Name: quantitative pcr of t7 bacteriophage from biopanning
Uploaded: 2018-09-27T14:00:00
Description: Watch this Scientific Journal Video about Quantitative PCR of T7 Bacteriophage from Biopanning at JoVE.com

这项工作得到了 PhRMA 基金会研究启动补助金和国家心脏, 肺和血液研究所的国家卫生研究院授予编号 R01HL138251。

1. T7 噬菌体基因组 DNA 引物设计与分析
<ol><li>T7 噬菌体基因组 DNA 扩增的设计底漆。 注: F (正向) 和 R (反向) 引物 (见材料表) 放大位于库变量区域上游的 T7 DNA 序列 (图 1)。</li>
	<li>选择合适的底漆分析仪来评估底漆的参数, 包括熔融温度 (Tm)、GC 含量 (GC%)、底漆脂肪酸、发夹形成和 PCR 适用性 (图 1)。</li>
	<li>命令引物寡核苷酸 (寡核苷酸),...

<ol>
	<li>Smith, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+fusion+phage:+novel+expression+vectors+that+display+cloned+antigens+on+the+virion+surface.">Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.</a> Science. 228 (4705), 1315-1317 (1985).</li><li>Smith, G. P., Petrenko, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Display.">Phage Display.</a> Chemical Reviews. 97 (96), 391-410 (1997).</li><li>Sergeeva, A., Kolonin, M. G., Molldrem, J. J., Pasqualini, R., Arap, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Display+technologies:+Application+for+the+discovery+of+drug+and+gene+delivery+agents.">Display technologies: Application for the discovery of drug and gene delivery agents.</a> Advanced Drug Delivery Reviews. 58 (15), 1622-1654 (2006).</li><li>Dias-Neto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+phage+display:+Integrating+and+comparing+available+molecular+tools+to+enable+costeffective+high-throughput+analysis.">Next-generation phage display: Integrating and comparing available molecular tools to enable costeffective high-throughput analysis.</a> PLoS ONE. 4 (12), 1-11 (2009).</li><li>Peng, X., Nguyen, A., Ghosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+M13+and+T7+bacteriophages+by+TaqMan+and+SYBR+green+qPCR.">Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.</a> Journal of Virological Methods. 252 (June 2017), 100-107 (2017).</li><li>Khan, M. S., Pande, T., Mvan de Ven, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+detection+of+T7+bacteriophages+using+paper+based+sandwich+ELISA.">Qualitative and quantitative detection of T7 bacteriophages using paper based sandwich ELISA.</a> Colloids and Surfaces B: Biointerfaces. 132, 264-270 (2015).</li><li>Brasino, M., Lee, J. H., Cha, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+highly+amplified+enzyme-linked+immunosorbent+assay+signals+from+genetically+engineered+bacteriophage.">Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage.</a> Analytical Biochemistry. 470, 7-13 (2014).</li><li>Yu, X., Burgoon, M., Shearer, A., Gilden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+phage+peptide+interaction+with+antibody+using+phage+mediated+immuno-PCR.">Characterization of phage peptide interaction with antibody using phage mediated immuno-PCR.</a> J immunol Methods. 326 (1-2), 33-40 (2007).</li><li>Lehmusvuori, A., Manninen, J., Huovinen, T., Soukka, T., Lamminm&#228;ki, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogenous+M13+bacteriophage+quantification+assay+using+switchable+lanthanide+fluorescence+probes.">Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes.</a> BioTechniques. 53 (5), 301-303 (2012).</li><li>Schlaman, H. R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+interactions+of+signaling+proteins+with+phage-displayed+ligands+by+fluorescence+correlation+spectroscopy.">Analysis of interactions of signaling proteins with phage-displayed ligands by fluorescence correlation spectroscopy.</a> Journal of Biomolecular Screening. 13 (8), 766-776 (2008).</li><li>Tjhung, K. F., Burnham, S., Anany, H., Griffiths, M. W., Derda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enumeration+of+phage+in+monodisperse+emulsions.">Rapid enumeration of phage in monodisperse emulsions.</a> Analytical Chemistry. 86 (12), 5642-5648 (2014).</li><li>Reitinger, S., Petriv, O. I., Mehr, K., Hansen, C. L., Withers, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+quantitation+of+bacteriophage+M13+using+desalting+spin+columns+and+digital+PCR.">Purification and quantitation of bacteriophage M13 using desalting spin columns and digital PCR.