数字PCR技术及其应用进展

数字PCR(Digital PCR,dPCR)是核酸绝对定量的新方法,该技术通过分液,将含有核酸模板的PCR反应体系分配到上万个反应器中进行PCR扩增,根据荧光信号的有或无,进行结果计数,通过泊松分布的统计处理,直接得出核酸的拷贝数。相比于实时荧光定量PCR(Quantitative PCR,qPCR),dPCR不需要建立标准曲线,应用前景更广。本文对dPCR的发展历史、原理及其应用进行了综述。     

数字PCR技术及其应用进展北大核心CSCD

数字PCR(Digital PCR,dPCR)是核酸绝对定量的新方法,该技术通过分液,将含有核酸模板的PCR反应体系分配到上万个反应器中进行PCR扩增,根据荧光信号的有或无,进行结果计数,通过泊松分布的统计处理,直接得出核酸的拷贝数。相比于实时荧光定量PCR(Quantitative PCR,qPCR),dPCR不需要建立标准曲线,应用前景更广。本文对dPCR的发展历史、原理及其应用进行了综述。     

数字PCR技术及其在检测领域的应用

随着分子生物学技术的不断发展和需求的多样化,用于核酸检测的各种PCR衍生技术应运而生。数字PCR是一种单分子水平的大规模分区扩增定量核酸检测技术。该技术以微腔室/微孔或微滴作为PCR反应器,无需校准物和绘制标准曲线即可实现对样品初始浓度的绝对定量,具有高灵敏度、高特异性和高精确度的特点。本文详细介绍了数字PCR的技术发展史、作用原理以及仪器平台类型,系统阐述了数字PCR在转基因检测、疾病诊断、环境及食品监管等方面的应用概况,并对该技术的应用前景进行了展望,以期对未来数字PCR的开发利用提供参考。     

实时荧光定量PCR的数据分析方法

实时荧光定量PCR是目前检测目的核酸拷贝数及分析靶基因在mRNA表达水平相对变化的主流技术。研究表明,分析结果的准确性依赖于数据分析方法的可靠性。我们简要综述实时荧光定量PCR的数据分析方法。     

实时荧光定量PCR(TaqMan)法测定外源基因的拷贝数

实时荧光定量PCR是近年新兴的一项技术,因其快速、方便、便宜,需要DNA样品量少,无需放射性检测等优点被广泛应用于基因的定量分析。该文就实时荧光定量PCR(TaqMan)技术的发展、基本原理及测定外源基因拷贝数的技术流程做一介绍。     

实时荧光定量PCR技术综述

实时荧光定量PCR技术是在PCR技术基础上发展起来的一种高度灵敏的核酸定量技术。与传统PCR相比,实时定量PCR能够更加快速、灵敏,并有效地对核酸进行定量检测。     

基于微滴式数字PCR技术的甲型流感病毒绝对定量方法的建立及应用

数字PCR(Digital PCR,dPCR)是核酸绝对定量的新方法,然而,基于dPCR的甲型流感病毒(Influenza A virus,FluA)绝对定量方法还未系统建立。本研究首先对微滴式dPCR的退火温度进行了梯度优化,确定了dPCR反应的最佳退火温度为64.4℃;利用FluA核酸标准品,确定了微滴式dPCR对FluA的检测范围为37.7~8.22104拷贝/#L,检测的检出限为3.77拷贝/反应。微滴式dPCR的检测结果与标准品拷贝数的相关系数为R2=0.9988,提示该方法检测结果具有较高的可信度。用建立好的微滴式dPCR方法可对待测临床样本中的FluA进行了拷贝数定量。因此本研究建立了基于微滴式dPCR的FluA绝对定量方法,可有效地对临床样本中甲型流感病毒载量进行绝对定量,为临床研究中病毒载量的测定提供了一种技术。     

