Name: digital pcr-based competitive index for high-throughput analysis of fitness in salmonella
Uploaded: 2019-05-13T15:00:00
Description: Watch this Scientific Journal Video about Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella at JoVE.com

本出版物中报告的研究得到了乔治·哈迪克斯总统学院研究基金和国家卫生研究院国家普通医学研究所的支持,奖励编号为GM103427。内容完全由作者负责,不一定代表国家卫生研究院的官方观点。

1. 将独特的DNA条形码纳入含有等位交换必要成分的质粒中
注:与现有的pKD13等位体交换质粒相比,创建了一种新的质粒,名为pSKAP,具有较高的拷贝数和更高的转换效率。这在步骤 1.1-1.12 中描述 (图 1.包含唯一DNA条形码和等位交换成分的最终质粒可通过质粒库(材料表)获得。
<ol><li>使用商业质粒小提具套件,纯化 pKD1314</su...

<ol>
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准确量化微生物的能力对微生物研究至关重要,从最初的混合种群中列举独特菌株的能力已被证明是评估微生物的适应性和毒性特征的宝贵工具。细菌。然而,实现这一目的的技术并没有随着分子生物学的现代发展而进步。该技术可轻松修改许多细菌的染色体,包括S.Typhimurium,已经可用了近20年14,然而这种能力很少用于分子标记菌株与独特的DNA序列。通过利用基于插入细菌染色...

评估致病微生物的适应性和毒性是微生物学研究的一个基本方面。它允许在菌株之间或突变的生物体之间进行比较,使研究人员能够确定特定条件下某些基因的重要性。传统上,毒性评估利用动物感染模型使用不同的细菌菌株,并观察受感染动物的结果(例如,传染性剂量50,致命剂量50,死亡时间,症状严重程度,缺乏症状等)。 这个程序提供了宝贵的毒性描述,但它需要菌株导致结果相当大的差异,以检测从野生类型的变化。此外,结果是半定量的,因为虽然疾病进展和症状严重性可以随着时间的推移主观量化,但与野生类型相比,对毒性的解释更定性(即更多、更少或同样具有毒性)。执行动物感染性测定的一种常见替代方法是生成竞争指数(CIs),该值直接将菌株的适应性或毒性与混合感染1中的野生型对应物进行比较。与传统的动物感染模式相比,该技术具有许多优点,通过标准化对野生型菌株的毒性,并确定可量化的值以反映衰减程度。这项技术还可以通过确定取消的竞争指数(COI)2来调整,以分析细菌的基因相互作用。计算一组突变生物的COI,使研究人员能够确定两个基因是否独立地导致发病机制,或者它们是否参与同一毒治途径并相互依赖。此外,计算 CI 需要对细菌进行枚举,从而对生物体的发病机制提供有价值的见解。CIs 和 COIs 还允许研究人员评估不会导致临床疾病但仍在健身方面存在差异的阿维鲁特菌株。这种技术受到使用传统抗生素耐药性标记来识别菌株的限制,从而将输入菌株的数量限制在一次只有一两个。由于这种限制,需要大量的实验组和复制,这除了增加劳动力和材料成本外,还增加了实验条件变异的机会和不准确的结果。(有关使用混合感染研究毒性、适应性和基因相互作用的好处和应用的透彻回顾,请参阅 C.R. Beuzón 和 D.W. Holden 1)
已经尝试克服这一限制,例如使用荧光标记的细胞量化通过流细胞学3,4,5。此技术使用标记的抗体对细胞进行量化,以表示型状标记或 2) 内分泌产生的荧光蛋白。使用标记抗体的检测量限制在1000个细胞/mL,因此需要大量细胞来分析3。表达荧光蛋白的细胞生理变化,易受高蛋白表达6引起的健身变化的影响。这两种方法都受使用流式细胞测定可检测的荧光标记的数量的限制。分子定量的进步是通过开发一种微阵列技术实现的,该技术在120个菌株中检测到120个菌株的衰减,最初混合感染了1000多个菌株,在鼠模型7中。该技术利用了突变菌株对RNA的微阵列分析,导致结果具有相当大的变异性。 然而,它确定,大量的混合感染池可以是一个有用的工具,并且通过利用敏感的检测技术,可以识别细菌毒性的差异。随着下一代测序的发展,Tn-seq扩展了转波龙突变的效用,为随机突变的8、9、10细菌提供了强有力的量化方法。 11.最近开发了一种替代方案,消除了转座子的需要,而是使用DNA条形码更容易地识别和跟踪基因组变化及其对适应性12的影响。这项技术是一项重大进步,但基因组条形码的插入仍是一个随机的过程。为了克服以往实验的随机性,Yoon等人开发了一种利用插入细菌13染色体精确位置的独特DNA条形码来计算沙门氏菌菌株的CIs的方法。使用基于 qPCR 的方法检测出独特的条形码菌株,该方法具有 SYBR 绿色和特定于每个唯一条形码的引油。该技术受到qPCR施加的限制,包括引漆效率和低灵敏度的差异,qPCR之前需要嵌套PCR就证明了这一点。然而,这种方法表明,靶向基因组修饰可用于检测和潜在量化多个细菌菌株池。
在下面的协议中,我们描述了一种新方法,用于使用大型混合接种池进行细菌竞争实验,然后使用高度敏感的数字PCR技术进行精确定量。该协议涉及基因标记的细菌菌株,在染色体的无害区域插入独特的DNA条形码。这种修饰允许使用现代分子技术快速准确地量化菌株,而不是传统的序列稀释、复制电镀和计数依赖表型标记的菌群形成单位(即抗生素耐药性)).这些修改允许在单个池接种中同时评估许多菌株,大大减少了实验变异的可能性,因为所有菌株都暴露在完全相同的条件下。此外,虽然这项技术是在沙门氏菌中开发的,但它对任何遗传可塑性有机体和几乎任何需要精确细菌计数的实验设计都具有很强的适应性,提供了新的工具,以提高微生物实验室的准确性和产量,不受以前方法的限制。

