SYBR Green I

摘要： 建立了基于核酸适配体的特异性识别和核酸外切酶Ⅰ(ExoⅠ)辅助目标物循环放大的非标记荧光检测方法,用于土霉素的定量测定.体系中无土霉素时,SYBR Green I(SGI)分子可以插入土霉素适配体与其互补链形成的双链DNA(dsDNA)中,并在495 nm光激发下产生强烈的荧光发射.在土霉素存在时,由于核酸适配体与土霉素之间的强亲和力而特异性结合,dsDNA被打开并释放出SGI,游离的SGI荧光明显减弱.为了增强土霉素存在时引起的SGI荧光猝灭效应,采用ExoⅠ辅助目标物循环放大的策略.丙烯酰胺凝胶电泳实验结果证实,ExoⅠ可选择性降解单链DNA以及与土霉素结合的核酸适配体.被释放的土霉素参与下一个循环,不断破坏dsDNA,游离的SGI增多,荧光逐渐减弱.基于ExoⅠ辅助目标物循环放大的策略,检测灵敏度得到了有效提高.在最优实验条件下,本方法检测土霉素的线性范围为0.01～10μg/mL,检出限(3σ)为6.77 ng/mL.采用本方法检测实际样品牛奶和蜂蜜中的土霉素,牛奶样品的加标回收率为93.0％ ～105.1％,相对标准偏差(RSD)为0.5％ ～6.2％,蜂蜜样品的加标回收率为94.0％ ～95.8％,RSD为0.3％ ～7.7％.此荧光传感器具有成本低、灵敏度高和特异性好等优点,在快速检测食品有害物残留方面具有良好的应用潜力.

摘要： 以哑铃型(DS)DNA链作信号探针,基于DNA连接反应调控核酸外切酶作用的原理,发展了一种高灵敏、非标记检测碱性磷酸酶(ALP)的荧光传感方法.无目标ALP(CIP)时,连接反应将DS探针缺口缝合,形成哑铃状闭合DNA,阻止外切酶的酶切,嵌入SYBR Green I(SG I)后,得到显著的荧光信号,荧光强度与CIP浓度成反比.CIP检测的线性范围为0.00004～0.002 U/mL,检测限为2×10-5 U/mL.该方法具有高灵敏、高选择性和低成本的特点,可适用于ALP活性抑制剂的筛选.

摘要： cqvip:真鲷虹彩病毒(Red Sea Bream Iridovirus, RSIV)一直被世界动物卫生组织(Office International Des Epizooties, OIE)列为必报疫病病原,为了精准快速检测该病原,本文选取真鲷虹彩病毒保守基因片段为靶序列,设计并合成SYBR Green I实时荧光PCR引物,将另行扩增获得的特异PCR目的基因片段克隆到pMD18-T载体,得到重组质粒标准品。通过优化反应条件,建立了RSIV SYBR Green I实时荧光PCR检测法,并对该方法进行了灵敏度、特异性和重复性分析。试验结果证明,该反应体系的最佳引物浓度为200 nmol/L;检测限最低可达到0.04 pg/反应,其灵敏度比普通PCR要高出10倍。特异性试验表明,该方法与IHNV、IPNV、SVCV和VHSV这4种水生动物病毒无交叉反应,只与目标病原有特异性扩增。重复性试验显示,该方法具备良好的重复性及稳定性。以上结果显示,研究建立的RSIV的SYBR Green I实时荧光PCR方法具有较高的敏感性、良好的特异性及重复性。适用于水生动物及产品中真鲷虹彩病毒的检测与监测,对加强进出口水产品中RSIV的检验检疫具有十分重要的意义。

摘要： 目的：探寻慢性荨麻疹患者血清中 miR-155 和 miR-203 的表达及临床意义。方法 收集本单位皮肤科诊断是慢性荨麻疹患者的血清为实验组，体检中心体检无皮肤病的正常健康人血清为对照组。用 miRAN 1st Strand cDNA Synthesis Kit（茎环法 miRNA cDNA 一链合成试剂盒），SYBR Green I 嵌合荧光法对 miR-155、miR-203进行检测。结果 慢性荨麻疹患者血清中 miR-155 的相对表达水平明显高于正常健康人血清中的相对表达水平，差异有统计学意义（P<0.05）；miR-203 在慢性荨麻疹患者血清的相对表达水平也高于正常健康人血清中的相对表达水平，差异有统计学意义（P<0.05）。结论 慢性荨麻疹患者血清中 miR-155、miR-203 的表达均存在差异，这种差异有望使之成为慢性荨麻疹实验室诊断的新生物学标志物。

