1。的Thermo Scientific的Solaris定量PCR检测设计算法<ol><li>在设计的Solaris qPCR的探针/引物对所使用的算法调整每个组件从而使通用循环条件的Tm。团的MGB探针，或轻微的凹槽约束力，在探头的基团增加以旧换新，并允许更短的设计更大的灵活性和性能检测的探头。顾名思义，MGBs是一类抗生素，可以容纳到DNA双螺旋的小沟。 MGBs连接到一个DNA探针的3或5月底。 FAM记者荧光探针在溶液中，是由Eclipse暗淬灭淬火。当探针结合到靶序列，在探头第3条的三维构象的变化，允许一个强烈的荧光信号。靶序列的DNA探针结合的稳定的MGB探针，它允许使用高度特异性的，更短的探针，而保持适当的熔融温度为PCR。因此，信号检测过程中发生的目标扩增的退火阶段。在延伸，MGB探针不赞成和荧光又是不灭的。因为探头不赞成，不影响PCR的效率。 </li><li>在Solaris算法的另一个特点是使用Superbases。 Superbases被修改，增强结合能力，以改善对AT债券的稳定和消除GG鸟嘌呤丰富的序列自我协会的基地。此外，他们不解渴5总站连接相邻的荧光团。这些基地选择性地，根据算法，扩大目标序列的可用性。这使得它可以使用，否则将避免在设计引物和探针的序列，如GC丰富的地区，。 </li><li>只要有可能，在Solaris算法采用外显子交界处，在目标网站跨越地区。这增加了避免污染物的基因组DNA扩增的特异性。 </li><li>重要的是，该算法还确定了所有已知的剪接变异体的共识序列，等，都是由一个单一的探针/引物覆盖。所以，只有一个实验是需要一个感兴趣的特定基因。 </li><li>最后，在Solaris的qPCR实验设计算法采用了非常严格的关键设计考虑的参数包括GC含量，长度，TM，和同质核苷酸的延伸。结果序列分析使用3种不同的数据库的特殊性的冲击：基因组，转录和假。 </li></ol> 2。获取的Solaris试剂<ol><li>要获得适当的Solaris试剂，请<a href="http://dharmacon.com">访问www.thermo.com / Solaris</a>的</li><li>一旦在网站上点击“GENEius产品搜索”。要直接去GENEius产品搜索，请访问<a href="http://www.dharmacon.com/catalogsearch/consolidatedsearch.aspx">www.thermo.com / SolarisSearch</a> </li><li>点击在搜索查询框，并选择适当的目标有机体。 </li><li> GENEius产品搜索，可以处理许多不同的基因标识。在这里，输入建议的搜索条件，寻找感兴趣的基因，其中包括加入参考序列，基因编号，基因符号，基因描述，桑格ID或加入号码，或目录号之一。然后，点击“搜索”。 </li><li>确定感兴趣的基因，并点击“选择”在左侧。这将打开一个窗口，显示不同的Thermo感兴趣的基因科技产品。根据“产品”，选择“定量PCR检测”选项卡。 </li><li>的“Solaris基因表达检测”的标题下，你会看到一个优化设计的Solaris探针/引物检测。此法将涵盖所有已知的基因目标的剪接变异体。选择所需的数量。他们在100，200，400， 1000 × 25μL的反应（100包大小，如果检测到，将无法使用）。点击“添加到购物车”。 </li><li>下一步，选择适当的热循环仪将用于主组合。要确定哪位高手组合将与现有的机器最好，单击“查找出最好与你的PCR扩增仪主混音作品！” </li><li>寻找定量PCR基因扩增仪，将在菜单上使用，以确定哪些组合使用。 </li><li>返回GENEius产品搜索窗口，并选择适当的主结构。主结构，100，200，400和1000的反应大小。点击“添加到购物车”。 </li><li>建议参考的基因和Verso cDNA合成试剂盒也可以订购一个完整的QRT - PCR分析。 </li><li>当Solaris定量PCR试剂后，储存在-20 ° C，直到他们准备使用。