CN105713966A - 一种快速检测玉米赤霉烯酮的方法 - Google Patents

一种快速检测玉米赤霉烯酮的方法 Download PDF

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CN105713966A
CN105713966A CN201610045201.2A CN201610045201A CN105713966A CN 105713966 A CN105713966 A CN 105713966A CN 201610045201 A CN201610045201 A CN 201610045201A CN 105713966 A CN105713966 A CN 105713966A
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曾云龙
任婕凤
刘婷
黄昊文
张起豪
邓克勤
易守军
唐春然
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Abstract

本发明涉及一种快速检测玉米赤霉烯酮的方法，该方法包括如下步骤：（A）使玉米赤霉烯酮核酸适体（Apt）和可与玉米赤霉烯酮核酸适体杂交的单链信号探针ssDNA杂交，形成杂交链；（B）使该杂交链与待测样品接触，当待测样品中有玉米赤霉烯酮存在时，杂交链与玉米赤霉烯酮反应释放出单链信号探针ssDNA；（C）利用DNA扩增，使杂交链成双链DNA，然后用核酸外切酶选择性地催化双链DNA水解成单核苷酸，体系中单链信号探针ssDNA不水解而保留下来；（D）在铜离子还原检测体系中检测体系荧光强度，从而测定待测样品中的真菌毒素玉米赤霉烯酮的含量。所述方法可以消除干扰，提高了玉米赤霉烯酮的检测灵敏度和精度。

