CN103409538B - One use hot start enzyme and dye GelgreenⅠ hTaq quantitative PCR kit comprising - Google Patents

One use hot start enzyme and dye GelgreenⅠ hTaq quantitative PCR kit comprising Download PDF

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CN103409538B
CN103409538B CN 201310360995 CN201310360995A CN103409538B CN 103409538 B CN103409538 B CN 103409538B CN 201310360995 CN201310360995 CN 201310360995 CN 201310360995 A CN201310360995 A CN 201310360995A CN 103409538 B CN103409538 B CN 103409538B
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htaq
solution
dye
enzyme
gelgreen
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CN 201310360995
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CN103409538A (en )
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蒋国成
陈旭
陈刚
吴小祝
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苏州吉泰生物科技有限公司
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Abstract

本发明公开了含有热启动hTaq酶和染料GelgreenⅠ的试剂盒在定量PCR技术中的用途,所述的试剂盒包括荧光染料Gelgreen? The present invention discloses the use of a hot start hTaq containing enzyme and dye GelgreenⅠ of quantitative PCR kit, said kit comprising a fluorescent dye GelGreen? I和热启动hTaq酶,所述的荧光染料Gelgreen? I hTaq and hot start enzyme, a fluorescent dye GelGreen? I能特异性地结合DNA双链而发出荧光信号。 I can specifically emit fluorescence signals binds to DNA duplexes. 本发明提供的含有热启动hTaq酶和染料GelgreenⅠ的试剂盒的用途,由于含有热启动hTaq酶,合适的镁离子浓度,能很好地抑制qPCR反应中的非特异性扩增,避免假阳性和高背景的结果,溶解曲线能获得单一峰;荧光染料Gelgreen? Containing the thermally present invention provides the use hTaq starting enzyme and dye kits GelgreenⅠ, since the enzyme contains a hot start hTaq suitable magnesium concentration, can well suppress nonspecific amplification qPCR reaction, to avoid false positives and high background of the results, the dissolution profile can be obtained as a single peak; fluorescent dye GelGreen? I? I? 价格低廉,使用方便,通用性好,灵敏度非常高,适用于qPCR技术;本发明为分子生物学研究提供了一种新的定量PCR试剂盒的用途。 Low cost, ease of use, versatility, sensitivity is very high, suitable for the qPCR; The present invention provides a new quantitative PCR kits for the use of molecular biological research.

Description

一种含有热启动hTaq酶和染料GeIgreenI的定量PCR试剂盒的用途 One use hot start enzyme and dye GeIgreenI hTaq quantitative PCR kit comprising

技术领域 FIELD

[0001] 本发明涉及定量PCR技术领域,具体地说,是一种含有热启动hTaq酶和染料GelgreenI的定量PCR试剂盒的用途。 [0001] The present invention relates to the field of quantitative PCR technology, particularly, a quantitative PCR comprising use hot start kit hTaq GelgreenI of enzyme and dye.

背景技术 Background technique

[0002] 实时定量PCR(qPCR)就是在PCR扩增过程中,通过实时检测PCR每一个循环扩增产物相对应的荧光信号,来实现对起始模板进行定量及定性分析的一种技术。 [0002] A technique real-time quantitative PCR (qPCR) is, during PCR amplification, a cycle of amplification product corresponding to the fluorescence signals for each PCR detection by real-time, to achieve the initial template for quantitative and qualitative analysis. qPCR技术与普通PCR技术的不同主要在于:普通的PCR人工参与的更多,数据可能不是特别准确,PCR 结束后还需琼脂糖电泳检测;而qPCR是实时监控检测,无需后期电泳,因此qPCR具有检测时间短、操作简便、特异性高、重复性好、灵敏度高、分辨率高、检测范围较广、定量精确等优点。 QPCR technology different from ordinary PCR technique include: PCR ordinary human intervention more, the data may not be particularly accurate, agarose gel electrophoresis after the PCR needed to detect; qPCR is a real-time monitoring and detection, without post electrophoresis, and therefore have qPCR the detection time is short, simple, high specificity, good repeatability, high sensitivity, high resolution, wide detection range, the advantages of precise quantification. 由于qPCR技术具备较多优点,现在已经应用到生命科学研究的各个领域,例如基因的差异表达分析、SNP检测、等位基因的检测、药物开发、临床诊断、转基因研究等。 Since the qPCR technology has many advantages, it is now applied to all areas of life science research, such as differential gene expression analysis, SNP detection, detection, drug discovery, clinical diagnostics, transgenic research allele.