</a> Journal of Virological Methods. 185 (1), 171-174 (2012).</li><li>. T7Select&#174; Biopanning Kit Available from: <a target="_blank" href="https://www.emdmillipore.com/US/en/product/T7Select-Biopanning-Kit">https://www.emdmillipore.com/US/en/product/T7Select-Biopanning-Kit</a> (2018)</li><li>Schneider, A., Hommel, G., Blettner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+Regression+Analysis.">Linear Regression Analysis.</a> Deutsches &#196;rzteblatt international. 107 (44), 776-782 (2010).</li><li>Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+Guidelines:+Minimun+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE Guidelines: Minimun information for publication of quantitative real-time PCR experiments.</a> Clinical Chemitry. 55 (4), 611-622 (2009).</li><li>Lock, M., Alvira, M. R., Chen, S. -. J., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+Determination+of+Single-Stranded+and+Self-Complementary+Adeno-Associated+Viral+Vector+Genome+Titers+by+Droplet+Digital+PCR.">Absolute Determination of Single-Stranded and Self-Complementary Adeno-Associated Viral Vector Genome Titers by Droplet Digital PCR.</a> Human Gene Therapy Methods. 25 (2), 115-125 (2014).</li><li>Fittipaldi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+of+infectious+bacteriophage+T4+virus+by+propidium+monoazide+real-time+PCR.">Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR.</a> Journal of Virological Methods. 168 (1-2), 228-232 (2010).</li><li>Lodder, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+bacteriophage+MS2+by+filtration+and+irradiation+determined+by+culture+and+quantitative+real-time+RT-PCR.">Reduction of bacteriophage MS2 by filtration and irradiation determined by culture and quantitative real-time RT-PCR.</a> Journal of Water and Health. 11 (2), 256-266 (2013).</li><li>Brown-Jaque, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+genes+in+phage+particles+isolated+from+human+feces+and+induced+from+clinical+bacterial+isolates.">Antibiotic resistance genes in phage particles isolated from human feces and induced from clinical bacterial isolates.</a> International Journal of Antimicrobial Agents. , (2017).</li><li>Naveca, F. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+reverse+transcription+real-time+polymerase+chain+reaction+for+simultaneous+detection+of+Mayaro,+Oropouche,+and+oropouche-like+viruses.">Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and oropouche-like viruses.</a> Memorias do Instituto Oswaldo Cruz. 112 (7), 510-513 (2017).</li><li>Jia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+differentiation+of+human+parvovirus+B19+and+human+parvovirus+4+by+an+internally+controlled+multiplex+quantitative+real-time+PCR.">Simultaneous detection and differentiation of human parvovirus B19 and human parvovirus 4 by an internally controlled multiplex quantitative real-time PCR.</a> Molecular and Cellular Probes. 36, 50-57 (2017).</li><li>Bliem, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+triplex+quantitative+pcr+strategy+for+quantification+of+toxigenic+and+nontoxigenic+Vibrio+cholerae+in+aquatic+environments.">A novel triplex quantitative pcr strategy for quantification of toxigenic and nontoxigenic Vibrio cholerae in aquatic environments.</a> Applied and Environmental Microbiology. 81 (9), 3077-3085 (2015).</li><li>Wan, Z., Goddard, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+Between+Conjugation+and+M13+Phage+Infection+in+Escherichia+coli+in+the+Absence+of+Selection+Pressure:+A+Kinetic+Study.">Competition Between Conjugation and M13 Phage Infection in Escherichia coli in the Absence of Selection Pressure: A Kinetic Study.</a> G3 Genes|Genomes|Genetics. 2 (10), 1137-1144 (2012).</li></ol>

我们开发了 qPCR 方法来量化噬菌体基因组 DNA5, 在这里我们描述和适应了一个 qPCR 的方法, 以列举 T7 噬菌体选择对类似 CF 粘液屏障。发布定量实时 PCR 实验 (MIQE) 指南的最低信息用于开发和验证 T7 噬菌体15的 qPCR 方法。我们从以此实验中量化噬菌体的协议是一种时间高效、可靠、经济的方法。我们的 qPCR 样品制备协议只使用高温处理 (100 °c, 15 分钟) 的噬菌体溶液...