Description

一种快速检测玉米赤霉烯酮的方法
技术领域
本发明属纳米生物传感以及生物检测技术领域，涉及玉米赤霉烯酮的检测方法及检测试剂盒。
背景技术
玉米赤霉烯酮主要由禾谷镰刀菌产生，粉红镰刀菌、窜珠镰刀菌、三线镰刀菌等多种镰刀菌也能产生这种毒素。李季伦1980年研究发现，许多农作物如小麦、大豆等植物中也存在玉米赤霉烯酮。玉米赤霉烯酮主要污染玉米、小麦、大米、大麦、小米和燕麦等谷物。其中玉米的玉米赤霉烯酮阳性检出率为45%，最高含毒量可达到2909mg/kg；小麦的检出率为20%，含毒量为0.364～11.05mg/kg。玉米赤霉烯酮的耐热性较强，110℃下处理1h才被完全破坏。玉米赤霉烯酮具有***样作用，能造成动物急慢性中毒，引起动物繁殖机能异常甚至死亡，可给畜牧场造成巨大经济损失。因此对玉米赤霉烯酮检测显得十分必要。
现有分析方法有色谱、免疫法，前者灵敏度不够，后者存在试剂贵、易失活等缺点。中国专利CN102443585A公开了一种玉米赤霉烯酮核酸适体及其应用，其可用于玉米赤霉烯酮的检测，但存在生物标记的DNA价格昂贵的问题。
发明内容
本发明的目的是针对现有技术中存在的问题，提供一种快速检测玉米赤霉烯酮的方法。
本发明采用的技术方案：一种快速检测玉米赤霉烯酮的方法，该方法包括如下步骤：
（A）使玉米赤霉烯酮核酸适体（Apt）和可与玉米赤霉烯酮核酸适体杂交的单链信号探针ssDNA杂交，形成杂交链；
（B）使该杂交链与待测样品作用，当待测样品中有玉米赤霉烯酮存在时，杂交链选择性地与玉米赤霉烯酮反应释放出单链信号探针ssDNA；
（C）为了消除杂交链的干扰，利用DNA扩增，使杂交链成双链DNA，然后用外切酶选择性地催化双链DNA水解成单核苷酸，体系中单链信号探针ssDNA未水解而保留下来；
（D）利用铜离子在玉米赤霉烯酮核酸适体-玉米赤霉烯酮结合体中不能被还原生成荧光发射峰在620nm左右的铜纳米粒子，铜离子只在单链信号探针ssDNA存在情况下被还原生成ssDNA修饰的在620nm左右（发射峰位置）有强的荧光发射的铜纳米粒子的原理，在铜离子还原检测体系中检测体系的荧光强度，从而测定待测样品中的真菌毒素玉米赤霉烯酮的含量。
所述铜离子还原检测体系包括先后添加的铜离子和使铜离子还原成铜纳米粒子的还原剂维生素C。步骤（D）的检测例如包括依次先后向体系中加入铜离子溶液和维生素C溶液，反应后生成荧光发射峰在620nm左右的铜纳米粒子。
所述玉米赤霉烯酮核酸适体为5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
所述的单链信号探针ssDNA为5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTG CACTTCACACGATT-3’。
本发明的检测原理如下：先让Apt与ssDNA杂交（下划线部分）；当有玉米赤霉烯酮存在时，杂交链与玉米赤霉烯酮反应释放出ssDNA；体系中存在Apt-ssDNA(剩余的)，Apt-玉米赤霉烯酮和ssDNA。铜离子在Apt-玉米赤霉烯酮中不能被还原生成荧光发射峰在620nm左右的铜纳米粒子，Apt-玉米赤霉烯酮的存在对测定无干扰；杂交链可能有干扰；为了消除杂交链的干扰：a.利用DNA扩增，使杂交链成双链DNA，b.用外切酶选择性地催化双链DNA水解成单核苷酸，体系中单链信号探针ssDNA未水解而保留下来；此时体系中只留下诱导铜离子还原成近红外铜纳米粒子的ssDNA，铜离子在ssDNA存在下还原并生成被ssDNA修饰的荧光发射峰在620nm左右的铜纳米粒子，以340nm的光为激发光，测定荧光发射（520nm-660nm）光谱的荧光强度，依据体系的荧光发射光谱的强度与玉米赤霉烯酮的量的关系，从而可以测定真菌毒素玉米赤霉烯酮的含量。由于消除体系中背景荧光的干扰，可以提高检测的灵敏度和精度。
本发明还提供了一种检测玉米赤霉烯酮的试剂盒，其至少包括：玉米赤霉烯酮核酸适体、单链信号探针DNA、DNA扩增体系、外切酶、铜离子还原检测体系。
所述玉米赤霉烯酮核酸适体为5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
所述可的单链信号探针DNA为5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3’。
所述DNA扩增体系包括缓冲溶液（缓冲溶液由Tris-HCl、MgCl2、(NH4)2SO4组成）、三磷酸脱氧单核苷酸混合溶液（dNTP）和Phi29DNA聚合酶。
所述外切酶为ExoIII核酸外切酶。
本发明还提供了一种可用于检测玉米赤霉烯酮的玉米赤霉烯酮核酸适体，其碱基序列为5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTT
GTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
本发明的优点
根据本发明的检测方法和试剂盒，可以消除干扰，提高玉米赤霉烯酮的检测灵敏度和精度。
附图说明
图1为ssDNA-铜纳米粒子荧光激发与发射光谱。
具体实施方式
实施例1
一种检测玉米赤霉烯酮的试剂盒至少包括：使玉米赤霉烯酮核酸适体、可与玉米赤霉烯酮核酸适体杂交的单链信号探针ssDNA、DNA扩增体系、外切酶、铜离子还原检测体系。所述玉米赤霉烯酮核酸适体为5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。所述的单链信号探针DNA为5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3’。所述DNA包括缓冲溶液（缓冲溶液由Tris-HCl、MgCl2、(NH4)2SO4组成）、三磷酸脱氧单核苷酸混合溶液dNTP和Phi29DNA聚合酶。所述外切酶为ExoIII核酸外切酶。所述的铜离子还原检测体系所用还原剂为维生素C。
实施例2
一种快速检测玉米赤霉烯酮的方法，具体操作过程如下：
将各DNA储备液在95℃下经加热处理5分钟，使用前，并在室温下放置30分钟。然后，分别取含2.5μmol玉米赤霉烯酮核酸适体Apt的杂交缓冲溶液50μL和2.5μmol信号探针ssDNA的杂交缓冲溶液50μL置于2ml离心管中，在37℃下杂交1小时，生成玉米赤霉烯酮核酸适体-信号探针杂交物（Apt-ssDNA）。
在37℃下，分别依次将浓度为0~50ng/mL的玉米赤霉烯酮添加到Apt-ssDNA溶液中，玉米赤霉烯酮与杂交链中玉米赤霉烯酮核酸适体段反应，生成核酸适体-玉米赤霉烯酮，释放出ssDNA。这时，体系中有ssDNA、剩余（未反应的）Apt-ssDNA和核酸适体-玉米赤霉烯酮等物质。
加入10μL缓冲溶液（缓冲溶液组成为50mMTris-HCl,10mMMgCl2，10mM(NH4)2SO4,pH7.5），然后加入dNTP(10mM)20μL。再向体系中加入2μLPhi29DNA聚合酶(10u/μl),在37℃下反应15分钟，使得以玉米赤霉烯酮核酸适体-信号探针杂交序列（Apt-ssDNA）为摸板扩增成双链DNA。在65℃下保持10分钟使Phi29DNA去活。
再向该反应体系中加入2μLExoIII核酸外切酶（20u/μL），在37℃下反应30分钟，ExoIII核酸外切酶选择性地催化双链DNA水解成单核苷酸，体系中单链信号探针ssDNA不水解而保留下来；溶液加热至95℃保持5分钟使核酸外切酶ExoIII失活，并将溶液放置于冰浴中迅速冷却至室温。
向反应液中加入25μL1mmol硝酸铜和200μLPBS缓冲液（10mM，pH7.8）。然后，混合物在室温下避光或暗室中放置10分钟后，在快速搅拌下，加入100μL浓度为1mM的新制备的维生素C溶液。然后在45℃下反应5min。将溶液转移至微量比色皿，以波长为340nm光为激发光，测定体系荧光发射（520-660nm）光谱的荧光强度，进行玉米赤霉烯酮的定量测定，结果如图1所示。
线性范围0.008–0.50ng/mL，线性方程为y=260.87+99.37lgC，线性相关系数r=0.9978，检测限1pg/mL，回收率在96.5~106.2%。其它真菌毒素等生物小分子对玉米赤霉烯酮检测无干扰。
<110>湖南科技大学
<120>一种快速检测玉米赤霉烯酮的方法
<160>2
<210>1
<211>73
<212>DNA
<213>玉米赤霉烯酮核酸适体
<400>1
5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’
<210>2
<211>55
<212>DNA
<213>单链信号探针
<400>2
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3’

