WO2019153677A1

WO2019153677A1 - New method for visual quantitative detection of dual heavy metal ions

Info

Publication number: WO2019153677A1
Application number: PCT/CN2018/099405
Authority: WO
Inventors: 罗云波; 许文涛; 黄昆仑; 杜再慧; 田晶晶
Original assignee: 中国农业大学
Priority date: 2018-02-08
Filing date: 2018-08-08
Publication date: 2019-08-15
Also published as: CA3059246C; CA3059246A1; CN108342456A

Abstract

Provided is a visual quantitative method for heavy metals, comprising an in vitro nucleic acid amplification and detection method, wherein during in vitro nucleic acid amplification, an ultra-fast PCR reaction is carried out by using primers composed successively of a complementary sequence, a linker arm, a complementary sequence, and a nucleic acid sequence that specifically amplifies the target to be tested, and during detection, self-assembly color development is realized by using a hairpin sequence composed of a complementary sequence and G-quadruplex.

Description

一种双重重金属离子的可视化定量检测新方法A new method for visual quantitative detection of double heavy metal ions

本申请要求于2018年02月08日提交中国专利局、申请号为201810129689.6、发明名称为“一种双重重金属离子的可视化定量检测新方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims priority to Chinese Patent Application No. 201101129689.6, entitled "A New Method for Visual Quantitative Detection of Double Heavy Metal Ions", filed on February 8, 2018, the entire contents of which are incorporated by reference. In this application.

技术领域Technical field

本发明属于生物检测技术领域，具体涉及一种双重金属的检测方法和生物传感器。The invention belongs to the technical field of biological detection, and particularly relates to a detection method and a biosensor of a double metal.

背景技术Background technique

自然界中的汞主要以三种形态存在，包括：单质汞(Hg)，有机汞(烷基汞、苯基汞)以及无机汞(Hg ⁺和Hg ²⁺盐及其配合物)，并且各种物质之间可以进行生物、化学转换。它是唯一一种在室温条件下以液体形式存在的金属，物理形状为银白色，熔点-38.87℃，沸点356.6℃，密度13.59克/立方厘米，元素符号为Hg。银有单质状态，但一般都以化合态存在于银矿石中。它呈现白色有光泽的形态，熔点961.78℃，沸点2212℃，密度10.49克/立方厘米，元素符号为Ag。 Mercury in nature exists mainly in three forms, including elemental mercury (Hg), organic mercury (alkyl mercury, phenyl mercury), and inorganic mercury (Hg ⁺ and Hg ²⁺ salts and their complexes), and various Biological and chemical conversions can be made between substances. It is the only metal present in liquid form at room temperature, the physical shape is silver white, melting point -38.87 ° C, boiling point 356.6 ° C, density 13.59 g / cm 3 , element symbol Hg. Silver has a simple state, but generally exists in a silver ore in a combined state. It has a white shiny form with a melting point of 961.78 ° C, a boiling point of 2212 ° C, a density of 10.49 g / cm 3 , and an element symbol of Ag.

传统的Hg ²⁺和Ag ⁺的检测方法主要包括原子吸收光谱法(AAS)、原子荧光光谱法(AFS)、电感耦合等离子体质谱法、电化学分析方法、原子吸收分光光度法等，这些检测方法灵敏度高、检测范围广、适合多种样品分析；但由于大型仪器的使用，需要较高的维护成本、专业人员操作等使得检测成本增加；同时样品预处理过程复杂且部分需要使用腐蚀性试剂；检测周期长；由于上述缺点，使得传统方法不适合缺乏精密仪器的现场分析等缺点，难以满足实际分析的要求等。 The traditional Hg ²⁺ and Ag ⁺ detection methods mainly include atomic absorption spectroscopy (AAS), atomic fluorescence spectroscopy (AFS), inductively coupled plasma mass spectrometry, electrochemical analysis, atomic absorption spectrophotometry, etc. The method has high sensitivity and wide detection range, and is suitable for analysis of various samples. However, due to the use of large instruments, high maintenance cost and professional operation are required to increase the detection cost; at the same time, the sample pretreatment process is complicated and some corrosive reagents are required. The detection period is long; due to the above shortcomings, the traditional method is not suitable for the lack of on-site analysis of precision instruments, etc., and it is difficult to meet the requirements of actual analysis.

发明内容Summary of the invention

本发明建立的检测方法和生物传感器，克服了现有检测技术的不足，实现了对重金属进行准确、快速、简单高效的检测和分析，是一种廉价、快速、灵敏、特异性强的双重重金属检测新方法。The detection method and the biosensor established by the invention overcome the deficiencies of the existing detection technology, realize accurate, rapid, simple and efficient detection and analysis of heavy metals, and are a double heavy metal which is cheap, fast, sensitive and specific. Detect new methods.

本发明的一个目的是提供一种金属的检测方法，所述方法包括体外核酸扩增技术,所述体外核酸扩增技术的反应体系包括下游引物、模板和至少两种上游引物；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；所述下游引物包括可特异性扩增所述模板的核苷酸序列。It is an object of the present invention to provide a method for detecting a metal, the method comprising an in vitro nucleic acid amplification technique, the reaction system of the in vitro nucleic acid amplification technique comprising a downstream primer, a template and at least two upstream primers; The base of at least one nucleotide of each of the upstream primers is different; each of the upstream primers of the at least two upstream primers comprises: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that specifically amplifies the template and a nucleotide that binds to the metal to be tested; the tether is located between the complementary sequence A and the complementary sequence B, the specific amplification of the The nucleotide sequence of the template and the nucleotide that can bind to the metal to be tested are located at the 5' or 3' end of the upstream primer; the nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and / Or a reverse complement; the linker comprises a structure that inhibits polymerase binding and/or a structure that inhibits elongation of a new strand during nucleic acid amplification in vitro; the downstream primer comprises a nucleoside that specifically amplifies the template Acid sequence.

所述模板至少包括互补序列G、互补序列H和序列I；其中，所述互补序列G、互补序列H分别与所述至少两种上游引物中的可特异性扩增所述模板的核苷酸序列互补和/或反向互补；所述序列I的核苷酸序列与所述下游引物中的可特异性扩增所述模板的核苷酸序列相同。The template comprises at least a complementary sequence G, a complementary sequence H and a sequence I; wherein the complementary sequence G, the complementary sequence H and the nucleotides of the at least two upstream primers capable of specifically amplifying the template, respectively The sequence is complementary and/or reverse complementary; the nucleotide sequence of Sequence I is identical to the nucleotide sequence of the downstream primer that specifically amplifies the template.

所述互补包括现有技术或公知常识所定义的互补或反向互补，和/或根据现有技术或公知常识所定义的互补原则进行互补或反向互补。The complementation includes complementary or reverse complementarity as defined by the prior art or common general knowledge, and/or complementarity or reverse complementation according to complementary principles as defined by the prior art or common general knowledge.

所述聚合酶包括可用于体外核酸扩增的聚合酶。The polymerase includes a polymerase that can be used for in vitro nucleic acid amplification.