数字PCR技术及应用研究进展

数字PCR是继实时定量PCR之后新兴发展起来的一种绝对定量分析技术。通过将单个DNA分子转移入独立的反应室,PCR扩增反应后,对荧光信号进行检测分析,实现单分子的绝对定量。数字PCR技术摆脱了对标准曲线的依赖,具有更高灵敏度和准确度,在基因突变检测、拷贝数变异检测、微生物检测、转基因食品检测以及下一代测序等方面均得到广泛应用。本文介绍数字PCR技术的定量方法,并评述该技术在主要应用领域的研究进展。     

单分子PCR技术及其应用

常规PCR技术大都以大量DNA分子(一般为105个以上)为模板进行DNA扩增。新近发展起来的单分子PCR技术是一种以极少量DNA分子(1、5或10个分子)为模板进行扩增的技术。在高保真度DNA聚合酶作用下,单分子PCR技术能扩增出高度一致的DNA;在低保真度DNA聚合酶作用下,单分子PCR技术又能构建出含有大量突变基因的DNA库。该将介绍单分子PCR的基本原理、实验步骤、特点及其应用。     

土壤中香蕉枯萎病菌FOC4的实时荧光定量PCR检测

香蕉枯萎病发病率与土壤中香蕉枯萎病菌FOC4的数量密切相关。为了在香蕉定植前准确检测土壤中FOC4的数量,本研究以本实验室克隆的FOC4特异性基因片段作为标准线性片段,设计特异性荧光定量引物:以标准线性片段为模板建立荧光定量标准曲线,以无FOC4的土壤配置FOC4孢子浓度梯度标准土样,采用MoBio土壤基因组DNA提取试剂盒提取总DNA,以各FOC4孢子浓度梯度(1.66×10~3～1.66×108)的标准土样所提取的总DNA为模板进行实时荧光定量PCR,测定各标准土样中FOC4数量。以标准土样FOC4线性片段基因拷贝数的结果与FOC4线性片段基因100%提取率时的理论拷贝数的比值作为该标准土样FOC4基因组的提取率,对待检测土样FOC4的定量检测值进行校正,建立了一套较准确检测土壤FOC4数量的实时荧光定量PCR技术,其检测下线可达400个孢子/克土。应用该项技术对南天黄品种蕉园10个枯萎病病株的土样和10个未发病植株的土样和巴西蕉园5个发病植株的土样进行实际定量检测。结果表明,南天黄发病植株、未发病植株和巴西蕉发病植株的土样中FOC4的数量分别为每克土5 100～11 000个孢子/克土,3 500～7 800个孢子/克土和11 800～101 000个孢子/克土。本研究可准确检测土壤中的FOC4含量,同时也为香蕉枯萎病的防控措施的制定提供了帮助。     

Detection of Stachybotrys chartarum using rRNA, tri5, and β-tubulin primers and determining their relative copy number by real-time PCR

Highly conserved regions are attractive targets for detection and quantitation by PCR, but designing species-specific primer sets can be difficult. Ultimately, almost all primer sets are designed based upon literature searches in public domain databases, such as the National Center for Biotechnology Information (NCBI). Prudence suggests that the researcher needs to evaluate as many sequences as available for designing species-specific PCR primers. In this report, we aligned 11, 9, and 16 DNA sequences entered for Stachybotrys spp. rRNA, tri5, and β-tubulin regions, respectively. Although we were able to align and determine consensus primer sets for the 9 tri5 and the 16 β-tubulin sequences, there was no consensus sequence that could be derived from alignment of the 11 rRNA sequences. However, by judicious clustering of the sequences that aligned well, we were able to design three sets of primers for the rRNA region of S. chartarum. The two primer sets for tri5 and β-tubulin produced satisfactory PCR results for all four strains of S. chartarum used in this study whereas only one rRNA primer set of three produced similar satisfactory results. Ultimately, we were able to show that rRNA copy number is approximately 2-log greater than for tri5 and β-tubulin in the four strains of S. chartarum tested.     