Claims (9)

1.一种快速检测玉米赤霉烯酮的方法，其特征是，该方法包括如下步骤：
（A）玉米赤霉烯酮核酸适体Apt与单链信号探针ssDNA杂交，形成杂交链；
（B）使该杂交链与待测样品作用，当待测样品中有玉米赤霉烯酮存在时，杂交链选择性地与玉米赤霉烯酮反应释放出单链信号探针ssDNA；
（C）为了消除杂交链的干扰，利用DNA扩增，使杂交链成双链DNA，然后用核酸外切酶选择性地催化双链DNA水解成单核苷酸，体系中单链信号探针ssDNA不水解而保留下来；
（D）利用铜离子不能在玉米赤霉烯酮核酸适体-玉米赤霉烯酮结合体中被还原生成荧光发射峰在620nm左右的铜纳米粒子，铜离子只在单链信号探针ssDNA中还原并生成被ssDNA修饰的在620nm左右有强的荧光发射的铜纳米粒子的原理，在铜离子还原检测体系中检测体系的荧光强度，从而测定待测样品中的真菌毒素玉米赤霉烯酮的含量。
2.根据权利要求1所述的检测玉米赤霉烯酮的方法，其特征是，所述玉米赤霉烯酮核酸适体为5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
3.根据权利要求1或2所述的检测玉米赤霉烯酮的方法，其特征是，所述的单链信号探针ssDNA为5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3’。
4.一种快速检测玉米烯酮的方法还提供检测玉米赤霉烯酮的试剂盒，其特征是，其至少包括：使玉米赤霉烯酮核酸适体、单链信号探针ssDNA、DNA扩增体系、外切酶、铜离子还原检测体系。
5.根据权利要求4所述的检测玉米赤霉烯酮的试剂盒，其特征是，所述玉米赤霉烯酮核酸适体为5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
6.根据权利要求4或5所述的检测玉米赤霉烯酮的试剂盒，其特征是，所述的单链信号探针ssDNA为5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3’。
7.根据权利要求4所述的检测玉米赤霉烯酮的试剂盒，其特征是，所述DNA扩增体系包括缓冲溶液、三磷酸脱氧单核苷酸混合溶液dNTP和Phi29DNA聚合酶；所述缓冲溶液由Tris-HCl、MgCl2、(NH4)2SO4组成。
8.根据权利要求4所述的检测玉米赤霉烯酮的试剂盒，其特征是，所述外切酶为ExoIII核酸外切酶。
9.一种可用于检测玉米赤霉烯酮的玉米赤霉烯酮核酸适体，其特征是，其碱基序列为5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
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