CN109609607A - 一种用于锌离子定量检测的方法 - Google Patents
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Abstract
本发明提供一种用于锌离子定量检测的方法,本发明检测主要包含两个反应步骤:依赖性切割反应和RT‑qPCR,在第一步中,将5μL Zn2+浓度的溶液、10μL Zn2+依赖性切割核酶溶液(17ES)和85μL切割缓冲液(300mM NaCl,40mM HEPES,pH 7.5)混合;在37℃下反应60min,之后,精准快速稀释105倍后进行RT‑qPCR实验,被切割的底物链无法作为模板进行扩增反应,而未被切割的底物链则可以通过扩增反应实现信号放大,最终结果以切割前后的Cq值变化(ΔCq)来表示切割效果,实现Zn2+的定量检测。本发明采用的Zn2+依赖性切割脱氧核酶17E是已知Zn2+依赖性切割脱氧核酶中切割速率最快的核酶。该酶能够催化切割底物链,并且对Zn2+具有高度亲和力和特异性。
Description
技术领域
本发明主要涉及锌离子检测技术领域,特别涉及一种用于锌离子定量检测的方法。
技术背景
锌是人体必需的微量元素,参与人体的代谢过程。人主要通过从食物和水中摄取锌。如果食物或水受到污染,锌的含量超过限定标准,则可能对人体健康造成危害。有研究者表明人体内锌含量过高可能导致细胞活性增加。因此,准确检测Zn2+浓度水平对维持人体健康至关重要。同时,锌元素的缺乏也是人们面临的共同问题。市场上充斥着大量的锌补充剂,但难以辨别产品的真假。因此,有必要开发一种精确的快速检测技术定量检测锌补充剂中Zn2+的浓度。
目前,Zn2+的传统检测方法包括电感耦合等离子体发射光谱法 (ICP-AES),电感耦合等离子体质谱法(ICP-MS)以及原子吸收光谱法(AAS)等。虽然这些方法提高了灵敏度和特异性,但它们高度依赖于精密仪器,并且样品检测的预处理和测定过程繁琐。此外,仪器和设备的安装环境和维护所需要求高,需要经过专业培训的操作人员来完成。以上方法均不适合快速检测。为了克服大规模仪器检测方法的局限,具有高灵敏度,简单和实时监测优势的基于有机分子(如酰腙,喹啉,蒽和罗丹明等)的Zn2+荧光化学传感器被开发出来。然而,大多数的这些化学传感器不可避免地涉及有机溶剂的发射,并受到荧光猝灭和水介质中选择性差的限制。
近年来,随着分子生物学技术的快速发展,具有特定生物学功能的核酸分子如切割DNA酶(cDNAzymes)和金属离子适配体 (Aptamers)等逐渐显现出来。已经报道的DNA酶包括Zn2+、Cu2+、 Pb2+和Hg2+等依赖性DNA酶。大多数DNA酶催化需要金属辅因子的协助,其中一些具有高选择性和高效性。其中Zn2+依赖性切割脱氧核酶17E,在Zn2+的存在下能够催化底物链切割,对Zn2+具有高度亲和力和特异性,是已知Zn2+依赖性切割脱氧核酶中切割速率最快的核酶。
实时荧光定量PCR(RT-qPCR)是一种简单且发展良好的技术,其在分子生物学中的应用极为普遍。RT-qPCR已被研究人员广泛用于基因检测,病原微生物检测,病毒检测,寄生虫检测和转基因食品检测等方面。与常规定性PCR相比,RT-qPCR实现了定性到定量的转变。使其不仅具有传统PCR技术的简单、快速特点,还具有指数扩增靶物质的能力,促进靶物质的检测。并且实现了更高的特异性和自动化程度。
发明内容
基于此,本将Zn2+依赖性切割脱氧核酶17E与RT-qPCR技术相结合建立了Zn2+的定量检测技术,实现了Zn2+的精确定量测定。具有简单、快速、高灵敏度、高特异性等特点,为痕量锌离子的精准检测提供了技术支持。
本发明解决上述技术问题采用的技术方案为:一种用于锌离子定量检测的方法,其特征在于,具体的检测步骤如下:
(1)准备实验材料:实验材料主要包括有级别为分析纯的氯化钠、氯化锌、氯化镁、氯化钙、氯化铜、氯化锡、氯化亚铁、色谱纯的4-羟乙基哌嗪乙磺酸(HEPES)和定量PCR扩增试剂 TransStartGreenqPCRSuperMix(2×);
(2)引物设计:根据17E核酸的原始序列,在不破坏酶的催化序列和切割位点的情况下在核酶底物链的两侧加入了引物结合序列。根据引物结合序列设计RT-qPCR的正向和反向引物;通过序列进行反应
表1实验所用锌离子依赖性切割核酶和引物序列:
(3)切割反应缓冲液配制:称取1.7532g NaCl(300mM)和 0.9532g HEPES(40mM)溶于100mL的超纯水中,并调pH至7.5,配制成切割缓冲液(300mM NaCl,40mM HEPES,pH 7.5),4℃保存备用;