摘要： 提出了一种检测三磷酸腺苷(ATP)的非标记荧光新方法,该方法基于T4多聚核苷酸激酶调控Lambda核酸外切酶活性的原理,利用DNA荧光染料SYBR Green I作信号报告基团.有ATP时,双链DNA底物能在T4 PNK作用下发生5'端磷酸化,进而被核酸外切酶降解成DNA碎片,得到较弱的荧光信号,信号强度与ATP浓度成反比.该方法对ATP检测的线性范围为0.04～4 mmol/L,检测限为20 nmol/L.实验结果表明本方法具有快速、成本低、灵敏度高和简单易操作等优点,可进一步应用于复杂生物样品的分析.

摘要： 目的建立超级耐药基因NDM-1的SYBR Green I荧光定量PCR检测方法。方法针对超级耐药基因NDM-1序列,合成其特异性引物,并对反应条件进行优化,建立基于SYBR Green I实时荧光定量PCR检测方法,观察该方法的灵敏度和重复性。结果标准品在5.21×10^(1)~5.21×10^(8) copies/μL内具有良好的线性关系,标准曲线R 2值均在0.99以上,最低检出量为5.21×10^(1) copies/μL,稳定性好。结论成功建立了超级耐药基因NDM-1的检测方法。

摘要： 利用氧化石墨烯(GO)的吸附、荧光猝灭性能结合外切酶反应,发展了一种非标记荧光检测碱性磷酸酶(ALP)活性的新方法.双链DNA的5'端磷酸根被ALP水解成羟基,抑制了Lambda核酸外切酶(λexo)的降解,再与SYBR Green I(SGⅠ)结合并加入GO,仍得到显著的荧光信号.无ALP时,5'磷酸化的双链DNA被λexo降解成单链DNA进而被GO吸附猝灭.结果表明,新方法对ALP的检测具有高灵敏度、高信噪比,线性范围为0.0001～0.01 U/mL,检测限低至0.00006 U/mL.

摘要： The study was to develop a real-time assay for detecting swine pro-platelet basic protein. According to PPBP gene sequence, specific primer was designed by primer design software Primer Premier5.0 The resulting fragments were cloned into the vector to construct recombinant plasmids of p3XFLAG-CMV-7.1-PPBP. The recombinant plasmid was used as standard products to establish standard curve of porcine PPBP and detect repeatability, sensitivity and specificity. The results show a precise linear relationship with a correlation coefficient R2＞0.99. The amplification efficiency is 102%. Dissolve curve appears one peak. The variation coefficient is less than 0.5% within and between assays. The SYBR GreenI real-time PCR assay was used to detect porcine PPBP from mock pigs and pigs infected with PRRSV. PPBP was found in this samples and was significant different between the infected and mock group.This SYBR Green I real-time PCR assay developed in this study has highly sensitivity, specificity, stability and repeatability, which could be a tool for detecting relationship between pig infectious diseases and porcine PPBP.%根据猪源干扰素刺激基因PPBP(pro-platelet basic protein)基因全长序列,使用引物设计软件Primer Premier5.0设计合成特异性引物,经过PCR扩增,将基因扩增的片段连接到相应的载体上,成功构建重组质粒p3XFLAG-CMV-7.1-PPBP.重组质粒经过筛选、鉴定以及纯化后,经10倍系列稀释作为质控样品,用于实时荧光定量PCR中PPBP标准曲线的构建,并检测重复性、灵敏性和特异性.结果显示标准曲线线性关系R2＞0.99;特异性试验中也能检测到PPBP扩增曲线;组间和组内变异系数均小于5%.使用建立的SYBR Green I荧光定量RT-PCR检测PRRSV感染组织和未感染组织中PPBP的表达情况,能够检测出组织中PPBP的含量存在差异.本研究初步建立了检测猪PPBP基因的SYBR Green I荧光定量RT-PCR的方法,为后续猪传染性疾病和猪PPBP之间相互关系的研究提供一个特异灵敏的检测和手段.