[0003]qPCR的定量原理涉及到几个重要的参数:Ct值,阙值,基线。 [0003] qPCR quantification principles involved several important parameters: Ct value threshold baseline. 一般来说,第3~15 个循环的荧光值就是基线。 In general, 3 to 15 cycles is the baseline fluorescence values. 阈值一般是基线的标准偏差的10倍。 Threshold is typically 10 times the baseline standard deviation. Ct值就是荧光值达到阈值时候的PCR循环次数。 Ct value is the PCR cycle fluorescence value reaches a threshold time. 由于在PCR扩增的指数时期,DNA模板的Ct值和该模板的起始拷贝数存在线性关系(线性方程为Ct=-KlogN。+B,其中N。是模板起始拷贝数),所以成为定量的依据。 Since the period of exponential PCR amplification, there is a linear relationship between Ct value starting copy number of the DNA template and the template (linear equation Ct = -KlogN. + B, where the template is the starting copy number N.), it becomes quantitative basis. qPCR的定量又分为:绝对定量和相对定量。 Quantitative qPCR is divided into: Absolute and relative quantification. 绝对定量的目的是测定目的基因在样本中的分子数目,即通常所说的拷贝数。 Absolute quantification purpose is the number of molecules in a sample assay target gene, commonly referred to as copy number. 相对定量的目的是测定目的基因在两个或多个样本中的含量的相对比例,而不需要知道它们在每个样本中的拷贝数。 Relative quantification of the relative proportions of the measurement object of the gene content in two or more samples, without knowing their copy number in each sample.

[0004]qPCR有两步法、二步法等多种方法,经典的二步法是变性、退火、延伸二步。 [0004] qPCR There are several ways two-step method, two-step method and the like, classical two-step denaturation, annealing, extension of two steps. 两步法是将退火与延伸采用同一个温度将二者合并为一步,缩短实验时间,该方法对酶的要求较高,三步法得到的数据相对更准确。 Is a two-step process using a single annealing and extension temperature both combined in one step, to shorten the time of the experiment, this method requires high enzyme to obtain data corresponding to the three-step method is more accurate. 具体用哪一种方法,要结合引物退火温度、酶的特性、具体试验方案进行选择。 Which particular method used, in conjunction with the primer annealing temperature, the characteristics of the enzyme, specific test programs selected.

[0005] 不管是绝对定量还是相对定量,用三步法还是两步法,qPCR技术的核心是扩增过程中有荧光发出,常用荧光染料有SYBRGreen、GelRed、GelGreen、GoldView™或GeneFinder™,只有保证荧光信号的增加与PCR产物的增加完全同步,才能确保检测灵敏度高,检测结果准确。 [0005] Whether absolute quantification or relative quantification, step process or a two step process, the core qPCR technology is fluorescence emitted during amplification, used fluorescent dyes SYBRGreen, GelRed, GelGreen, GoldView ™ or GeneFinder ™, only to ensure that the increase in the fluorescence signal is fully synchronized with the increase of PCR product in order to ensure high detection sensitivity, accurate detection results. 在qPCR检测过程中经常碰到非特异性扩增问题,非特异性扩增就是扩增出了目的条带以外的条带,出现假阳性和高背景的结果。 In qPCR detection process often encounter the problem of non-specific amplification of non-specific amplification is amplified bands other than the target band, false-positive results and high background appears.

发明内容 SUMMARY

[0006] 本发明的目的是针对现有技术中的不足,提供一种含有热启动hTaq酶和染料GelgreenI的定量PCR试剂盒的用途。 [0006] The object of the present invention is directed to the prior art is insufficient, there is provided the use of a hot start hTaq containing enzyme and dye GelgreenI quantitative PCR kits.