所述可特异性扩增所述模板的核苷酸序列具体包括根据所述模板的特征序列所设计的引物序列；所述特征序列包括现有技术或公知常识所定义的特征序列；所述设计包括现有技术或公知常识所记载的设计方法。The nucleotide sequence capable of specifically amplifying the template specifically includes a primer sequence designed according to a characteristic sequence of the template; the characteristic sequence includes a characteristic sequence defined by prior art or common knowledge; the design The design method described in the prior art or common knowledge is included.

所述A、B、G、H、I只用于区别不同的互补序列或序列，不用于排序。The A, B, G, H, and I are only used to distinguish different complementary sequences or sequences, and are not used for sorting.

具体的，所述方法还包括下述1)-3)中的至少一种：Specifically, the method further includes at least one of the following 1)-3):

1)所述体外核酸扩增技术包括超快速PCR反应，所述超快速PCR反应的反应过程包括：90-98℃，2-6s；50-60℃，2-8s；共20-40个循环；1) The in vitro nucleic acid amplification technique comprises an ultra-fast PCR reaction comprising: 90-98 ° C, 2-6 s; 50-60 ° C, 2-8 s; a total of 20-40 cycles ;

具体的，所述超快速PCR反应的反应过程包括：95℃，2s；58℃，3s；共30个循环；Specifically, the reaction process of the ultra-fast PCR reaction comprises: 95 ° C, 2 s; 58 ° C, 3 s; a total of 30 cycles;

再具体的，所述超快速PCR反应的反应体系中上游引物和下游引物的浓度为普通PCR浓度的10倍以上；还具体的，为20倍；所述超快速PCR反应的反应体系中还包括DNA聚合酶，所述DNA聚合酶的浓度为普通PCR浓度的10倍以上，还具体的，为60倍；More specifically, the concentration of the upstream primer and the downstream primer in the reaction system of the ultra-fast PCR reaction is 10 times or more of the ordinary PCR concentration; specifically, 20 times; the reaction system of the ultra-fast PCR reaction further includes DNA polymerase, the concentration of the DNA polymerase is 10 times or more of the ordinary PCR concentration, and specifically, 60 times;

2)所述连接臂包括具长链结构的化合物；2) the connecting arm comprises a compound having a long chain structure;

3)所述可与待测金属结合的核苷酸具体包括含胸腺嘧啶或胞嘧啶的核苷酸。3) The nucleotide which can bind to the metal to be tested specifically includes a thymine- or cytosine-containing nucleotide.

具体的，所述方法还包括下述1)-8)中的至少一种：Specifically, the method further includes at least one of the following 1)-8):

1)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列通过连接臂连接后得到的引物；1) one of the at least two upstream primers comprising: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing through a tether;

2)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；2) One of the at least two upstream primers comprises: substituting a nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing by one or several nucleotides and/ Or a primer obtained by deleting and/or adding a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing, which are ligated by a tether;

3)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物；3) one of the at least two upstream primers comprises: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether;

4)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；4) One of the at least two upstream primers comprises: substituting a nucleotide sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing by one or several nucleotides and/ Or a primer obtained by deleting and/or adding a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing;

5)所述下游引物包括序列表中SEQ ID NO：3所示的核苷酸序列；5) the downstream primer comprises the nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing;

6)所述下游引物包括将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；6) The downstream primer comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing and SEQ ID NO: 3 in the Sequence Listing a nucleotide sequence having the same function as the nucleotide sequence shown;

7)所述模板包括序列表中SEQ ID NO：6所示的核苷酸序列；7) the template comprises the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;

8)所述模板包括将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列。8) The template comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing and SEQ ID NO: 6 in the Sequence Listing. A nucleotide sequence having the same function as a nucleotide sequence.

所述功能包括可实现所述模板的扩增。The function includes enabling amplification of the template.

具体的，所述连接臂为oxyethyleneglycol bridge，oxyethyleneglycol的化学结构为：Specifically, the connecting arm is an oxyethyleneglycol bridge, and the chemical structure of the oxyethyleneglycol is:

再具体的，所述至少两种上游引物中的一种包括：AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTC TCTTT CCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT或TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTT TTTTTTGCACATAACACCCC；More specifically, one of the at least two upstream primers comprises: AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTC TCTTT CCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT or TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTT TTTTTTGCACATAACACCCC;

还具体的，所述至少两种上游引物包括：AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTC CCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT和TGAGGTAGTAGG TTGTATAGTT-oxyethyleneglycol bridge-AACTATA CAACCTACTACCTCATTTTT TTTTTTGCACATAACACCCC；Further specifically, the at least two upstream primers include: AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTC CCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT and TGAGGTAGTAGG TTGTATAGTT-oxyethyleneglycol bridge-AACTATA CAACCTACTACCTCATTTTT TTTTTTGCACATAACACCCC;

具体的，所述下游引物还包括在所述下游引物上标记免疫标记；所述免疫标记包括生物素；所述生物素标记在所述下游引物的5’端第一个核苷酸上；所述标记方法属于现有技术；再具体的，所述下游引物为Biotin-TCATCGCACCGTCAAAGGAACC；Specifically, the downstream primer further comprises marking an immunolabel on the downstream primer; the immunolabel comprises biotin; the biotin label is on the first nucleotide of the 5' end of the downstream primer; The labeling method belongs to the prior art; more specifically, the downstream primer is Biotin-TCATCGCACCGTCAAAGGAACC;

具体的，所述方法还包括核酸自组装显色反应,所述核酸自组装显色反应的反应体系包括发卡序列，所述发卡序列包括发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补和/或反向互补。Specifically, the method further includes a nucleic acid self-assembly color reaction, the reaction system of the nucleic acid self-assembly color reaction comprises a hairpin sequence, and the card issue sequence comprises a card issue sequence 1 and/or a card issue sequence 2, and the card issue sequence 1 The invention comprises: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, wherein the three or more arbitrary nucleotides are located at the 5' end and/or the 3' end of the card issue sequence; Included is a complementary sequence E and a complementary sequence F; said complementary sequence D is complementary and/or inversely complementary to a complementary sequence F; said complementary sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B The complementary sequence C is complementary and/or inversely complementary to the complementary sequence E.

所述一和二用于区别不同的发卡序列，不用于排序；所述C、D、E、F只用于区别不同的互补序列，不用于排序。The ones and twos are used to distinguish different card-issuing sequences, and are not used for sorting; the C, D, E, and F are only used to distinguish different complementary sequences, and are not used for sorting.

具体的，所述发卡序列包括下述1)-4)中的至少一种：Specifically, the card issue sequence includes at least one of the following 1)-4):

1)所述发卡序列一包括序列表中SEQ ID NO：7所示的核苷酸序列和/或将SEQ ID NO：7所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：7所示核苷酸序列具有相同功能的核苷酸序列；1) The hairpin sequence 1 comprises the nucleotide sequence shown in SEQ ID NO: 7 in the Sequence Listing and/or the nucleotide sequence shown in SEQ ID NO: 7 is substituted by one or several nucleotides and/or Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing;

2)所述发卡序列二包括序列表中SEQ ID NO：8所示的核苷酸序列和/或将SEQ ID NO：8所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：8所示核苷酸序列具有相同功能的核苷酸序列；2) The card-issuing sequence 2 comprises the nucleotide sequence shown by SEQ ID NO: 8 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 8 is substituted by one or several nucleotides and / Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing;

3)所述发卡序列一包括将序列表中SEQ ID NO：9所示的核苷酸序列和/或将SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；3) the card-issuing sequence one comprises the nucleotide sequence shown by SEQ ID NO: 9 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 9 is substituted by one or several nucleotides and a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;

4)所述发卡序列二包括将序列表中SEQ ID NO：10所示的核苷酸序列和/或将SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列。4) The hairpin sequence two comprises the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 10 is substituted by one or several nucleotides and And a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing.