(4)17E和底物链溶解与杂交:将装有合成序列粉末的试管放入高速离心机中,4℃、13400×g离心10min后取出加入超纯水溶解序列至10μM,4℃保存备用。分别取100μL的底物链(10μM), 100μL的催化酶链(10μM)加入800μL切割缓冲液中,60℃加热5 min,然后缓慢降至室温,配制成1μM的锌离子依赖性切割核酶溶液(17ES),4℃保存备用;
(5)Zn2+依赖性切割反应:
表2切割反应体系表:
将该反应体系在37℃下反应60min;
(6)RT-qPCR反应:样品经Zn2+切割完成后,精准快速稀释105倍后进行RT-qPCR实验;
(7)Zn2+的检测:将5μL Zn2+浓度的溶液、10μL Zn2+依赖性切割核酶溶液(17ES)和85μL切割缓冲液(300mM NaCl,40mM HEPES,pH 7.5)混合;在37℃下反应60min。之后,精准快速稀释 105倍后进行RT-qPCR实验,被切割的底物链无法作为模板进行扩增反应,而未被切割的底物链则可以通过扩增反应实现信号放大。最终结果以切割前后的Cq值变化(ΔCq)来表示切割效果,实现Zn2+的定量检测。
与现有技术相比,本发明的有益效果为:
(1)本发明采用的Zn2+依赖性切割脱氧核酶17E是已知Zn2+依赖性切割脱氧核酶中切割速率最快的核酶。该酶能够催化切割底物链,并且对Zn2+具有高度亲和力和特异性。
(2)本发明中Zn2+依赖性切割反应可在恒定温度下完成,在恒温下即可完成信号放大,将Zn2+转化为核酸信号。
(3)本发明在不破坏酶17E的催化序列和切割位点的情况下在其底物链的两侧加入了引物结合序列。在切割缓冲液中加入Zn2+催化 17E切割底物链后,被切割的底物链无法作为模板进行RT-qPCR扩增反应,而未被切割的底物链则可以通过扩增反应实现信号放大。
(4)本发明的快速检测方法不需要大型的仪器设备和专业的操作人员,检测灵敏度高、特异性强,检测限可低至58.61pM。
附图说明
图1为可行性分析荧光光谱结果图,其中,曲线E-S1为切割体系中含有酶链和底物链,曲线E-S2为切割体系中含有酶链、底物链和Zn2+,曲线ES51为切割体系中含有ES5,曲线ES52为切割体系中含有ES5和Zn2+。
图2为生物传感器锌离子切割反应体系优化实验结果图,图A 为pH值对ΔCq值的影响,图B为HEPES浓度对ΔCq值的影响,图C为NaCl浓度对ΔCq值的影响,图D为切割反应时间对ΔCq值的影响。
图3为锌离子浓度的线性范围为80pM至1280pM时的标准曲线结果图。
具体实施案例
下面,举实施例说明本发明,但是,本发明并不限于下述的实施例。
本发明中选用的所有原辅材料、试剂和仪器、设备都为本领域熟知选用的,但不限制本发明的实施,其他本领域熟知的一些试剂和设备都可适用于本发明以下实施方式的实施。
实施例一:
本发明采用的用于锌离子定量检测的方法具体的步骤如下:
(1)准备实验材料:实验材料主要包括有级别为分析纯的氯化钠、氯化锌、氯化镁、氯化钙、氯化铜、氯化锡、氯化亚铁、色谱纯的4-羟乙基哌嗪乙磺酸(HEPES)和定量PCR扩增试剂 TransStartGreenqPCRSuperMix(2×);
(2)引物设计:根据17E核酸的原始序列,在不破坏酶的催化序列和切割位点的情况下在核酶底物链的两侧加入了引物结合序列。根据引物结合序列设计RT-qPCR的正向和反向引物;
表1实验所用锌离子依赖性切割核酶和引物序列:
(3)切割反应缓冲液配制:称取1.7532g NaCl(300mM)和 0.9532g HEPES(40mM)溶于100mL的超纯水中,并调pH至7.5,配制成切割缓冲液(300mM NaCl,40mM HEPES,pH 7.5),4℃保存备用;
(4)17E和底物链溶解与杂交:将装有合成序列粉末的试管放入高速离心机中,4℃、13400×g离心10min后取出加入超纯水溶解序列至10μM,4℃保存备用。分别取100μL的底物链(10μM), 100μL的催化酶链(10μM)加入800μL切割缓冲液中,60℃加热5 min,然后缓慢降至室温,配制成1μM的锌离子依赖性切割核酶溶液(17ES),4℃保存备用;
(5)Zn2+依赖性切割反应:
表2切割反应体系表:
将该反应体系在37℃下反应60min;
(6)RT-qPCR反应:样品经Zn2+切割完成后,精准快速稀释105倍后进行RT-qPCR实验;
(7)Zn2+的检测:将5μL Zn2+浓度的溶液、10μL Zn2+依赖性切割核酶溶液(17ES)和85μL切割缓冲液(300mM NaCl,40mM HEPES,pH 7.5)混合;在37℃下反应60min。之后,精准快速稀释 105倍后进行RT-qPCR实验,被切割的底物链无法作为模板进行扩增反应,而未被切割的底物链则可以通过扩增反应实现信号放大。最终结果以切割前后的Cq值变化(ΔCq)来表示切割效果,实现Zn2+的定量检测。