摘要： Objective:To establish a Real-time quantitative RT-qPCR detection method of IL-17 and detect the IL-17 mRNA level of human three negative breast cancer (TNBC).Methods:TNBC was the experimental group while the fibroadenoma adjacent normal breast was control group.Both groups were stained by HE in order to confirme.Total RNA was extracted by Trizol and reverse transcribed into cDNA.β-actin was chose as an internal control.Establish SYBR Green I Real-time RT qPCR method and detect the initial template amount of two groups of IL-17.The relative expression of mRNA IL-17 was calculated by IL-17/β-actin.Results:IL-17 amplification efficiency was 98.6%,correlation coefficient was 0.997.Melting curve was specific unimodal.Coefficient of variation was less than 2.0%.IL-17 mRNA expressed in each group as follow:TNBC group [(0.64±0.12)×10-2] was higher than normal control group [(0.43±0.07)×10-2].The difference was statistically significant (P=0.025).Conclusion:The RT-qPCR Real-time detection method was successfully established.The high expression of IL-17 in the TNBC group suggested that it may be related to the TNBC,which laid a theoretical foundation for the study of the pathogenesis of TNBC.%目的:建立Real-time RT-qPCR(实时荧光定量RT-PCR)检测IL-17的方法,应用该方法检测三阴性乳腺癌(three negatⅣe breast cancer,TNBC)组织IL-17 mRNA的水平.方法:以TNBC组织为实验组,纤维腺瘤旁正常乳腺组织为对照组,并经HE染色病理分析确诊,Trizol法提取总RNA并反转录为cDNA.选β-actin作为内参,建立SYBR GreenⅠReal-time RT-qPCR检测法.利用该方法检测两组IL-17和β-actin的初始模板量,IL-17/β-actin计算IL-17 mRNA 的相对表达量.结果:IL-17扩增效率为98.6%,相关系数0.997,溶解曲线为特异单峰,变异系数小于2.0%,IL-17 mRNA在TNBC组[(0.64±0.12)×10-2]的相对表达高于正常对照组[(0.43±0.07)×10-2],差异有统计学意义(P=0.025).结论:成功建立了人源IL-17的Real-time RT-qPCR检测方法,TNBC组IL-17的高表达提示其可能与TNBC有一定关系,为研究TNBC的发病机制奠定了理论基础.

摘要： 目的：建立Poly（A）加尾SYBR Green I实时荧光定量PCR法检测血清微RNA⁃122（miR⁃122）水平，并初步探讨其临床应用价值。方法选取2013年9月至2014年9月本院就诊的糖尿病患者和健康体检者120例为研究对象，分为糖尿病脂质代谢紊乱组（n=40）、糖尿病非脂质代谢紊乱组（n=40）和对照组（n=40）。提取血清总RNA，miR⁃122 Poly（A）聚合酶加尾逆转录获得cDNA，进行SYBR Green I实时荧光定量PCR扩增。制作C.elegans⁃miR⁃39模拟物浓度梯度稀释的标准曲线定量检测血清miR⁃122水平，进行3组血清miR⁃122水平的组间比较。结果该方法可定量检测血清miR⁃122水平，PCR扩增产物特异性好，熔解曲线呈单峰；检测灵敏度为102拷贝/μl，检测线性范围102~107拷贝/μl。高、中、低浓度样本重复性检测的批内变异系数（CV）分别为2.23%、2.48%和2.75%，其批间CV分别为5.35%、5.88%和5.72%，显示该方法具有较好的重复性和稳定性。该方法检测糖尿病脂质代谢紊乱组、糖尿病非脂质代谢紊乱组和对照组的血清miR⁃122水平分别为（4.85±3.68）×103拷贝/μl、（3.06±1.86）×103拷贝/μl和（1.03±0.82）×103拷贝/μl，糖尿病脂质代谢紊乱组血清miR⁃122水平高于糖尿病非脂质代谢紊乱组和对照组（均P<0.01），糖尿病非脂质代谢紊乱组血清miR⁃122水平高于对照组（P<0.05）。结论成功建立Poly（A）加尾SYBR Green I实时荧光定量PCR方法检测血清miR⁃122水平，该方法的灵敏度高、特异性和重复性均较好。%Objective To establish a method of SYBR Green I real⁃time fluorescence quantitative PCR(FQ⁃PCR)by Poly(A)tailing for determination of serum microRNA⁃122(miR⁃122)level and to preliminarily explore its clinical value. Methods One hundred and twenty subjects who were treated for diabetes or received health checkup in our hospital between September 2013 and September 2014 were included in this study. The subjects were divided into the dyslipidemia diabetic group (n=40),non⁃dyslipidemia diabetic group(n=40)and control group(n=40). Total RNA was extracted from serum samples. MiR⁃122 was reversely transcribed into cDNA by Poly(A) polymerase tailing,and then the cDNA was amplified by SYBR Green I real⁃time FQ⁃PCR. Standard curve of serially diluted concentrations of C.elegans⁃miR⁃39 mimics was generated and used for quantitative measurement of serum miR⁃122 levels. The serum miR⁃122 levels were compared among the 3 groups. Results FQ⁃PCR method can quantitatively measure the serum miR⁃122 levels. The PCR amplification yielded specific products with a single peak on melting curves. The detection sensitivity was 102 copies/μl,and the linear range of detection was 102 to 107 copies/μl. The coefficients of variation(CV)for repetitive measurements of high⁃,medium⁃and low⁃concentration samples were 2.23%,2.48% and 2.75% within⁃runs,5.35%,5.88% and 5.72% between⁃runs,respectively, suggesting good reproducibility and stability by using this method. The serum levels of miR⁃122 as detected by this method were (4.85 ± 3.68)× 103 copies/μl in the dyslipidemia diabetic group,(3.06 ± 1.86)× 103 copies/μl in the non⁃dyslipidemia diabetic group,and(1.03±0.82)×103 copies/μl in the control group. The serum levels of miR⁃122 in dyslipidemia diabetic group were higher than that in non⁃dyslipidemia diabetic group or in the control group(both P<0.01),and it’s higher in non⁃dyslipidemia diabetic group than that in the control group (P<0.05). Conclusion Poly (A) tailing⁃based SYBR Green I real⁃time FQ⁃PCR was successfully established for determining serum miR⁃122 levels,which was with high sensitivity,good specificity and reproducibility.