所述功能包括：所述上游引物通过碱基互补可打开所述发卡序列一的发卡结构，并且所述发卡序列一能在TDT酶的作用下实现自身核苷酸链的延伸；所述发卡序列二可通过碱基互补打开所述发卡序列一的发卡结构；所述发卡序列一和发卡序列二可通过碱基互补互相循环打开彼此的发卡结构，并彼此相互连接；所述发卡序列一和发卡序列二形成的连接体中，发卡序列一同时还能在TDT酶的作用下实现自身核苷酸链的延伸。The function comprises: the upstream primer can open the hairpin structure of the hairpin sequence one by base complementation, and the hairpin sequence can realize the extension of the self nucleotide chain under the action of the TDT enzyme; the card issue sequence The card issuer structure of the card issue sequence 1 can be opened by base complementation; the card issue sequence 1 and the card issue sequence 2 can be mutually cyclically opened to each other to open each other's card issue structure, and connected to each other; the card issue sequence and card issuance In the linker formed by the sequence two, the hairpin sequence can also realize the extension of the self-nucleotide chain under the action of the TDT enzyme.

具体的，所述核酸自组装显色反应还包括下述1)-2)中所述的至少一种：Specifically, the nucleic acid self-assembly color reaction further includes at least one of the following 1) to 2):

1)将本发明所述方法中的至少一种制备得到是反应体系与本发明所述方法中的至少一种发卡序列混合，于37℃孵育20min；再加入TdT酶反应体系，37℃反应20min后，于75℃加热10min；然后再加入氯高铁血红素和G-四链体诱导缓冲液于37℃孵育20min；最后加入ABTS2-和过氧化氢进行显色反应；1) Preparing at least one of the methods of the present invention, the reaction system is mixed with at least one hairpin sequence in the method of the present invention, and incubated at 37 ° C for 20 min; then adding a TdT enzyme reaction system, and reacting at 37 ° C for 20 min. After heating at 75 ° C for 10 min; then adding hemin and G-quadruplex induction buffer for 20 min at 37 ° C; finally adding ABTS2- and hydrogen peroxide for color reaction;

具体的，在所述加入TdT酶反应体系前，还包括纯化步骤；具体的，所述纯化步骤包括通过在所述下游引物上连接生物素，利用生物素与亲酶链核素相结合的方法纯化带有生物素的目标产物。Specifically, before the adding the TdT enzyme reaction system, a purification step is further included; specifically, the purification step includes a method of combining biotin and avidin by binding biotin to the downstream primer. The target product with biotin is purified.

2)所述核酸自组装显色反应的反应体系中，所述发卡序列一或发卡序列二的终浓度均为2μM。2) In the reaction system of the nucleic acid self-assembly color reaction, the final concentration of the hairpin sequence or the hairpin sequence 2 is 2 μM.

具体的，所述方法还包括下述1)-4)中至少一种：Specifically, the method further includes at least one of the following 1)-4):

1)通过最终反应体系的颜色变化判断待测物中是否含有待测目标；具体的，当反应体系的颜色发生变化时，判断待测物中含有待测目标；再具体的，当反应体系的颜色为蓝绿色时，判断待测物中含有待测目标；1) judging whether the object to be tested contains the object to be tested by the color change of the final reaction system; specifically, when the color of the reaction system changes, it is judged that the object to be tested contains the object to be tested; and specifically, when the reaction system When the color is blue-green, it is judged that the object to be tested contains the object to be tested;

2)通过最终反应体系的颜色来制作标准曲线的方法来计算待测物中的待测目标的浓度；2) a method of preparing a standard curve by the color of the final reaction system to calculate the concentration of the target to be tested in the test object;

3)通过增加上游引物和发夹序列一、发卡序列二的种类来实现双重或多重检测。3) Double or multiple detection is achieved by adding upstream primers and hairpin sequences, and the type of hairpin sequence two.

具体的，当所述检测为双重或多重检测时，可通过微阵列法判断待测物中是否含有待测目标或含有几种待测目标；所述微阵列法包括，将所述不同种类的发夹序列分别单独放置于不同的微孔中进行反应，然后根据反应结果判断是否含有待测目标或含有几种待测目标，当微孔中的反应液颜色发生变化或变为蓝绿色时，判断含有待测目标；发生颜色变化或变为蓝绿色的微孔总数为待测物中含有的待测目标的种类总数。Specifically, when the detection is dual or multiple detection, the microarray method may be used to determine whether the object to be tested contains the object to be tested or contains several objects to be tested; the microarray method includes: The hairpin sequence is separately placed in different micropores for reaction, and then according to the reaction result, it is judged whether the object to be tested is contained or contains several objects to be tested. When the color of the reaction liquid in the micropore changes or turns blue-green, The total number of micropores containing the target to be tested; the color change or the blue-green color is the total number of types of the target to be tested contained in the test object.

所述发夹序列一的种类包括：所述发夹序列一中的互补序列C与同一个所述上游引物中的互补序列A和/或B互补或反向互补的发夹序列为同一种类的发夹序列一，否则，为不同种类的发夹序列一；所述上游引物核苷酸序列相同的为同一个上游引物。The type of the hairpin sequence one comprises: the complementary sequence C in the hairpin sequence one is the same type as the hairpin sequence complementary or reverse complementary to the complementary sequence A and/or B in the same upstream primer. The hairpin sequence is one, otherwise, it is a different type of hairpin sequence; the upstream primer nucleotide sequence is the same as the same upstream primer.

4)在核酸自组装显色反应前，还包括对体外核酸扩增产物进行纯化。4) Purification of the in vitro nucleic acid amplification product prior to the nucleic acid self-assembly color reaction.

具体的，所述纯化包括试剂盒纯化或磁珠纯化方法；所述纯化包括去除反应体系中除体外核酸扩增产物和发卡序列链接产物外的其他杂质。Specifically, the purification includes a kit purification or a magnetic bead purification method; the purification includes removing impurities other than the in vitro nucleic acid amplification product and the hairpin sequence linking product in the reaction system.

