源有限的环境中应用。我们展示了一种基于等温扩增技术并结合靶标特异性链置换探针的诊断方法，可高灵敏度、高特异性地检测并区分这些病毒。...

基于多重等温扩增的诊断平台用于检测寨卡病毒、基孔肯雅病毒和登革病毒1型

本方案可在封闭环境中测定埃及伊蚊（Aedes...

媒介能力分析 埃及伊蚊 蚊子传播寨卡病毒

本方案描述了一种用于检测蚊虫唾液腺子孢子的夹心酶联免疫吸附测定法。该方法利用易于获取的单克隆抗体，可实现对携带恶性疟原虫（Plasmodium falciparum）或间日疟原虫（Plasmodium...

检测 疟原虫 子孢子在 按蚊属 利用酶联免疫吸附测定法检测蚊子

标准膜饲法（SMFA）被认为是评估和鉴定潜在抗疟化合物的金标准。该人工饲喂系统用于感染蚊子，以进一步评价这些化合物对Plasmodium...

标准膜饲法检测 恶性疟原虫 感染 按蚊属 蚊媒

多重荧光免疫测定正成为血清学检测的参考方法。然而，其应用受限于复杂且耗时的微球-分析物偶联过程。本报告介绍了一种半自动化、可重复的多重微球偶联方案，其效果与标准方法相当，但显著缩短了获得结果所需的时间。...

一种用于检测虫媒病毒抗体反应的多重血清学检测方法

了解基于MALDI-TOF的SNP基因分型平台如何同时检测疟疾媒介蚊子的物种鉴定、杀虫剂抗性和寄生虫感染等多种关键特征，为疟疾防控策略提供重要的分子技术支持。

一种用于检测传播疟疾蚊虫的多指标检测方法

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