应收到以下试剂：一个20X的解决方案，其中包含两个引物在800 nm的每一个基因特异性探针在200纳米，色曲提供提供探针和引物，和Solaris的qPCR基因表达的2倍浓度的预混ENCE信息。至少12个月的Solaris qPCR的基因表达分析稳定。避免重复冷冻解冻。 </li></ol> 3。 qPCR的设置<ol><li>开始与基因的qPCR协议。应使用离心柱方法提取总RNA。建议的Thermo Scientific Verso公司cDNA合成试剂盒进行反转录生成的cDNA。 </li><li>解冻的引物，探针，并掌握冰混合准备的qPCR。另外手头上有PCR级水和白色不透明的qPCR板，它可以提高实时PCR的敏感性。 </li><li>主结构包含在内的所有实时PCR的组成部分：1）热启动DNA聚合酶，Thermoprime DNA聚合酶的热启动版本。这种类型的聚合酶是用来防止非特异性扩增反应的成立过程中和需要激活步骤在95 ° C 15分钟。 2）在Solaris预混dNTPs，dTTP的替代的dUTP最大限度地扩增效率3）专有反应​​缓冲液，优化工作与Solaris引物/探针检测。这个反应缓冲液含有蓝色染料增加试剂和塑料之间的对比。 4）ROX，如果您的qPCR仪器需要ROX。 </li><li>一旦解决方案已经解冻，混合弹管（不涡）。分拆下来，以防止试剂损失。 </li><li>下一步，准备一个15毫升无菌猎鹰管成立的感兴趣的基因，加上一个参照基因扩大取决于样本数，反应组合。这样三个复制可以为每个样品准备，包括无模板对照（NTC）和标准曲线的样品，如果将供分析用的标准曲线法，准备足够的。 </li><li>对于每个样品的96孔板上运行，混合以下内容：5μL2X的Solaris qPCR的预混，0.5μL的Solaris一套引物/探针（20X），PCR级水等，最终反应体积，一旦的cDNA，将25μL。对于每个样品384孔板上运行组合如下：12.5μL2X的Solaris qPCR的预混的Solaris一套引物/探针（20X），1.25μL，PCR级水等，最终反应体积，一旦基因添加10μL。 </li><li>使用多通道移液器，传输中，主机结构的钢板适当的水井。主结构中的蓝色染料，将有助于跟踪上的白色板吹打进展。然后，添加1-5μLcDNA模板（96孔板）或1-2μLcDNA模板（384孔板）。移液器上下搭配。板放置在离心机和自旋向下，以消除任何气泡，因为这些都会干扰与荧光读数。 </li><li>计划的热循环仪：必须激活这种酶与95 ° C，15分钟（1个周期）。 40周期95℃，15秒的变性，退火和延伸，在60 ° C为60秒。 </li><li>将在qPCR的仪器板。启动程序。 </li></ol>使用Solaris的代表qPCR的结果<img src="/files/ftp_upload/1700/1700fig1.jpg" alt="图1" /> 图1。PCR的效率和R2的值来判断的Solaris检测提供可靠的检测，即使在非常低的输入浓度。在ABI 7900HT使用的Solaris qPCR的基因表达检测仪器分别为F2RL1或CDC20，10个10倍稀释的合成，合成的RNA扩增序列或DNA扩增序列的cDNA扩增。日志规模的扩增曲线和标准曲线显示随着每次检测的性能，包括效率，R2的值，动态范围10 LOG10稀释，检测下限。 <img src="/files/ftp_upload/1700/1700fig2.jpg" alt="图2" /> 图2的Solaris分析给出一致的结果，甚至不同的研究人员和实验室之间。 100，10，1和0.1 nm的最终浓度的siRNA靶向ALDOA和PPIB，以及非针对控制（NTC），分别转染HeLa细胞。收获细胞总RNA隔离治疗后48小时。合成cDNA，使用Thermo Scientific的Verso公司cDNA合成试剂盒。在地理上分开的两个实验室使用罗氏的LightCycler 480（384孔）平台的Solaris qPCR的基因表达分析检测ALDOA，PPIB和GAPDH扩增的cDNA液。计算使用ΔΔCq法（归参照基因GAPDH的和负温度系数（NTC）处理的细胞）击倒。击倒相同的水平进行了论证研究人员在两个站点之间的靶基因。