本发明的另外一个目的是提供一种用于检测金属的试剂盒和/或生物传感器，所述试剂盒和/或生物传感器包括下述1)-6)所述中的至少一种：Another object of the present invention is to provide a kit and/or biosensor for detecting metal, the kit and/or biosensor comprising at least one of the following 1) to 6):

1)包括至少两种上游引物，所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；1) comprising at least two upstream primers, the bases of at least one of the nucleotide sequences of the at least two upstream primers being different; each of the at least two upstream primers being upstream The primer comprises: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence which can specifically amplify the template, and a nucleotide which can bind to the metal to be tested; the tether is located in the complementary sequence A and Between the complementary sequences B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be detected are located at the 5' end or the 3' end of the upstream primer; the complementary sequence A and the nucleotide sequence of the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or a structure that inhibits new strand extension during nucleic acid amplification in vitro;

2)包括模板，所述模板至少包括互补序列G、互补序列H和序列I；其中，所述互补序列G、互补序列H分别与至少两种上游引物中的可特异性扩增所述模板的核苷酸序列互补和/或反向互补；所述序列I的核苷酸序列与下游引物中的可特异性扩增所述模板的核苷酸序列相同；2) comprising a template comprising at least a complementary sequence G, a complementary sequence H and a sequence I; wherein the complementary sequence G, the complementary sequence H and the at least two upstream primers respectively specifically amplify the template The nucleotide sequence is complementary and/or reverse complementary; the nucleotide sequence of the sequence I is identical to the nucleotide sequence of the downstream primer that specifically amplifies the template;

3)包括下游引物、模板和至少两种上游引物；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；所述下游引物包括可特异性扩增所述模板的核苷酸序列；3) comprising a downstream primer, a template and at least two upstream primers; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; the at least two upstream primers Each of the upstream primers includes: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; Between the complementary sequence A and the complementary sequence B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be tested are located at the 5' end or the 3' end of the upstream primer. The nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits new strands during nucleic acid amplification in vitro; An extended structure; the downstream primer comprising a nucleotide sequence that specifically amplifies the template;

4)包括至少两种上游引物和发卡序列；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和 /或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；4) comprising at least two upstream primers and a hairpin sequence; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; of the at least two upstream primers Each upstream primer comprises: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; the tether is located at the complementary Between sequence A and complementary sequence B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be detected are located at the 5' end or the 3' end of the upstream primer; The nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits new strand extension during nucleic acid amplification in vitro; structure;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补。The card-issuing sequence includes: a card-issuing sequence one and/or a card-issuing sequence two, the card-issuing sequence one comprising: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, and the three or more arbitrary nucleotides Located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, which is complementary or inversely complementary to said complementary sequence E.

5)包括至少两种上游引物、模板和发卡序列；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；5) comprising at least two upstream primers, a template and a hairpin sequence; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; the at least two upstream primers Each of the upstream primers includes: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; Between the complementary sequence A and the complementary sequence B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be tested are located at the 5' end or the 3' end of the upstream primer. The nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits new strands during nucleic acid amplification in vitro; Extended structure

所述模板至少包括互补序列G、互补序列H和序列I；其中，所述互补序列G、互补序列H分别与至少两种上游引物中的可特异性扩增所述模板的核苷酸序列互补和/或反向互补；所述序列I的核苷酸序列与下游引物中的可特异性扩增所述模板的核苷酸序列相同；The template comprises at least a complementary sequence G, a complementary sequence H and a sequence I; wherein the complementary sequence G and the complementary sequence H are complementary to nucleotide sequences of at least two upstream primers that specifically amplify the template, respectively And/or reverse complement; the nucleotide sequence of the sequence I is identical to the nucleotide sequence of the downstream primer that specifically amplifies the template;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；The card-issuing sequence includes: a card-issuing sequence one and/or a card-issuing sequence two, the card-issuing sequence one comprising: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, and the three or more arbitrary nucleotides Located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, said complementary sequence C being complementary or oppositely complementary to said complementary sequence E;

6)包括下游引物、模板、至少两种上游引物和发卡序列；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；6) comprising a downstream primer, a template, at least two upstream primers, and a hairpin sequence; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; the at least two Each of the upstream primers includes: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; An arm is located between the complementary sequence A and the complementary sequence B, and the nucleotide sequence capable of specifically amplifying the template and the nucleotide which can bind to the metal to be detected are located at the 5' end of the upstream primer or a 3' end; the nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits nucleic acid amplification in vitro The structure of the extension of the new chain;

所述下游引物包括可特异性扩增所述模板的核苷酸序列；The downstream primer includes a nucleotide sequence that specifically amplifies the template;

具体的，所述可与待测金属结合的核苷酸具体包括含胸腺嘧啶或胞嘧啶的核苷酸；Specifically, the nucleotide that can bind to the metal to be tested specifically includes a thymine- or cytosine-containing nucleotide;

具体的，所述至少两种上游引物中的一种包括下述1)-4)中的至少一种：Specifically, one of the at least two upstream primers includes at least one of the following 1)-4):

1)将序列表中SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列通过连接臂连接后得到的引物；1) a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing via a tether;

2)将序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；2) subjecting the nucleotide sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing to one or several nucleotide substitutions and/or deletions and/or additions and to the SEQ ID in the Sequence Listing a primer obtained by linking a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 2 to the nucleotide sequence shown by SEQ ID NO: 2;

3)将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物；3) a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether;

4)将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；4) subjecting the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing to one or several nucleotide substitutions and/or deletions and/or additions and to the SEQ ID in the Sequence Listing a primer obtained by linking a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 5 to the nucleotide sequence shown by SEQ ID NO: 5;

具体的，所述下游引物包括序列表中SEQ ID NO：3所示的核苷酸序列，和/或将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；Specifically, the downstream primer comprises the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, and/or the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing is subjected to one or several nucleotides. a nucleotide sequence having a substitution and/or deletion and/or addition and having the same function as the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

具体的，所述模板包括序列表中SEQ ID NO：6所示的核苷酸序列,和/或将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列。Specifically, the template includes the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing, and/or the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing is subjected to one or several nucleotides. A nucleotide sequence substituted and/or deleted and/or added and having the same function as the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing.

具体的，所述连接臂包括具长链结构的化合物；Specifically, the connecting arm comprises a compound having a long chain structure;

具体的，所述至少两种上游引物中的一种包括：AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCT CTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT或TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATA CAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；Specifically, one of the at least two upstream primers comprises: AGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCT CTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT or TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATA CAACCTACTACCTCATTTTTTTTTGCACATAACACCCC;

具体的，所述至少两种上游引物包括：AGAGAGAGAGAGGGAAAGAGAG AG-oxyethyleneglycol bridge-CTCTC TCTTTCCCTCTCTCTCTCTGTGAAATTATCGC CACGTTCGGTTTT和TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；Specifically, the at least two upstream primers include: AGAGAGAGAGAGGGAAAGAGAG AG-oxyethyleneglycol bridge-CTCTC TCTTTCCCTCTCTCTCTCTGTGAAATTATCGC CACGTTCGGTTTT and TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTGCACATAACACCCC;

具体的，所述下游引物还包括在所述下游引物上标记免疫标记；所述免疫标记包括生物素；所述生物素标记在所述下游引物的5’端第一个核苷酸上；所述标记方法属于现有技术；具体的，所述下游引物为Biotin-TCATCGCACCGTCAAAGGAACC；Specifically, the downstream primer further comprises marking an immunolabel on the downstream primer; the immunolabel comprises biotin; the biotin label is on the first nucleotide of the 5' end of the downstream primer; The labeling method belongs to the prior art; specifically, the downstream primer is Biotin-TCATCGCACCGTCAAAGGAACC;