噬菌体 (噬菌体) 显示技术, 由乔治·史密斯在1985年开发, 是一个强大的高通量的方法, 以识别肽或蛋白质配体的目标或受体从细胞膜, 疾病抗原, 细胞器或特定器官和组织在过去二年1,2。这里, 在噬菌体 (通常是 M13 或 T7) 的外壳蛋白上显示多肽或单链抗体的随机库, 在体外或体内对固定蛋白的平移可以识别出特定的配体。生物系统通过一个迭代选择过程。然后, 配体可以与影像或治疗药物结合, 以诊断和治疗疾病3,4。在以此的多个步骤中准确地列举噬菌体是非常关键的: (1) 量化噬菌体与基底和 (2) 量化噬菌体, 以确定是否有丰富的每一轮选择 (噬菌体浓缩表明 biopanned目标的噬菌体亲和性)。目前对定量、双层斑块检测的黄金标准提出了多重挑战;它是繁琐的, 繁琐的, 可能是不准确的大量样本。因此, 我们小组开发了一种灵敏、可重复、准确、高效的定量聚合酶链反应 (qPCR) 方法, 用于从以此5中列举 M13 和 T7 噬菌体粒子。
qPCR 是准确定量 T7 和 M13 噬菌体的一种具有吸引力和可行性的方法。由于每个单独的噬菌体粒子只能包含一份基因组 DNA (dsDNA 或 ssDNA), 一个噬菌体基因组相当于一个噬菌体粒子;通过量化噬菌体基因组的数量, 可以量化噬菌体的数量。在 qPCR 期间, 荧光记者染料在 PCR 扩增过程中, 不专门或具体通过序列特异引物结合噬菌体基因组 DNA, 荧光信号随着每轮放大而增加。当荧光信号达到阈值时, 放大的圆形/循环被指出为阈值周期 (Ct)。已知浓度的参考噬菌体 DNA 被绘制反对他们的 Ct 值, 以建立一个标准曲线。将标准曲线与 DNA 样本的 Ct 值结合, 可以对噬菌体的浓度进行插补。
虽然以前已经制定了许多战略, 并广泛地用于从以此中定量噬菌体, 但每一个都有具体的挑战。最常用和最常用的方法是双层斑块法。在这里, 宿主细菌感染了在串行稀释制备的噬菌体, 被镀在固体琼脂基体上, 并与琼脂覆盖;噬菌体被列举的斑块的数量 (即斑块形成单位或 pfu) 在琼脂板块。斑块检测是敏感但繁琐, 耗时和不准确的, 特别是对许多样品和高浓度5。此外, 酶联免疫吸附检测 (ELISA) 已经适应了列举 M13 和 T7 噬菌体粒子6,7,8。在这里, 不同稀释的噬菌体被绑定并捕获在固体基底上 (即微板块), 用噬菌体特异抗体进行探测, 并使用记者 (例如,酶敏感的显色基质, 显影) 来确定存在的噬菌体粒子数量。样品的读数 (如荧光、吸光度) 可以用来量化已知浓度下的噬菌体标准的未知浓度。不同的噬菌体为基础的 ELISAs 已经开发, 但它们有潜在的局限性。一个小组开发了以纸为基础的三明治 ELISA, 其量化范围跨越七级 (102-109 pfu/毫升);然而, 这种方法需要多步骤的抗体涂层, 并采取了整整一天的化验8。我们的小组还开发了一种 ELISA 方法, 以量化 M13 噬菌体粒子, 但有一个不太敏感的 检测范围为 10 6 至 1011 pfu/毫升5。已开发出可切换的镧系荧光探针, 用于枚举 M13, 可在20分钟内获得量化数据;然而, 这种检测有一个狭窄的动态范围为 109至 1012 pfu/毫升9。一组使用原子力显微镜在溶液中列举 M13 噬菌体粒子, 但这需要先进的电子显微镜, 只有在10的浓度范围内工作。另一项研究使用单分散乳液诱捕荧光 M13 和 T4 报告噬菌体和计数噬菌体的数量的荧光液滴;然而, 这种方法也显示了一个狭窄的量化范围从 102到 106 pfu/毫升11。当液滴数字 PCR 用于定量 M13 噬菌体时, 这种方法无法区分传染性和非传染性噬菌体粒子的数量12。
我们的小组最近开发了 qPCR 方法, 以列举 T7 和 M13 噬菌体从以此识别的血脑屏障细胞模型使用两种不同的荧光记者染料5。与上述量化方法相比, 我们开发的 qPCR 方法是高通量和时间效率的数量众多的样本, 并能够区分传染性和非传染性噬菌体与 DNase I 预处理的噬菌体样本。重要的是, 这种方法可以重现性和准确地量化 M13 和 T7 噬菌体从以此样本。为指导在噬菌体 qPCR 领域的新手研究人员, 这里我们描述了一个详细的 qPCR 方法来列举 T7 噬菌体颗粒从半胱氨酸约束的图书馆 (CX7C) 对体外囊性纤维化 (CF) 粘液屏障。从这项工作, qPCR 方法可以扩展, 以量化的噬菌体粒子从其他类型的以此和其他来源, 包括水, 土壤和体液。