[0007] 为实现上述目的,本发明采取的技术方案是: [0007] To achieve the above object, the present invention takes the following technical solution:

[0008] 含有热启动hTaq酶和染料GelgreenI的试剂盒在定量PCR技术中的用途,所述的试剂盒包括荧光染料GelgreenI和热启动hTaq酶,所述的荧光染料GelgreenI能特异性地结合DNA双链而发出荧光信号,而不结合DNA链的GelgreenI染料分子不会发射任何荧光信号,从而保证荧光信号的增加与PCR产物的增加完全同步。 [0008] hTaq containing hot-start enzyme and dye GelgreenI use of quantitative PCR kit, said kit comprising a fluorescent dye and a hot start hTaq GelgreenI enzyme, a fluorescent dye that specifically bind DNA GelgreenI bis chain emit fluorescence signals and does not bind DNA strand GelgreenI dye molecule does not emit any fluorescent signals, thereby ensuring an increase in fluorescence signal with a PCR product increases completely synchronized.

[0009] 所述的荧光染料GelgreenI的工作浓度为0• 5X~IX,所述热启动hTaq酶的工作浓度为〇. 01~〇. 1U/uL。 [0009] The working concentration of the fluorescent dye is GelgreenI 0 • 5X ~ IX, the working concentration hTaq hot start enzyme is square. ~ 01 billion. 1U / uL.

[0010] 所述的试剂盒还包括PCR缓冲液、dATP溶液、dUTP溶液、dGTP溶液、dCTP溶液、PCR 保护剂、镁离子和无菌双蒸水。 [0010] The kit further comprises a PCR buffer, dATP solution, dUTP solution, dGTP solution, dCTP solution, PCR protectant, magnesium ions and sterile double distilled water.

[0011] 所述的试剂盒还包括PCR缓冲液、正向引物、反向引物、dATP溶液、dUTP溶液、dGTP 溶液、dCTP溶液、PCR保护剂、镁离子和无菌双蒸水。 [0011] The kit further comprises a PCR buffer, a forward primer, a reverse primer, dATP solution, dUTP solution, dGTP solution, dCTP solution, PCR protectant, magnesium ions and sterile double distilled water.

[0012] 所述的dATP溶液、dUTP溶液、dGTP溶液或dCTP溶液的工作浓度为100~ 200ymol/L〇 [0012] The solution of dATP, dUTP solution, dGTP dCTP working strength solution, or solution is 100 ~ 200ymol / L〇

[0013] 所述的PCR保护剂是小牛血清白蛋白、明胶、Tween20或二硫苏糖醇。 PCR protective agent [0013] is the bovine serum albumin, gelatin, or Tween20-dithiothreitol.

[0014] 所述的PCR保护剂更优选为小牛血清白蛋白。 [0014] PCR of the protecting agent is more preferably bovine serum albumin.

[0015] 应用本发明试剂盒的PCR反应体系中DNA模板量为:以25yL反应体系计,基因组10~lOOOng,或者质粒1~30ng,或者RT-PCR反应后的cDNA1~2yL;镁离子浓度为L5 ~2. 0mmol/L〇 [0015] PCR reaction system application kits of the present invention, DNA templates in an amount of: at 25yL reaction system meter, cDNA1 the genome of 10 ~ lOOOng, or plasmid 1 ~ 30ng, or RT-PCR reactions ~ 2yL; magnesium ion concentration L5 ~ 2. 0mmol / L〇

[0016] 本发明优点在于: [0016] The advantages of the present invention comprising:

[0017] 本发明提供的含有热启动hTaq酶和染料GelgreenI的试剂盒的用途,由于含有热启动hTaq酶,合适的镁离子浓度,能很好地抑制qPCR反应中的非特异性扩增,避免假阳性和高背景的结果,溶解曲线能获得单一峰;焚光染料GelgreenI价格低廉,使用方便,通用性好,灵敏度非常高,适用于qPCR技术;本发明为分子生物学研究提供了一种新的定量PCR试剂盒的用途。 [0017] The present invention provides a thermal comprising starting use of the kit of enzyme and dye GelgreenI hTaq, since the enzyme contains a hot start hTaq suitable magnesium concentration, can well suppress nonspecific amplification qPCR reaction, to avoid false the positive result of the high background, the dissolution profile can be obtained as a single peak; low burning light dye GelgreenI price, ease of use, versatility, sensitivity is very high, suitable for the qPCR; the present invention provides a new molecular biology of the use of quantitative PCR kit.