1)所述发卡序列一包括序列表中SEQ ID NO：7所示的核苷酸序列，和/或将SEQ ID NO：7所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：7所示核苷酸序列具有相同功能的核苷酸序列；1) the hairpin sequence 1 comprises the nucleotide sequence shown in SEQ ID NO: 7 in the Sequence Listing, and/or the nucleotide sequence shown in SEQ ID NO: 7 is substituted by one or several nucleotides and a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing;

2)所述发卡序列二包括序列表中SEQ ID NO：8所示的核苷酸序列，和/或将SEQ ID NO：8所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：8所示核苷酸序列具有相同功能的核苷酸序列；2) The card-issuing sequence II comprises the nucleotide sequence shown in SEQ ID NO: 8 in the Sequence Listing, and/or the nucleotide sequence shown in SEQ ID NO: 8 is substituted by one or several nucleotides and a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing;

3)所述发卡序列一包括将序列表中SEQ ID NO：9所示的核苷酸序列，和/或将SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；3) The card-issuing sequence one comprises the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, and/or the nucleotide sequence shown in SEQ ID NO: 9 is substituted by one or several nucleotides. And/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;

4)所述发卡序列二包括将序列表中SEQ ID NO：10所示的核苷酸序列，和/或将SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列。4) The hairpin sequence 2 comprises the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing, and/or the nucleotide sequence shown by SEQ ID NO: 10 is substituted by one or several nucleotides. And/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing.

所述互补包括现有技术或公知常识所定义的互补或反向互补，和/或根据现有技术或公知常识所定义的互补原则进行互补或反向互补；The complementation includes complementary or reverse complementarity as defined by the prior art or common general knowledge, and/or complementarity or reverse complementation according to complementary principles as defined by the prior art or common general knowledge;

所述聚合酶包括可用于体外核酸扩增的聚合酶；The polymerase includes a polymerase that can be used for in vitro nucleic acid amplification;

所述可特异性扩增所述模板的核苷酸序列具体包括根据所述模板的特征序列所设计的引物序列；所述特征序列包括现有技术或公知常识所定义的特征序列；所述设计包括现有技术或公知常识所记载的设计方法；The nucleotide sequence capable of specifically amplifying the template specifically includes a primer sequence designed according to a characteristic sequence of the template; the characteristic sequence includes a characteristic sequence defined by prior art or common knowledge; the design Including the design methods described in the prior art or common knowledge;

所述A、B、G、H、I只用于区别不同的互补序列或序列，不用于排序；The A, B, G, H, and I are only used to distinguish different complementary sequences or sequences, and are not used for sorting;

具体的，所述试剂盒和/或生物传感器包括至少两种上游引物，并包括下述1)-8)所述中的至少一种：Specifically, the kit and/or biosensor comprises at least two upstream primers and includes at least one of the following 1)-8):

所述功能包括可实现所述模板的扩增；Said function comprising enabling amplification of said template;

还具体的，所述至少两种上游引物中的一种包括：AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTC TCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT或TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATAC AACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；Further specifically, one of the at least two upstream primers comprises: AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTC TCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT or TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATAC AACCTACTACCTCATTTTTTTTTGCACATAACACCCC;

还具体的，所述至少两种上游引物包括：AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTC TCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT和TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATAC AACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；Further specifically, the at least two upstream primers include: AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTC TCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT and TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATAC AACCTACTACCTCATTTTTTTTTGCACATAACACCCC;

具体的，所述试剂盒和/或生物传感器还包括发卡序列，所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’ 端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；Specifically, the kit and/or the biosensor further includes a card issue sequence, the card issue sequence includes: a card issue sequence one and/or a card issue sequence two, the card issue sequence one comprises: a complementary sequence C, a complementary sequence D and three Any of the above nucleotides, the three or more arbitrary nucleotides are located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; Sequence D is complementary and/or inversely complementary to a complementary sequence F; said complementary sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, said complementary sequence C and said complementary sequence E Complementary or reverse complementarity;

所述发卡序列包括下述1)-4)中的至少一种：The card issue sequence includes at least one of the following 1)-4):

具体的，所述试剂盒和/或生物传感器还包括TdT反应缓冲液、dATP、dGTP、TdT酶、氯高铁血红素、G-四链体诱导缓冲液、ABTS ^2-、过氧化氢、home-made XP beads、表面修饰了一层亲酶链核素的磁珠中的至少一种。 Specifically, the kit and/or biosensor further comprises TdT reaction buffer, dATP, dGTP, TdT enzyme, hemin, G-quadruplex induction buffer, ABTS ^2- , hydrogen peroxide, home- Made XP beads, at least one of the magnetic beads whose surface is modified with a layer of pro-ligase.

具体的，所述所述试剂盒和/或生物传感器包括下述1)-5)：Specifically, the kit and/or biosensor includes the following 1)-5):

1)TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AAC TATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；1) TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AAC TATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC;

2)AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT；2) AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT;

3)序列表中SEQ ID NO：6所示的核苷酸序列；3) the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;

4)序列表中SEQ ID NO：3所示的核苷酸序列；4) the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

5)序列表中SEQ ID NO：7所示的核苷酸序列、序列表中SEQ ID NO：8所示的核苷酸序列、序列表中SEQ ID NO：9所示的核苷酸序列和序列表中SEQ ID NO：10所示的核苷酸序列。5) the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing, and The nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing.

所述oxyethyleneglycol的化学结构为：The chemical structure of the oxyethyleneglycol is:

本发明的再一个目的是提供一种银的检测方法，所述方法包括体外核酸扩增技术, 所述体外核酸扩增技术的反应体系包括上游引物、下游引物和模板，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；It is still another object of the present invention to provide a method for detecting silver, the method comprising an in vitro nucleic acid amplification technique, the reaction system of the in vitro nucleic acid amplification technique comprising an upstream primer, a downstream primer and a template, the upstream primer comprising: Primers obtained by ligating the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing by ligation arms, or SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing The nucleotide sequence is substituted and/or deleted and/or added by one or several nucleotides and has the same function as the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing. a primer obtained by ligating a nucleotide sequence through a tether; the tether comprises a structure that inhibits polymerase binding and/or a structure that inhibits elongation of a new strand during nucleic acid amplification in vitro;

所述下游引物包括：序列表中SEQ ID NO：3所示的核苷酸序列，或将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；The downstream primer comprises: the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing is substituted by one or several nucleotides and / Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

所述模板包括：序列表中SEQ ID NO：6所示的核苷酸序列，或将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列。The template includes: the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing is substituted by one or several nucleotides and/or A nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing.

