基于数字PCR的沙门氏菌健康高通量分析竞争指数
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 使用商业质粒小提具套件,纯化 pKD1314</su…
Representative Results
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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1.5 mL microcentrifuge tubes | Eppendorf | 22600028 | Procure from any manufacturer |
| 16 mL culture tubes | MidSci | 8599 | Procure from any manufacturer |
| 5-200 μL pipette tips | RAININ | 30389241 | Procure alternative tip brands with caution based on manufacturing quality |
| 5-50 μL multichannel pipette | RAININ | 17013804 | Use alternative multichannel pipettes with caution |
| Agarose | ThermoFisher Scientific | BP160-500 | Procure from any manufacturer |
| BLAST Analysis | NCBI | N/A | https://blast.ncbi.nlm.nih.gov/Blast.cgi |
| C1000 Touch Thermocycler with 96-Deep Well Reaction Module | Bio Rad | 1851197 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| Chemically competent DH5α | Invitrogen | 18258012 | Procure from any manufacturer or prepare yourself |
| Chloramphenicol | ThermoFisher Scientific | BP904-100 | Procure from any manufacturer |
| Cytation5 Microplate reader | BioTek | CYT5MF | Procure from any manufacturer, use any system capable of accurately quantifying DNA |
| Data Analysis Software (QuantaSoft and QuantaSoft Data Analysis Pro) | Bio Rad | N/A | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| ddPCR 96-Well Plates | Bio Rad | 12001925 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| ddPCR Droplet Reader Oil | Bio Rad | 1863004 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| ddPCR Supermix for Probes (No dUTP) | Bio Rad | 1863024 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| DG8 Cartridges for QX200/QX100 Droplet Generator | Bio Rad | 1864008 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| DG8 Gaskets for QX200/QX100 Droplet Generator | Bio Rad | 1863009 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| Droplet Generation Oil for Probes | Bio Rad | 1863005 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| Kanamycin | ThermoFisher Scientific | BP906-5 | Procure from any manufacturer |
| Luria-Bertani agar | ThermoFisher Scientific | BP1425-2 | Procure from any manufacturer or make it yourself from agar, tryptone, yeast digest, and NaCl |
| Luria-Bertani broth | ThermoFisher Scientific | BP1426-2 | Procure from any manufacturer or make it yourself from tryptone, yeast digest, and NaCl |
| PCR Plate Heat Seal, foil, pierceable | Bio Rad | 1814040 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| PCR Tubes | Eppendorf | 951010022 | Procure from any manufacturer |
| Petri dishes | ThermoFisher Scientific | FB0875712 | Procure from any manufacturer |
| pPCR Script Cam SK+ | Stratagene/Agilent | 211192 | No longer available commercially |
| Primer/Probe Design | IDT | N/A | https://www.idtdna.com/Primerquest/Home/Index |
| pSKAP and pSKAP_Barcodes | Addgene | Plasmid numbers 122702-122726 | www.addgene.org |
| PX1 PCR Plate Sealer | Bio Rad | 1814000 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| QX200 Droplet Generator | Bio Rad | 1864002 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| QX200 Droplet Reader | Bio Rad | 1864003 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
| S. Typhimurium strain ATCC 14028s | ATCC | ATCC 14028s | www.atcc.org |
| Take3 Micro-Volume Plate | BioTek | TAKE3 | Procure from any manufacturer, use any system capable of accurately quantifying DNA |
| Thermo Scientific FastDigest BamHI | ThermoFisher Scientific | FERFD0054 | Procure from any manufacturer |
| Thermo Scientific FastDigest DpnI | ThermoFisher Scientific | FERFD1704 | Procure from any manufacturer |
| Thermo Scientific FastDigest HindIII | ThermoFisher Scientific | FERFD0504 | Procure from any manufacturer |
| Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro Kit | ThermoFisher Scientific | FERK0832 | Procure from any manufacturer |
| Thermo Scientific GeneJet Miniprep Kit | ThermoFisher Scientific | FERK0503 | Procure from any manufacturer |
| Thermo Scientific Phusion High-Fidelity DNA Polymerase | ThermoFisher Scientific | F534L | Procure from any manufacturer |
| Thermo Scientific T4 DNA Ligase | ThermoFisher Scientific | FEREL0011 | Procure from any manufacturer |
| Thermocycler | Bio Rad | 1861096 | Procure from any manufacturer |
| UVP Visi-Blue Transilluminator | ThermoFisher Scientific | UV95043301 | Or other transiluminator that allows visualization of DNA |
| Water, Molecular Biology Grade | ThermoFisher Scientific | BP28191 | Procure from any manufacturer |
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Tags
Digital PCRCompetitive IndexHigh-throughput AnalysisFitnessSalmonellaQuantificationMixed PopulationMolecular-based TechniquesGenetic MarkersBacterial ChromosomesGenomic DNADilution SeriesDigital PCR SupermixProbe-based ChemistryNo Template ControlsNegative ControlsPositive ControlsDroplet GeneratorThermal CyclerData Analysis Software
Citazione di questo articolo
Shaw, J. A., Bourret, T. J. Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella. J. Vis. Exp. (147), e59630, doi:10.3791/59630 (2019).