附图说明 BRIEF DESCRIPTION

[0018] 附图1是qPCR反应的程序设置图。 [0018] Figure 1 is a program provided in FIG qPCR reaction.

[0019] 附图2是qPCR的扩增曲线 [0019] Figure 2 is the qPCR amplification curve

[0020] 附图3是qPCR的融解曲线。 [0020] Figure 3 is a melting curve of qPCR.

具体实施方式 detailed description

[0021] 下面结合附图对本发明提供的具体实施方式作详细说明。 [0021] DETAILED DESCRIPTION OF THE DRAWINGS Embodiment of the present invention provides detailed description.

[0022] 本发明实施例中所用焚光染料GelgreenI购自上海吉泰远成生物科技有限公司;热启动hTaq酶购自NewEnglandBiolabs(NEB)公司;PCR缓冲液、dATP溶液、dUTP 溶液、dGTP溶液、dCTP溶液均购自上海吉泰远成生物科技有限公司;正向引物、反向引物、 PCR保护剂等均购自上海吉泰远成生物科技有限公司。 [0022] Example embodiments of the present invention, the dye used in the light burning GelgreenI SGT far as available from Biotechnology Limited; hot-start enzymes were purchased from hTaq NewEnglandBiolabs (NEB) Company; the PCR buffer, dATP solution, dUTP solution, dGTP solution, dCTP solutions were purchased from SGT far as biotechnology Ltd.; forward primer, reverse primer, protective agent were purchased from the PCR SGT far as biotechnology company. 本发明含热启动hTaq酶和染料GelgreenI的定量PCR试剂盒,能解决非特异性扩增这个难题,并且提供了一种新的性能较好的焚光物质Gelgreen1〇 The present invention containing hot start enzyme and dye GelgreenI hTaq quantitative PCR kit, non-specific amplification can solve this problem, and provides a better performance of a new light burning substance Gelgreen1〇

[0023] 实施例1 [0023] Example 1

[0024] -、本实施例的定量PCR试剂盒成份如下: [0024] -, quantitative PCR components of the kit according to the present embodiment is as follows:

[0025] lOXBuffer(Mg2+free); [0025] lOXBuffer (Mg2 + free);

[0026] 10XGelgreenI; [0026] 10XGelgreenI;

[0027] 10XPCR保护剂(小牛血清白蛋白); [0027] 10XPCR protecting agent (bovine serum albumin);

[0028] 各10mmol/L的dATP、dUTP、dGTP和dCTP溶液; [0028] Each 10mmol / L of dATP, dUTP, dGTP, and dCTP solution;

[0029] 25mmol/L的Mg2+; [0029] 25mmol / L of Mg2 +;

[0030] 5U/yL的hTaq酶; [0030] 5U / yL of hTaq enzyme;

[0031] 无菌双蒸水。 [0031] sterile double distilled water.

[0032] 二、本实施例定量PCR试剂盒的应用 [0032] Second, the application of quantitative PCR kit embodiment according to the present embodiment

[0033] (1)制备待测DNA:按常规方法从小鼠血液中抽提DNA,扩增EGF基因。 [0033] (1) Preparation of test DNA: DNA is extracted by conventional methods from mouse blood, EGF gene was amplified.

[0034] (2)引物的合成。 [0034] (2) Synthesis of primers.

[0035] 正向引物:TATAGATATCATGAATAGTTATCCAGGATGCCCA [0035] Forward primer: TATAGATATCATGAATAGTTATCCAGGATGCCCA

[0036] 反向引物:TATAGAAITCTCAACGCAGITCCCACCATCGTAG。 [0036] Reverse primer: TATAGAAITCTCAACGCAGITCCCACCATCGTAG.

[0037] (3)PCR反应体系的建立,反应体积共25yL,参照下表添加各试剂。 [0037] establishment (3) PCR reaction system, a total reaction volume 25yL, see following table for the respective reagents.