所述连接臂包括具长链结构的化合物；The connecting arm comprises a compound having a long chain structure;

具体的，所述连接臂为oxyethyleneglycol，oxyethyleneglycol的化学结Specifically, the connecting arm is an oxyethyleneglycol, a chemical knot of oxyethyleneglycol

构为：Constructed as:

具体的，所述上游引物为TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTT TTGCACATAACACCCC；Specifically, the upstream primer is TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTT TTGCACATAACACCCC;

具体的，所述方法还包括下述1)-2)中的至少一种：Specifically, the method further includes at least one of the following 1)-2):

再具体的，所述超快速PCR反应的反应体系中上游引物和下游引物的浓度为普通PCR浓度的10倍以上；还具体的，为20倍；所述超快速PCR反应的反应体系中还包括DNA聚合酶，所述DNA聚合酶的浓度为普通PCR浓度的10倍以上，还具体的，为60倍。More specifically, the concentration of the upstream primer and the downstream primer in the reaction system of the ultra-fast PCR reaction is 10 times or more of the ordinary PCR concentration; specifically, 20 times; the reaction system of the ultra-fast PCR reaction further includes The DNA polymerase has a concentration of 10 times or more of the ordinary PCR concentration, and specifically, 60 times.

2)所述方法还包括核酸自组装显色反应,所述核酸自组装显色反应的反应体系包括发卡序列，所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补。2) The method further comprises a nucleic acid self-assembly color reaction, the nucleic acid self-assembly color reaction reaction system comprising a hairpin sequence, the card issue sequence comprising: a card issue sequence one and/or a card issue sequence two, the card issue sequence The invention comprises: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, wherein the three or more arbitrary nucleotides are located at the 5' end and/or the 3' end of the card issue sequence; Included is a complementary sequence E and a complementary sequence F; said complementary sequence D is complementary and/or inversely complementary to a complementary sequence F; said complementary sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B And the complementary sequence C is complementary or oppositely complementary to the complementary sequence E.

具体的，所述发卡序列包括下述1)-2)中至少一种：Specifically, the card issuance sequence includes at least one of the following 1)-2):

1)所述发卡序列一包括将序列表中SEQ ID NO：9所示的核苷酸序列和/或SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；1) The hairpin sequence 1 comprises a nucleotide sequence represented by SEQ ID NO: 9 in the sequence listing and/or a nucleotide sequence represented by SEQ ID NO: 9 by one or several nucleotide substitutions and/or Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;

2)所述发卡序列二包括将序列表中SEQ ID NO：10所示的核苷酸序列和/或SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列。2) The hairpin sequence two comprises substituting one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 10. Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing.

具体的，所述方法还包括：将本发明所述的至少一种银的检测方法制备得到的反应体系与本发明所述的至少一种银的检测方法中的发卡序列混合，于37℃孵育20min；再加入TdT酶反应体系，37℃反应20min后，于75℃加热10min；然后再加入氯高铁血红素和G-四链体诱导缓冲液于37℃孵育20min；最后加入ABTS ^2-和过氧化氢进行显色反应； Specifically, the method further comprises: mixing the reaction system prepared by the detection method of at least one silver according to the present invention with the hairpin sequence in the at least one silver detection method of the present invention, and incubating at 37 ° C 20 min; further add TdT enzyme reaction system, react at 37 ° C for 20 min, then heat at 75 ° C for 10 min; then add hemin and G-quadruplex induction buffer for 20 min at 37 ° C; finally add ABTS ^2- and Hydrogen peroxide undergoes a color reaction;

具体的，所述核酸自组装显色反应的反应体系中，所述发卡序列一或发卡序列二的终浓度均为2μM。Specifically, in the reaction system of the nucleic acid self-assembly color reaction, the final concentration of the hairpin sequence or the hairpin sequence 2 is 2 μM.

通过最终反应体系的颜色变化判断待测物中是否含有待测目标；Judging whether the object to be tested contains the object to be tested by the color change of the final reaction system;

具体的，当反应体系的颜色发生变化时，判断待测物中含有待测目标；再具体的，当反应体系的颜色为蓝绿色时，判断待测物中含有待测目标；Specifically, when the color of the reaction system changes, it is determined that the object to be tested contains the object to be tested; and specifically, when the color of the reaction system is blue-green, it is determined that the object to be tested contains the object to be tested;

通过最终反应体系的颜色来制作标准曲线的方法来计算待测物中的待测目标的浓度；Calculating the concentration of the target to be tested in the test object by a method of preparing a standard curve by the color of the final reaction system;

在核酸自组装显色反应前，还包括对体外核酸扩增产物进行纯化。The nucleic acid amplification product in vitro is also purified prior to the nucleic acid self-assembly color reaction.

本发明的还一个目的是提供一种用于检测银的试剂盒和/或生物传感器，所述试剂盒和/或生物传感器包括下述1)-4)所述中的至少一种：It is still another object of the present invention to provide a kit and/or biosensor for detecting silver, the kit and/or biosensor comprising at least one of the following 1) to 4):

1)包括上游引物，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；1) comprising an upstream primer comprising: a primer obtained by ligating the nucleotide sequence shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing via a tether, or SEQ ID in the Sequence Listing The nucleotide sequence shown by NO: 4 and/or SEQ ID NO: 5 is substituted and/or deleted and/or added by one or several nucleotides and is SEQ ID NO: 4 and/or SEQ ID in the Sequence Listing. NO: a primer obtained by ligating a nucleotide sequence having the same function as a nucleotide sequence of 5, which is linked by a tether; the linker comprises a structure which inhibits polymerase binding and/or inhibits an in vitro nucleic acid amplification process Chain extended structure;

2)包括上游引物、下游引物和模板，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；2) comprising an upstream primer, a downstream primer, and a template, the upstream primer comprising: a primer obtained by ligating the nucleotide sequence represented by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether, or The nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing is substituted and/or deleted and/or added by one or several nucleotides and is SEQ ID NO: 4 in the Sequence Listing. And/or a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown in SEQ ID NO: 5 by a ligation arm; the linker comprises a structure capable of inhibiting polymerase binding and/or inhibiting nucleic acid in vitro a structure in which a new chain extends during amplification;

所述模板包括：序列表中SEQ ID NO：6所示的核苷酸序列，或将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列；The template includes: the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing is substituted by one or several nucleotides and/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing;

3)包括上游引物和发卡序列，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；3) comprising an upstream primer and a hairpin sequence, the upstream primer comprising: a primer obtained by ligating the nucleotide sequence represented by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether, or a sequence listing The nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 is substituted and/or deleted and/or added by one or several nucleotides and is SEQ ID NO: 4 and / in the Sequence Listing. Or a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown in SEQ ID NO: 5 by a ligation arm; the linker comprises a structure which inhibits polymerase binding and/or inhibits nucleic acid amplification in vitro The structure of the new chain extension in the process;

4)包括上游引物、下游引物、模板和发卡序列，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；4) comprising an upstream primer, a downstream primer, a template and a hairpin sequence, the upstream primer comprising: a primer obtained by ligating the nucleotide sequence shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the sequence listing through a tether Or the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing is subjected to one or several nucleotide substitutions and/or deletions and/or additions and to the SEQ ID in the Sequence Listing. a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 5 by a ligation arm; the linker comprises a structure capable of inhibiting polymerase binding and/or Inhibiting the structure of new strand extension during in vitro nucleic acid amplification;