<table>
	<tbody>
		<tr>
			<td>Materials and reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primers for T7 genomic DNA</td>
			<td>IDT</td>
			<td></td>
			<td>F: CCTCTTGGGAGGAAGAGATTTG 
			R: TACGGGTCTCGTAGGACTTAAT</td>
		</tr>
		<tr>
			<td>T7Select packaging control DNA</td>
			<td>EMD Millipore</td>
			<td>69679-1UG</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp optical 96-well reaction plate</td>
			<td>ThermoFisher Scientific</td>
			<td>N8010560</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR master mix--Power up SYBR Green master mix</td>
			<td>Applied biosystems</td>
			<td>A25742</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp optical adhesive film kit</td>
			<td>ThermoFisher Scientific</td>
			<td>4313663</td>
			<td></td>
		</tr>
		<tr>
			<td>T7Select 415-1 Cloning Kit</td>
			<td>EMD Millipore</td>
			<td>70015</td>
			<td>User protocols : http://www.emdmillipore.com/US/en/product/T7Select-415-1-Cloning-Kit,EMD_BIO-70015#anchor_USP</td>
		</tr>
		<tr>
			<td>DNase I solution</td>
			<td>ThermoFisher Scientific</td>
			<td>90083</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well transwell plate</td>
			<td>Corning</td>
			<td>3472</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled H2O</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>Corning</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ViiA7 Real-Time PCR System with Fast 96-Well Block</td>
			<td>ThermoFisher Scientific</td>
			<td>4453535</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Pico 21 Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75002415</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Digital Vortex Mixer</td>
			<td>Fisher Scientific</td>
			<td>02-215-370</td>
			<td></td>
		</tr>
		<tr>
			<td>HERMO SCIENTIFIC Multi-Blok Heater</td>
			<td>ThermoFisher Scientific</td>
			<td>Model:2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend X1 Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75004220</td>
			<td></td>
		</tr>
		<tr>
			<td>M-20 Microplate Swinging Bucket Rotor</td>
			<td>ThermoFisher Scientific</td>
			<td>75003624</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio Real-time PCR software</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>v1.2</td>
		</tr>
		<tr>
			<td>Real-time qPCR primer design</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OligoAnalyzer 3.1</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative pcr of t7 bacteriophage from biopanning

该协议描述了定量 PCR (qPCR) 从噬菌体选择实验 (即"以此") 中枚举 T7 噬菌体的应用。qPCR 是一种基于荧光的方法来量化 DNA, 在这里, 它被用来量化噬菌体基因组作为噬菌体粒子的代理。在该协议中, 用高温加热技术描述了一种简便的噬菌体 dna 制备方法, 无需额外的 DNA 纯化。该方法只需要少量的热处理噬菌体和小体积的 qPCR 反应。qPCR 是高通量和快速, 能够处理和获取数据从96井的反应在 2–4 h. 与其他噬菌体枚举方法相比, qPCR 更有时间效率。在这里, qPCR 用于列举从以此中识别出的 T7 噬菌体对体外囊性纤维样粘液模型。qPCR 方法可以扩展, 以量化 T7 噬菌体从其他实验, 包括其他类型的以此 (e.,固定化蛋白结合, 体内噬菌体筛选) 和其他来源 (例如,水系统或体液)。总之, 这个协议可以修改, 以量化任何 DNA 封装病毒。

不同的底漆设计工具可用于设计 qPCR 底漆。通常, 底漆设计程序都有自己的内置算法来计算和验证引物的关键参数,如GC%, Tm, 底漆二聚体或发夹形成等.一般情况下, 关键标准是相似的在不同底漆设计工具, 和底漆可以按照他们的指示设计。底漆爆破可用于确定引物的特异性。图 1显示了 T7 基因组 DNA 的可变区域上游的一个引物对。为了确定...