这篇文章的作者是Thermo Scientific的员工。

Thermo Scientific的的Solaris qPCR实验提供了一个简单的，执行的qPCR分析方法的改进。这些套引物和探针使用高紧缩算法，结合MGB探针技术和Superbases，最大化的qPCR实验的保真度和鲁棒性。一个引物和探针组认识到所有已知的一个特定基因的剪接变异体。目标站点，只要有可能，包括跨越外显子区域，以确保未扩增，基因组DNA。这些特性使得可以同时运行众多的样品，大大减少了执行复杂的实验，所需的时间量。由于主结构是蓝色的，它有可能使用白色不透明板，这对提高定量PCR的灵敏度，而不会失去跟踪的吹打，的。 的Solaris定量PCR检测的设计特点，采取一切在一起，使这本小说的qPCR检测一个简单的，但具体和可靠的高性能的检测。

quantitative real-time pcr using the thermo scientific solaris qpcr assay

在Solaris qPCR的基因表达检测是一种新型的引物/探针设置，设计简化的qPCR的过程，同时保持检测的灵敏度和准确性。这些引物/探针集是预先设计&gt; 98％的人类和小鼠基因组和功能显着改善，从以前提供的技术。这些改进可能凭借Thermo Scientific的生物信息学专家的发展，一种新的设计方法。 几个方便的特点已被纳入到Solaris定量PCR检测，以简化过程进行实时定量PCR。首先，该协议是类似的普遍采用的替代品，所以在定量PCR所使用的方法很可能要熟悉。二，主结构是蓝色的，这使得设置的qPCR反应更容易追踪。三，热循环条件是所有检测（基因），从而有可能同时运行的许多样品和减少潜在的错误。最后，探针和引物序列信息提供，简化出版过程。 在这里，我们将演示如何使用产品搜索功能上的订购网站（www.thermo.com / Solaris）的发现和如何使用的Solaris试剂使用标准曲线法执行的qPCR GENEius获得适当的Solaris试剂。

Watch this Scientific Journal Video about Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay at JoVE.com

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

的Solaris qPCR的基因表达分析小说预先设计的定量PCR引物/探针组合，旨在简化不牺牲检测的特异性和稳健性的qPCR过程。

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 ...

quantitative-real-time-pcr-using-thermo-scientific-solaris-qpcr

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

49.7K 次观看.  Thermo Scientific Solaris qPCR Products.  日本語 

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

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

科研

遗传学

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

用便携式实时 PCR 系统进行土壤载体 Phytopathogens 的现场分子检测

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

环境科学

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

医学

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

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 locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

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 ...

免疫与感染

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

生物学

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

一个定量聚合酶重组酶扩增法的发展与内部阳性对照

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

使用数字微滴PCR定量环状RNA

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

Quantifying Environmental Microorganisms and Viruses Using qPCR

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Bradley Schmitz
Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a widely-used molecular technique for enumerating microorganisms in the environment. Prior to this approach, quantifying microorganisms was limited largely to classical culture-based techniques. However, the culturing of microbes from environmental samples can be particularly challenging, and it is generally held that as few as 1 to 10% of the microorganisms present within environmental samples are detectable using these techniques. The advent of qPCR in environmental microbiology research has therefore advanced the field greatly by allowing for more accurate determination of concentrations of microorganisms such as disease-causing pathogens in environmental samples. However, an important limitation of qPCR as an applied microbiological technique is that living, viable populations cannot be differentiated from inactive or non-living populations.
This video demonstrates the use of qPCR to detect pepper mild mottle virus from an environmental water sample.

量化环境微生物和病毒使用 qPCR

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Bradley Schmitz
Quantitative polymerase ...