所述下游引物包括：序列表中SEQ ID NO：3所示的核苷酸序列，或将序列表中 SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；The downstream primer comprises: the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing is substituted by one or several nucleotides and / Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；The card issue sequence includes: a card issue sequence one and/or a card issue sequence two, the card issue sequence one includes: a complementary sequence C, a complementary sequence D and more than one arbitrary nucleotide, and the three or more arbitrary nucleotides are located a 5' end and/or a 3' end of the hairpin sequence; the card issue sequence 2 comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; C is complementary and/or inversely complementary to the complementary sequence A and/or the complementary sequence B, which is complementary or oppositely complementary to the complementary sequence E;

具体的，所述连接臂为oxyethyleneglycol，oxyethyleneglycol的化学结构为：Specifically, the connecting arm is oxyethyleneglycol, and the chemical structure of oxyethyleneglycol is:

具体的，所述上游引物为TGAGGTAGTAGGTTGTATAGTT–oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTT TTGCACATAACACCCC；Specifically, the upstream primer is TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTT TTGCACATAACACCCC;

具体的，所述下游引物还包括在所述下游引物上标记免疫标记；所述免疫标记包括生物素；所述生物素标记在所述下游引物的5’端第一个核苷酸上；Specifically, the downstream primer further comprises marking an immunolabel on the downstream primer; the immunolabel comprises biotin; the biotin label is on the first nucleotide of the 5' end of the downstream primer;

所述标记方法属于现有技术；再具体的，所述下游引物为Biotin-TC ATCGCACCGTCAAAGGAACC；The labeling method belongs to the prior art; more specifically, the downstream primer is Biotin-TC ATCGCACCGTCAAAGGAACC;

具体的，所述试剂盒和/或生物传感器包括下述1)-4)：Specifically, the kit and/or biosensor includes the following 1)-4):

1)TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AA CTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；1) TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AA CTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC;

2)序列表中SEQ ID NO：6所示的核苷酸序列；2) the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;

3)序列表中SEQ ID NO：3所示的核苷酸序列；3) the nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing;

4)序列表中SEQ ID NO：7所示的核苷酸序列、序列表中SEQ ID NO：8所示的核苷酸序列、序列表中SEQ ID NO：9所示的核苷酸序列和序列表中SEQ ID NO：10所示的核苷酸序列；4) the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing, and a nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing;

其中，oxyethyleneglycol的化学结构为：Among them, the chemical structure of oxyethyleneglycol is:

本发明的还一个目的是提供本发明任意所述方法中的至少一种、本发明任意所述试剂盒和/或生物传感器中的至少一种的应用。It is still another object of the present invention to provide at least one of any of the methods of the present invention, the use of at least one of the kits and/or biosensors of any of the present invention.

具体的，所述应用包括下述1)-4)中的至少一种应用：Specifically, the application includes at least one of the following 1)-4):

1)检测金属；1) detecting metal;

2)制备检测金属的产品和/或相关产品中的应用；2) preparing an application for detecting metal products and/or related products;

3)双重或多重金属的检测；3) detection of double or multiple metals;

4)制备用于双重或多重金属检测的产品和/或相关产品中的应用。4) Preparation of products for use in dual or multiple metal detection and/or related products.

具体的，所述金属包括Hg ²⁺和/或Ag ⁺。 Specifically, the metal includes Hg ²⁺ and/or Ag ⁺ .

可选的，所述任一应用不包括中国专利法第二十五条所述的疾病的诊断和治疗方法。Optionally, the application does not include the diagnosis and treatment of the disease described in Article 25 of the Chinese Patent Law.

本发明建立的一种基于超快速PCR的双重比色传感新方法：A new method of dual colorimetric sensing based on ultra-fast PCR established by the invention:

(1)该方法建立了超快速PCR反应体系，将耗时3小时左右的传统PCR过程缩减到2.5min，显著减少了PCR反应的用时；(1) The method establishes an ultra-fast PCR reaction system, which reduces the conventional PCR process, which takes about 3 hours, to 2.5 minutes, which significantly reduces the time of the PCR reaction;

(2)将超快速PCR反应体系搭载酶联核酸自组装显色模块，解决了传统PCR 难于可视化定量检测的难题；(2) The ultra-fast PCR reaction system is equipped with an enzyme-linked nucleic acid self-assembly color-developing module, which solves the problem that the traditional PCR is difficult to visualize quantitative detection;

(3)将双重超快速PCR反应体系搭载双重酶联核酸自组装显色模块，实现了双重重金属的超快速、定量、可视化检测。(3) The dual ultra-fast PCR reaction system was equipped with a dual enzyme-linked nucleic acid self-assembly color-developing module to realize ultra-fast, quantitative and visual detection of double heavy metals.

本发明的一个具体实施例提供了一种基于双重超快速PCR的双重比色传感新方法，用于Hg ²⁺和Ag ⁺的可视化超灵敏检测。该新方法根据汞离子胸腺嘧啶错配，银离子胞嘧啶错配，设计超快速聚合酶链式反应(Polymerase Chain Reaction,PCR)的双重扩增引物，同时通过该引物可结合酶联核酸自组装显色模块，整合建立一种新兴的基于错配的汞离子和银离子双重靶标功能核酸检测新方法。 A specific embodiment of the present invention provides a novel dual colorimetric sensing method based on dual ultra-fast PCR for visual hypersensitive detection of Hg ²⁺ and Ag ⁺ . The new method is based on a mercury ion thymine mismatch, a silver ion cytosine mismatch, and a dual amplification primer designed by a polymerase chain reaction (PCR), which can be combined with an enzyme-linked nucleic acid self-assembly. The color development module integrates an emerging new method for nucleic acid detection based on mismatched mercury ion and silver ion dual target function.

本发明具有下述有益技术效果：The invention has the following beneficial technical effects:

1)本发明所建立的检测方法和生物传感器，比传统方法更快捷、更灵敏，具备特异性强、灵敏度高、检测结果可靠等优点，可以简化分析检测步骤，缩短分析时间，更重要的是使在线实时检测成为可能，便于携带和野外作业，在重金属快速检测领域，具有非常好的应用前景。1) The detection method and the biosensor established by the invention are faster and more sensitive than the traditional method, and have the advantages of high specificity, high sensitivity, reliable detection result, etc., which can simplify the analysis and detection steps, shorten the analysis time, and more importantly, It makes online real-time detection possible, easy to carry and field work, and has a very good application prospect in the field of rapid detection of heavy metals.

2)本发明所建立的检测方法和生物传感器可同时实现对Hg ²⁺和Ag ⁺的双重特异性检测，检测的特异性好、灵敏度高，检测结果可靠、肉眼即可辨别，检测过程快捷方便，在日常监控或市场筛查等方面具有重要意义。具体的，灵敏度实验结果表明，本发明所建立的检测方法和生物传感器对Hg ²⁺的检测限为1.3pM，对Ag ⁺的检测限为2.5pM，检测的灵敏度高； 2) The detection method and the biosensor established by the invention can simultaneously realize the dual specific detection of Hg ²⁺ and Ag ⁺ , the detection has good specificity, high sensitivity, reliable detection result, can be discerned by the naked eye, and the detection process is quick and convenient. It is of great significance in daily monitoring or market screening. Specifically, the sensitivity experiment results show that the detection method and the biosensor of the invention have a detection limit of 1.3 pM for Hg ^{2+ and} a detection limit of 2.5 pM for Ag ⁺ , and the detection sensitivity is high;

特异性试验结果表明本发明所建立的检测方法和生物传感器对Cu2 ⁺、Mg ²⁺无交叉反应，可同时实现Hg ²⁺和Ag ⁺的双重特异性检测；加标回收检测实验结果表明，本发明所建立的检测方法和生物传感器对Hg ²⁺和Ag ⁺的检测值接近于实际加标值，检测结果可靠、准确度高。 The specificity test results show that the detection method and biosensor established by the invention do not cross-react with Cu2 ⁺ and Mg ²⁺ , and can achieve dual specific detection of Hg ²⁺ and Ag ⁺ at the same time; The detection method and biosensor of the invention have the detection values of Hg ²⁺ and Ag ⁺ close to the actual spiked value, and the detection result is reliable and accurate.