[0038] 表1PCR反应体系 [0038] Table 1PCR reaction system

[0039] [0039]

Figure CN103409538BD00051

[0040] (4)qPCR反应 [0040] (4) qPCR reaction

[0041] 采用三步法PCR反应程序,热启动hTaq酶需要热激活处理以恢复酶活,所以设置PCR反应预变性条件为95 °C、10分钟,程序设置如图1所示。 [0041] The three-step PCR reaction procedure, hot start hTaq enzymes require thermal activation process to recover enzyme activity, the set conditions for the PCR reaction denaturation 95 ° C, 10 min program settings as shown in FIG.

[0042] Segment1 :PCR反应预变性阶段。 [0042] Segment1: PCR denaturation reaction stage.

[0043] Segment2:qPCR反应阶段。 [0043] Segment2: qPCR reaction stage.

[0044] Segment3 :扩增产物融解曲线信息采集阶段。 [0044] Segment3: amplification product melting curve information acquisition phase.

[0045] (5)实验结果 [0045] (5) Test Results

[0046] qPCR的扩增曲线请见图2,从图中可以看出荧光信号的采集非常好,说明本发明所选用的荧光染料GelgreenI用于qPCR非常合适,方便实用、灵敏性高且荧光染料成本低。 Amplification curve [0046] Please see FIG. 2 qPCR, can be seen from FIG collected the fluorescence signal is very good, the choice of the present invention described fluorescent dyes for qPCR GelgreenI very suitable, convenient and practical, high sensitivity and fluorescent dyes low cost.

[0047] qPCR的融解曲线请见图3,图片上只有一个单一峰,说明使用的试剂盒扩增效果很好,因含有合适浓度热启动hTaq酶,很好地解决了qPCR技术中存在的非特异性扩增难题。 [0047] qPCR melting curve See Figure 3, the picture is only a single peak, indicating that use of the kit to amplify well, for containing an appropriate concentration of enzyme hTaq hot start, a good solution to the art that non-specific qPCR specifically amplify problems.

[0048] 实施例2 [0048] Example 2

[0049] 本实施例的定量PCR试剂盒成份如下: [0049] Quantitative PCR components of the kit according to the present embodiment is as follows:

[0050] lOXBuffer(Mg2+free); [0050] lOXBuffer (Mg2 + free);

[0051] lOXGelgreenI; [0051] lOXGelgreenI;

[0052] 10XPCR保护剂(Tween20); [0052] 10XPCR protecting agent (Tween20-);

[0053] 各5mmol/L的dATP、dUTP、dGTP和dCTP溶液; [0053] Each 5mmol / L of dATP, dUTP, dGTP, and dCTP solution;

[0054] 20mmol/L的Mg2+; [0054] 20mmol / L of Mg2 +;

[0055] 5U/yL的hTaq酶; [0055] 5U / yL of hTaq enzyme;

[0056] 10ymol/L的正向引物; [0056] 10ymol / L forward primer;

[0057] 10ymol/L的反向引物; [0057] 10ymol / L reverse primer;

[0058] 无菌双蒸水。 [0058] sterile double distilled water.

[0059] 所述的荧光染料GelgreenI的工作浓度为0• 5X~1X。 [0059] The fluorescent dye GelgreenI working concentration of 0 • 5X ~ 1X. 所述热启动hTaq酶的工作浓度为〇. 01~〇. 1U/yL。 The hot-start enzyme hTaq working concentration of square. ~ 01 billion. 1U / yL. 所述的dATP溶液、dUTP溶液、dGTP溶液或dCTP溶液的工作浓度为100~200umol/L。 The solution of dATP, dUTP solution, dGTP dCTP working strength solution, or solution is 100 ~ 200umol / L.

[0060] 按常规方法制备待测DNA,建立PCR反应体系,进行qPCR反应。 [0060] Such DNA prepared by conventional methods, PCR reaction system, the reaction qPCR was performed.