Watch this Scientific Journal Video about Quantitative PCR of T7 Bacteriophage from Biopanning at JoVE.com

Quantitative PCR of T7 Bacteriophage from Biopanning

以此 T7 噬菌体的定量 PCR 研究

本文介绍了一种可重现的、准确的、时间有效的定量 PCR (qPCR) 方法来枚举 T7 噬菌体。该协议明确描述了噬菌体基因组 DNA 制备、PCR 反应制备、qPCR 循环条件和 qPCR 数据分析。

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

这种方法可以帮助回答病毒学和微生物学领域与从复杂样品和基于噬菌体的诊断和治疗中列举噬菌体相关领域的关键问题。该技术的主要优点是，它能够对噬菌体显示生物平移实验中的大量样品进行时间高效、准确的定量，这与传统测定相比是行不通的。演示这个程序将是拉什米莫汉蒂和贾斯米姆莱尔，研究生从我的实验室。在设计底剂并创建库存浓度后，以每微升0.1微克的浓度获取参考T7噬菌体DNA。使用超纯水，连续稀释该DNA十倍，从每微升一百万倍图到每微升一个女性图，在0.2毫升PCR管中。准备每个浓度的20微升，确保改变移液器尖端和涡流管之间的稀释阶段。然后将T7噬菌体参考DNA浓度从微升的femttogram转换为每微升的基因组拷贝。首先，移液器200微升样品T7噬菌群克隆成1.5毫升的微型离心管，用于10个所需样品。在每个管中添加两个DNase的微升。在加热干燥浴中，在37摄氏度下将管子盖住并孵育10分钟。然后在100摄氏度下在加热干燥浴中孵育15分钟。将含有热处理噬菌体的管在18，534次g下离心10秒。将离心管放在触摸激活模式下的涡流混合器上 10 秒钟，以混合噬菌体。在 18，534 次 g 下再次旋转，10 秒。在此之后，将样品留在实验室长椅上约一小时，以冷却至室温。为10个浓度不详的生物活化噬菌板样品和7个三酸钠标准浓度样品准备qPCR预混料。为 51 个样品提供预混，包含 255 微升 qPCR 主混合、51 微升底料和 102 微升水。将8微升的已准备好的PCR混合成96孔qPCR板的51孔。接下来，在每口井中加入两个经过热处理的T7噬菌口样品微升，将其分配到 qPCR 预混料的表面之下。使用胶膜密封板。将板包裹在铝箔中，以尽量减少荧光光漂白，然后在 qPCR 设备上运行板。设置循环条件和融化曲线设置，如文本协议中概述的。运行 qPCR 后，从原始数据中分析放大图、熔体曲线图和多分点图。放大图指示每个样品的 PCR 放大是否成功。多组分图表示荧光信号在整个放大周期中的放大和衰减。虽然熔体曲线图用于验证底像的特异性，因为每个 PCR 产品都有一个独特的熔化温度。然后从参考DNA生成标准曲线。在这里，扩增效率计算为92.4%的代表性数据从qPCR研究然后比较相同的噬菌群，由双层斑块测定量化在测试范围内1000万至1000万基因组拷贝每微升。如这里所见，qPCR定量中的噬菌体数量高于斑块测定。qPCR方法以方差系数表示的可重复性在尝试此程序时，其值为2.85至27.2%，重要的是要记住，在开发和使用qPCR时有许多因素可以准确量化噬菌体，包括仔细处理和制备样品以及校准相关设备。按照这种方法，可以执行其他技术，以枚举在维他生物平移、噬菌体库DNA制备以及复杂环境样品中下一代测序和噬菌体检测的各种应用中的噬菌体颗粒。不要忘记，噬菌体在实验室中被视为生物危害，在执行手术时应始终佩戴个人防护设备（包括手套和实验室外套）等预防措施。

Research

JoVE Journal

Immunology and Infection

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

定量成像小样本人体血液中单核细胞和树突状细胞系特定的Toll样受体介导的信号

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

科研

免疫与感染

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

为马科动物疱疹病毒2诊断的呼吸道液体定量PCR检测方法的开发和验证

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Optimization of a Quantitative Micro-neutralization Assay

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

小鼠特拉切斯刷细胞的分离与定量评价

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

用于狂犬病诊断的泛源病毒实时RT-PCR

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR

利用基于表观遗传学的定量PCR确定免疫细胞特性和纯度

Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...