摘要： SYBR Green I是一种荧光染料，与双链DNA结合后，能产生增强的荧光信号。在Gene Amp5700扩增仪或Roch Lightcycler扩增仪上，SYBR Green I的荧光可以被实时检测。近年来器官移植在世界乃至我国广泛开展，快速准确地完成临床移植HLA分型，是确保器官移植成功至关重要的环节。本实验利用SYBR Green I与双链DNA结合发射荧光的特性，将其用于实时荧光PCR检测，以HLADRB1*12位点为例，进行HLA分型。

摘要： SYBR Green I是一种荧光染料，与双链DNA结合后，能产生增强的荧光信号。在Gene Amp5700扩增仪或Roch Lightcycler扩增仪上，SYBR Green I的荧光可以被实时检测。近年来器官移植在世界乃至我国广泛开展，快速准确地完成临床移植HLA分型，是确保器官移植成功至关重要的环节。本实验利用SYBR Green I与双链DNA结合发射荧光的特性，将其用于实时荧光PCR检测，以HLADRB1*12位点为例，进行HLA分型。

摘要： SYBR Green I是一种荧光染料，与双链DNA结合后，能产生增强的荧光信号。在Gene Amp5700扩增仪或Roch Lightcycler扩增仪上，SYBR Green I的荧光可以被实时检测。近年来器官移植在世界乃至我国广泛开展，快速准确地完成临床移植HLA分型，是确保器官移植成功至关重要的环节。本实验利用SYBR Green I与双链DNA结合发射荧光的特性，将其用于实时荧光PCR检测，以HLADRB1*12位点为例，进行HLA分型。

摘要： SYBR Green I是一种荧光染料，与双链DNA结合后，能产生增强的荧光信号。在Gene Amp5700扩增仪或Roch Lightcycler扩增仪上，SYBR Green I的荧光可以被实时检测。近年来器官移植在世界乃至我国广泛开展，快速准确地完成临床移植HLA分型，是确保器官移植成功至关重要的环节。本实验利用SYBR Green I与双链DNA结合发射荧光的特性，将其用于实时荧光PCR检测，以HLADRB1*12位点为例，进行HLA分型。

摘要： 本发明建立了一种结合SYBR GreenⅠ的环介导等温扩增技术定量检测斱法，具体是在定性LAMP斱法的基础上，对影响定量的各个因素进行调整，最终确定了一个新的定量LAMP反应体系。利用本发明检测沙门氏菌以及单增李斯特菌得到了良好的检测结果，结果重现性好。