<table>
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22600028</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>16 mL culture tubes</td>
			<td>MidSci</td>
			<td>8599</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>5-200 &#956;L pipette tips</td>
			<td>RAININ</td>
			<td>30389241</td>
			<td>Procure alternative tip brands with caution based on manufacturing quality</td>
		</tr>
		<tr>
			<td>5-50 &#956;L multichannel pipette</td>
			<td>RAININ</td>
			<td>17013804</td>
			<td>Use alternative multichannel pipettes with caution</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>BP160-500</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>BLAST Analysis</td>
			<td>NCBI</td>
			<td>N/A</td>
			<td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermocycler with 96-Deep Well Reaction Module</td>
			<td>Bio Rad</td>
			<td>1851197</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>Chemically competent DH5&#945;</td>
			<td>Invitrogen</td>
			<td>18258012</td>
			<td>Procure from any manufacturer or prepare yourself</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>ThermoFisher Scientific</td>
			<td>BP904-100</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Cytation5 Microplate reader</td>
			<td>BioTek</td>
			<td>CYT5MF</td>
			<td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td>
		</tr>
		<tr>
			<td>Data Analysis Software (QuantaSoft and QuantaSoft Data Analysis Pro)</td>
			<td>Bio Rad</td>
			<td>N/A</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates</td>
			<td>Bio Rad</td>
			<td>12001925</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil</td>
			<td>Bio Rad</td>
			<td>1863004</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP)</td>
			<td>Bio Rad</td>
			<td>1863024</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>DG8 Cartridges for QX200/QX100 Droplet Generator</td>
			<td>Bio Rad</td>
			<td>1864008</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>DG8 Gaskets for QX200/QX100 Droplet Generator</td>
			<td>Bio Rad</td>
			<td>1863009</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes</td>
			<td>Bio Rad</td>
			<td>1863005</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>BP906-5</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Luria-Bertani agar</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1425-2</td>
			<td>Procure from any manufacturer or make it yourself from agar, tryptone, yeast digest, and NaCl</td>
		</tr>
		<tr>
			<td>Luria-Bertani broth</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1426-2</td>
			<td>Procure from any manufacturer or make it yourself from tryptone, yeast digest, and NaCl</td>
		</tr>
		<tr>
			<td>PCR Plate Heat Seal, foil, pierceable</td>
			<td>Bio Rad</td>
			<td>1814040</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>PCR Tubes</td>
			<td>Eppendorf</td>
			<td>951010022</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>ThermoFisher Scientific</td>
			<td>FB0875712</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>pPCR Script Cam SK+</td>
			<td>Stratagene/Agilent</td>
			<td>211192</td>
			<td>No longer available commercially</td>
		</tr>
		<tr>
			<td>Primer/Probe Design</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>https://www.idtdna.com/Primerquest/Home/Index</td>
		</tr>
		<tr>
			<td>pSKAP and pSKAP_Barcodes</td>
			<td>Addgene</td>
			<td>Plasmid numbers 122702-122726</td>
			<td>www.addgene.org</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>Bio Rad</td>
			<td>1814000</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator</td>
			<td>Bio Rad</td>
			<td>1864002</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio Rad</td>
			<td>1864003</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>S. Typhimurium strain ATCC 14028s</td>
			<td>ATCC</td>
			<td>ATCC 14028s</td>
			<td>www.atcc.org</td>
		</tr>
		<tr>
			<td>Take3 Micro-Volume Plate</td>
			<td>BioTek</td>
			<td>TAKE3</td>
			<td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td>
		</tr>
		<tr>
			<td>Thermo Scientific FastDigest BamHI</td>
			<td>ThermoFisher Scientific</td>
			<td>FERFD0054</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific FastDigest DpnI</td>
			<td>ThermoFisher Scientific</td>
			<td>FERFD1704</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific FastDigest HindIII</td>
			<td>ThermoFisher Scientific</td>
			<td>FERFD0504</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>FERK0832</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific GeneJet Miniprep Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>FERK0503</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific Phusion High-Fidelity DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>F534L</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific T4 DNA Ligase</td>
			<td>ThermoFisher Scientific</td>
			<td>FEREL0011</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Bio Rad</td>
			<td>1861096</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>UVP Visi-Blue Transilluminator</td>
			<td>ThermoFisher Scientific</td>
			<td>UV95043301</td>
			<td>Or other transiluminator that allows visualization of DNA</td>
		</tr>
		<tr>
			<td>Water, Molecular Biology Grade</td>
			<td>ThermoFisher Scientific</td>
			<td>BP28191</td>
			<td>Procure from any manufacturer</td>
		</tr>
	</tbody>
</table>