实施例二:本发明将Zn2+依赖性切割核酶17E的酶链(Enzyme) 和底物链(Substrate)通过5个A碱基串联成一条链ES5(如说明书附图1),以1mM Zn2+和100nM ES5进行切割实验,经37℃,60min 切割后,稀释105倍,进行RT-qPCR验证,并与原来的酶链和底物链 (如说明书附图1)组成的切割酶(E-S)的催化活性进行对比。得到E-S的ΔCq值比ES5的ΔCq值更大,具有较高催化切割效率。
实施例三:Zn2+切割体系反应条件的优化:以1mM Zn2+为切割浓度,经过37℃,60min的切割反应后,均稀释105倍,进行RT-qPCR 验证。为了提高该荧光定量生物传感器的灵敏度,通过比较切割缓冲液的pH值、HEPES浓度、NaCl浓度、切割反应时间来系统地分析。结果证明切割缓冲液pH为7.5时传感器性能更好(说明书附图2A), HEPES浓度为40mM时ΔCq值最大(说明书附图2B),NaCl浓度为300mM时Zn2+依赖性切割脱氧核酶17E的催化活性最高(说明书附图2C)。此外,60min为最佳切割时间(说明书附图2D);其中, (说明书附图2A)pH值对ΔCq值的影响;(说明书附图2B)HEPES 浓度对ΔCq值的影响;(说明书附图2C)NaCl浓度对ΔCq值的影响; (说明书附图2D)切割反应时间对ΔCq值的影响。
实施例四:
荧光定量生物传感器灵敏度验证:为了评定该荧光定量生物传感器的灵敏度,在最佳实验条件下测量含有不同浓度的Zn2+标准溶液 (0~1280pM)的ΔCq值,每个浓度三个平行。在定量PCR体系中, Zn2+浓度为80pM至1280pM时,参照说明书附图三所作图为线性的,并且相关方程为:ΔCq=4.3449lg[C(Zn2+)]–7.7719,相关系数R2为 0.9953,适合定量检测;其中说明书附图3为锌离子浓度的线性范围为80pM至1280pM时的标准曲线。
以上所述实施例仅表达了本发明的某种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求。
Claims (1)
1.一种用于锌离子定量检测的方法,其特征在于,具体的检测步骤如下:
(1)准备实验材料:实验材料主要包括有级别为分析纯的氯化钠、氯化锌、氯化镁、氯化钙、氯化铜、氯化锡、氯化亚铁、色谱纯的4-羟乙基哌嗪乙磺酸(HEPES)和定量PCR扩增试剂TransStartGreenqPCRSuperMix(2×);
(2)引物设计:根据17E核酸的原始序列,在不破坏酶的催化序列和切割位点的情况下在核酶底物链的两侧加入了引物结合序列,根据引物结合序列设计RT-qPCR的正向和反向引物;
(3)切割反应缓冲液配制:称取1.7532g NaCl(300mM)和0.9532g HEPES(40mM)溶于100mL的超纯水中,并调pH至7.5,配制成切割缓冲液(300mM NaCl,40mM HEPES,pH 7.5),4℃保存备用;
(4)17E和底物链溶解与杂交:将装有合成序列粉末的试管放入高速离心机中,4℃、13400×g离心10min后取出加入超纯水溶解序列至10μM,4℃保存备用,分别取100μL的底物链(10μM),100μL的催化酶链(10μM)加入800μL切割缓冲液中,60℃加热5min,然后缓慢降至室温,配制成1μM的锌离子依赖性切割核酶溶液(17ES),4℃保存备用;
(5)Zn2+依赖性切割反应:将Zn2+依赖性切割核酶17E的酶链(Enzyme)和底物链(Substrate)通过5个A碱基串联成一条链ES5,以1mM Zn2+和100nM ES5进行切割实验,经37℃,60min切割后,稀释105倍,进行RT-qPCR验证;
(6)RT-qPCR反应:样品经Zn2+切割完成后,精准快速稀释105倍后进行RT-qPCR实验;
(7)Zn2+的检测:将5μL Zn2+浓度的溶液、10μL Zn2+依赖性切割核酶溶液(17ES)和85μL切割缓冲液(300mM NaCl,40mM HEPES,pH 7.5)混合;在37℃下反应60min,之后,精准快速稀释105倍后进行RT-qPCR实验,被切割的底物链无法作为模板进行扩增反应,而未被切割的底物链则可以通过扩增反应实现信号放大,最终结果以切割前后的Cq值变化(ΔCq)来表示切割效果,实现Zn2+的定量检测。
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