[0061] 实施例3 [0061] Example 3

[0062] 本实施例的定量PCR试剂盒成份如下: [0062] Quantitative PCR components of the kit according to the present embodiment is as follows:

[0063] lOXBuffer(Mg2+free); [0063] lOXBuffer (Mg2 + free);

[0064] lOXGelgreenI; [0064] lOXGelgreenI;

[0065] 10XPCR保护剂(二硫苏糖醇); [0065] 10XPCR protecting agent (dithiothreitol);

[0066] 各lOmmol/L的dATP、dUTP、dGTP和dCTP溶液; [0066] Each lOmmol / L of dATP, dUTP, dGTP, and dCTP solution;

[0067] 30mmol/L的Mg2+; [0067] 30mmol / L of Mg2 +;

[0068] 5U/yL的hTaq酶; [0068] 5U / yL of hTaq enzyme;

[0069] 10ymol/L的正向引物; [0069] 10ymol / L forward primer;

[0070] 10ymol/L的反向引物; [0070] 10ymol / L reverse primer;

[0071] 无菌双蒸水。 [0071] sterile double distilled water.

[0072] 所述的荧光染料GelgreenI的工作浓度为0• 5X~1X。 [0072] The fluorescent dye GelgreenI working concentration of 0 • 5X ~ 1X. 所述热启动hTaq酶的工作浓度为〇. 01~〇. 1U/yL。 The hot-start enzyme hTaq working concentration of square. ~ 01 billion. 1U / yL. 所述的dATP溶液、dUTP溶液、dGTP溶液或dCTP溶液的工作浓度为100~200umol/L。 The solution of dATP, dUTP solution, dGTP dCTP working strength solution, or solution is 100 ~ 200umol / L.

[0073] 按常规方法制备待测DNA,建立PCR反应体系,进行qPCR反应。 [0073] Such DNA prepared by conventional methods, PCR reaction system, the reaction qPCR was performed.

[0074] 以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。 [0074] The above are only preferred embodiments of the present invention, it should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, can make various improvements and additions, modifications and additions of these also it is considered the scope of the present invention.

Claims (1)

  1. 1.含有热启动hTaq酶和染料Gelgreen I的试剂盒在定量PCR技术中的用途,其特征在于,所述的试剂盒包括上海吉泰远成生物科技有限公司的焚光染料Gelgreen I和New England Biolabs公司的热启动hTaq酶,所述的焚光染料Gelgreen I能特异性地结合DNA 双链而发出荧光信号,所述的荧光染料Gelgreen I的工作浓度为0.5X,所述热启动hTaq 酶的工作浓度为〇. 05U/ y L,所述的试剂盒包括正向引物、反向引物、PCR缓冲液、dATP溶液、dUTP溶液、dGTP溶液、dCTP溶液、PCR保护剂、镁离子和无菌双蒸水,所述的dATP溶液、 dUTP溶液、dGTP溶液和dCTP溶液的工作浓度为200 ymol/L,所述的正向引物的序列是TA TAGATATCATGAATAGTTATCCAGGATGCCCA,所述的反向引物的序列是TATAGAAITCTCAACGCAGITC CCACCATCGTAG,所述的PCR保护剂是小牛血清白蛋白。 1. Use hTaq containing hot-start enzyme and dye Gelgreen I kit is a quantitative PCR technique, wherein said kit comprises SGT far as light burning Biotechnology Co., Ltd. and New England dye Gelgreen I hTaq hot start enzyme Biolabs company, the burning light Gelgreen I dye which specifically bind double-stranded DNA and emits a fluorescence signal, the working concentration of the fluorescent dye is Gelgreen I 0.5X, the hot-start enzyme hTaq working concentration of square. 05U / y L, said kit comprising a forward primer, a reverse primer, the PCR buffer, dATP solution, dUTP solution, dGTP solution, dCTP solution, the PCR protectant, magnesium and sterile double distilled water, dATP said solution, dUTP solution, dGTP dCTP working strength solution and a solution of 200 ymol / L, the forward primer sequence is TA TAGATATCATGAATAGTTATCCAGGATGCCCA, the reverse primer sequence is TATAGAAITCTCAACGCAGITC CCACCATCGTAG, the protecting agent is a PCR bovine serum albumin.
CN 201310360995 2013-08-19 2013-08-19 One use hot start enzyme and dye GelgreenⅠ hTaq quantitative PCR kit comprising CN103409538B (en)

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