digital pcr-based competitive index for high-throughput analysis of fitness in salmonella

竞争指数是一种常用方法,用于评估细菌的适应性和/或毒性。这种方法的效用体现在其易于执行和标准化许多菌株对野生型生物体的适用性的能力。然而,该技术受限于可用的型型标记和可同时评估的菌株数量,因此需要进行大量的复制实验。在进行大量实验的同时,基于型状标记对细菌进行量化的人工和材料成本并非微不足道。为了克服这些消极方面,同时保留积极方面,我们开发了一种基于分子的方法,在将基因标记工程到细菌染色体上后,直接量化微生物。独特的是 , 25 个碱基对 DNA 条形码入在野生型和突变菌株的染色体上的无害位点。 体外竞争实验使用由池菌株组成的接种。比赛结束后,使用数字PCR量化了每个菌株的绝对数量,并计算了每种菌株的竞争指数。我们的数据表明,这种量化沙门氏菌的方法对于检测高度丰富(高适应性)和稀有(低适应性)微生物具有极其灵敏、准确和精确的检测方法。此外,这种技术很容易适应几乎任何具有染色体的有机体能够修改,以及各种需要绝对定量微生物的实验设计。

使用这种方法需要执行适当的控制反应,以验证用于识别目标DNA的每个探针的敏感性和特异性。在这个具有代表性的实验中,我们验证了8个独特的DNA条形码与8个相应的探针进行鉴定。所有八个探针在NTC和阴性对照反应中的假阳性率都很低(表3),即使在高度相似的DNA序列中,也突出了它们的特异性。为了评估每种情况的敏感性,包含唯一条形码的 gDNA 在包含其余七个?...