Comparative mitochondrial and nuclear quantitative PCR of historical marine mammal tissue,bone, baleen,and tooth samples

The use of historical and ancient tissue samples for genetic analysis is increasing, with ever greater numbers of samples proving to contain sufficient mitochondrial and even nuclear DNA for multilocus analysis. DNA yield, however, remains highly variable and largely unpredictable based solely on sample morphology or age. Quantification of DNA from historical and degraded samples can greatly improve efficiency of screening DNA extracts prior to attempting sequencing or genotyping, but requires sequence‐specific quantitative polymerase chain reaction (qPCR) based assays to detect such minute quantities of degraded DNA. We present two qPCR assays for marine mammal DNA quantification, and results from analysis of DNA extracted from preserved soft tissues, bone, baleen, and tooth from several cetacean species. These two assays have been shown to amplify DNA from 26 marine mammal species representing 12 families of pinnipeds and cetaceans. Our results indicate that different tissues retain different ratios of mitochondrial to nuclear DNA, and may be more or less suitable for analysis of nuclear loci. Specifically, historical bone and tooth samples average 60‐fold higher ratio of mitochondrial DNA to nuclear DNA than preserved fresh soft tissue, and the ratio is almost 8000‐fold higher in baleen.     

转基因猪中外源基因拷贝数和整合位点的研究

主要采用了绝对定量PCR和热不均一交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR),检测了体细胞核移植技术生产的绿色荧光蛋白转基因猪中外源基因拷贝数和整合位点,并利用旁侧PCR(Junction PCR)对整合位点进行确定,同时进一步分析了整合位点的纯合性．结果表明,绝对定量PCR可以准确有效地检测外源基因拷贝数,标准曲线为：log₂N (拷贝数) =-0.935 4ΔCt + 3.411 6 (R²=0.997 4,P < 0.001),两只转基因猪中外源基因拷贝数分别为30.85 ± 1.77和18.87 ± 1.34;TAIL-PCR能成功地克隆转基因猪中外源基因整合位点,得到25条特异性条带,经BLAST比对,共获得TgInS1 (1 440 bp)、TgInS2 (1 263 bp)和TgInS3 (1 861 bp) 3个整合位点．以整合位点侧翼序列特异性引物与外源基因特异性引物的组合引发Junction PCR,得到预计大小的特异性片段,确定了整合位点上、下游侧翼序列的准确性．采用整合位点5′上游和3′下游侧翼序列特异性引物与外源基因特异性引物的组合,进行Junction PCR,在两只转基因猪中都得到与野生型猪一致的侧翼序列特异性引物扩增片段,表明我们获得的转基因猪都为整合位点杂合子．初步建立了绝对定量PCR和TAIL-PCR对外源基因拷贝数和整合位点检测的体系,为今后研究外源基因在转基因猪中遗传和表达的稳定性打下了基础．     

利用实时荧光定量比较Ct法检测转基因小鼠外源基因拷贝数

目的：在建立转基因小鼠模型时,外源基因拷贝数是影响其表达水平和遗传稳定性的重要因素之一。外源基因拷贝数的精确测定,是建立转基因动物模型的重要环节。方法：合成cagA基因和内参基因GAPDH的引物,用标准曲线法测得cagA和GAPDH基因的扩增效率分别为97．6％和98．6％;将128拷贝阴性小鼠基因组和128拷贝c0鲥打靶质粒的混合物作为参照样品,取6只来自同一母本的F2阳性小鼠的128拷贝基因组作为待测样品;选取GAPDH作为内源参照基因,用比较Ct法对待测样品进行定量。结果：经计算,6只待测小鼠的cagA基因拷贝数平均值为8。结论：利用实时荧光定量PCR仪,呆用改良后的比较Ct法对转基因小鼠的外源基因拷贝数进行了精确测定。     

High-throughput transgene copy number estimation by competitive PCR

Transgene copy number affects the level and stability of gene expression. Therefore, it is important to determine the copy number of each transgenic line. Polymerase chain reaction (PCR) is widely employed to quantify amounts of target sequences. Although PCR is not inherently quantitative, various means of overcoming this limitation have been devised. Recent real-time PCR methods are rapid; however, they typically lack a suitable internal standard, limit the size of the target sequence, and require expensive specialized equipment. Competitive PCR techniques avoid these problems, but traditional competitive methods are time consuming. Here we apply mathematical modeling to create a rapid, simple, and inexpensive copy number determination method that retains the robustness of competitive PCR.