Watch this Scientific Journal Video about Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella at JoVE.com

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

基于数字PCR的沙门氏菌健康高通量分析竞争指数

这种基于分子的确定细菌适应性的方法,使用通过数字PCR量化的独特基因组DNA条形码,有助于精确、准确地检测微生物。该协议描述了计算沙门氏菌菌株的竞争指数;然而,该技术很容易适应需要绝对定量任何基因可塑性有机体的协议。

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

该技术使用快速和高通量分子基技术，能够准确准确地定量地定量来自高度相似菌株的混合种群的微生物。归根结底，在能够比较菌株之间的健身。同时评估单个池中多个菌株的能力可显著减少井对井或有机体对有机体的变异性，因为所有菌株都暴露在完全相同的条件下。该技术的适应性使得它几乎可用于任何基因可塑性生物体和几乎任何需要精确定量微生物的实验设计。如文本协议所述，从对细菌染色体进行遗传标记的工程开始。创建包含除一个条形码以外的所有条形码的基因组 DNA 池。使用此池作为稀释剂，使用含有单个剩余条形码的基因组 DNA 执行稀释序列。根据制造商关于使用专为探头化学设计的数字 PCR 超级混合的建议，为数字 PCR 重复准备反应。使用池基因组DNA作为模板DNA。此外，添加 20 X 底像探头主混合，如文本协议中所述。根据制造商的建议为数字 PCR 准备复制控制反应。每个探针组合的控制反应必须包含无模板控件、负控件和正控制。重复这些步骤，为每个条形码基因组DNA样本创建数字PCR反应。根据制造商的说明，使用液滴发生器针对每种反应条件生成液滴。将新创建的液滴转移到适当的 96 井板中。在 5 至 50 微升多通道移液器上使用 200 微升移液器提示。生成并转移所有液滴后，使用铝箔板密封器密封板。使用制造商推荐的热循环器执行循环条件。在进行热循环时，使用板设置信息（如样品名称、实验类型和使用的 Supermix）对数据分析软件进行编程。还包括目标 1 名称和目标 1 类型，以及目标 2 名称和目标 2 类型。热循环完成后，根据制造商的说明，将完成的反应转移到液滴读卡器，并开始读取过程。读取所有孔并完成运行后，使用数据分析软件打开 QLP 数据文件。选择使用相同底像探头母版混合的所有孔。在"液滴"选项卡上，检查每个井中分析的液滴数量，包括正液滴和负液滴。从分析中排除任何总液滴少于 10，000 的井。移动到 1D 振幅选项卡并检查正和负液滴的振幅。确保它们由两个不同的群体组成。在软件中，使用阈值功能为使用的每个探头进行正极和负液滴之间的截止。一旦将适当的阈值应用于所有油井，软件将计算每次反应中的DNA拷贝数。将数据导出到电子表格，以便于进一步分析。使用这些值，计算样本中每个唯一基因组条形码的初始拷贝数。从负控制反应中确定平均误报率，并从实验反应中获得的值中减去此值。根据设置每个实验时执行的稀释值，必要时将值相乘。现在，通过计算竞争指数或取消基于数字PCR的条形码应变定量中的竞争指数来确定生物体的相对健康。在所有时间点对每个条形码应变执行此步骤。含有 AA 条形码的基因组 DNA 在所有其他条形码基因组 DNA 的背景中稀释。通道 1 表示 AA 条形码的 FAM 探头，而通道 2 表示 AO 条形码的 HEX 探头。每个探针的结果均作为单个液滴荧光振幅和表示选定孔中所有液滴荧光强度频率的直方图。对于每种情况，正液和负液滴应形成2个不同的种群。应使用阈值特征分隔种群，以定义正液滴和负液滴。阈值会因使用的探测器而异，但使用相同探头组合的所有孔应具有相同的阈值。在被稀释的 AA 中，直方图似乎只描绘了单个总体，因为正液滴的数量大大超过负液滴。然而，通过检查左侧面板中的液滴荧光振幅，仍然可以看到 2 个不同的群体。随着 AA 条形码基因组 DNA 被稀释，阳性液滴减少，而阳性 AO 液滴的数量保持不变。一旦将适当的阈值应用于所有油井，软件将计算每次反应中的DNA拷贝数。如果您常规执行 PCR 作为研究的一部分，您可以执行此技术来评估模型生物体的适合性。此过程通常用于确定上游实验的最终结果。它的真正用途是促进任何依赖于细菌定量的实验的易用性和多功能性。我们使用这种技术来评估沙门氏菌在感染的莫林模型中的体能。此外，这项技术正在被调整，用于我们的实验室中的其他致病微生物。

Research

JoVE Journal

Genetics

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

温度敏感致死突变体的高通量机器人辅助隔离<em&gt;莱茵衣藻</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...