3)本发明所建立的检测方法和生物传感器，解决了传统PCR难于可视化定量检测的难题，实现了双重重金属Hg ²⁺和Ag ⁺的超快速、定量、可视化检测。 3) The detection method and the biosensor established by the invention solve the problem that the traditional PCR is difficult to visualize and quantitatively detect, and realize the ultra-fast, quantitative and visual detection of the double heavy metals Hg ²⁺ and Ag ⁺ .

附图说明DRAWINGS

图1为超快速PCR装置的结构图；Figure 1 is a structural diagram of an ultra-fast PCR device;

图2为双重超快速PCR反应的扩增效果验证结果图；其中，泳道1为反应体系中只添加Ag ⁺的结果；泳道2为反应体系中只添加Hg ²⁺的结果；泳道3和泳道4为反应体系中同时添加Hg ²⁺和Ag ⁺的结果；泳道5为阴性对照(反应体系中不添加Hg ²⁺和Ag ⁺)的结果；图2a为经磁珠纯化后的结果图；图2b为经磁珠纯化前和后的对照结果图，其中泳道3为未经磁珠纯化的结果图，泳道4为经过磁珠纯化的结果图； Figure 2 is a graph showing the results of the amplification effect of the double ultra-fast PCR reaction; wherein lane 1 is the result of adding only Ag ⁺ in the reaction system; lane 2 is the result of adding only Hg ²⁺ in the reaction system; lane 3 and lane 4 The results of simultaneous addition of Hg ²⁺ and Ag ⁺ in the reaction system; Lane 5 is the result of the negative control (no addition of Hg ²⁺ and Ag ^{+ in the} reaction system); Figure 2a is the result after purification by magnetic beads; Figure 2b For the control results before and after purification by magnetic beads, wherein lane 3 is the result of the non-magnetic bead purification, and lane 4 is the result of the magnetic bead purification;

图3为Hg ²⁺的标准曲线图； Figure 3 is a standard curve of Hg ²⁺ ;

图4为Ag ⁺的标准曲线图； Figure 4 is a standard curve of Ag ⁺ ;

图5为特异性实验结果图，其中1为微孔1,2为微孔2,3为微孔3,4为微孔4；a为对含Hg ²⁺与Cu ²⁺的样品进行检测的结果图；b为对含Ag ⁺和Mg ²⁺的样品进行检测的结果图；c为对含Hg ²⁺和Ag ⁺的样品进行检测的结果图。 Figure 5 is a graph showing the results of specific experiments, in which 1 is

micropores

1, 2 is

micropores

2, 3 is

micropores

3, 4 is micropores 4; a is a sample containing Hg ²⁺ and Cu ²⁺ Results graph; b is a graph showing the results of detection of samples containing Ag ⁺ and Mg ²⁺ ; c is a graph showing the results of detecting samples containing Hg ²⁺ and Ag ⁺ .

具体实施方式Detailed ways

下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。The molecular biology experimental methods not specifically described in the following examples are all carried out according to the specific methods listed in the book "Molecular Cloning Experimental Guide" (third edition) J. Sambrook, or according to kits and products. The instructions are carried out.

下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

实施例1、一种用于检测重金属Hg ²⁺和Ag ⁺的超快速PCR的双重比色传感新方法的建立 Example 1. Establishment of a new dual colorimetric sensing method for ultrafast PCR for detection of heavy metals Hg ²⁺ and Ag ⁺

(一)实验材料(1) Experimental materials

本实施例所设计的引物的核苷酸序列见表1和序列表。The nucleotide sequences of the primers designed in this example are shown in Table 1 and the Sequence Listing.

表1Table 1

表1中，上游引物Primer 1的连接臂(oxyethyleneglycol bridge)左侧的核苷酸序列为序列表中SEQ ID NO：1所示的核苷酸序列，连接臂右侧的核苷酸序列为序列表中SEQ ID NO：2所示的核苷酸序列，连接臂的化学结构为：In Table 1, the nucleotide sequence on the left side of the oxyethyleneglycol bridge of the upstream primer Primer 1 is the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence on the right side of the tether is in the order of In the nucleotide sequence shown by SEQ ID NO: 2 in the list, the chemical structure of the tether is:

表1中，下游引物Primer 2由序列表中SEQ ID NO：3所示的核苷酸在5’的第一个核苷酸标记了生物素后得到的。In Table 1, the downstream primer Primer 2 was obtained by labeling biotin with the nucleotide shown by SEQ ID NO: 3 in the Sequence Listing at the first nucleotide of 5'.

表1中，上游引物Primer 3的连接臂(oxyethyleneglycol bridge)左侧的核苷酸序列为序列表中SEQ ID NO：4所示的核苷酸序列，连接臂右侧的核苷酸序列为序列表中SEQ ID NO：5所示的核苷酸序列，连接臂的化学结构与Primer 1的连接臂的化学结构相同。In Table 1, the nucleotide sequence on the left side of the oxyethyleneglycol bridge of the upstream primer Primer 3 is the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, and the nucleotide sequence on the right side of the tether is in the order of In the nucleotide sequence shown by SEQ ID NO: 5 in the list, the chemical structure of the tether is the same as that of Primer 1.

表1中，模板Template的核苷酸序列为序列表中SEQ ID NO：6所示的核苷酸序列。In Table 1, the nucleotide sequence of the template Template is the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing.

表1中，发卡序列1-4(Hairpin 1、Hairpin 2、Hairpin 3、Hairpin 4)分别依次为序列表中SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10所示的核苷酸序列。In Table 1, the hairpin sequences 1-4 (Hairpin 1, Hairpin 2, Hairpin 3, Hairpin 4) are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: The nucleotide sequence shown in 10.

表1中所列的序列均为人工合成。The sequences listed in Table 1 are all synthetic.

SYBR Gold核酸染料、末端脱氧转移酶(TdT)、10×TdT buffer、三磷酸脱氧腺苷(dATP)、三磷酸脱氧鸟嘌呤(dGTP)、dNTP、Ex Taq DNA聚合酶、10×Taq buffer、氯高铁血红素、氯化汞、硝酸银、磁珠Sera-mag SpeedBeads、磁珠

MyOne ^TM Streptavidin T1,核酸分子量标准ultra-low range DNA ladder，均购自赛默飞科技(Thermo Scientific Life Technologies)。实验用水均来自Milli-Q纯水***。 SYBR Gold nucleic acid dye, terminal deoxygenase (TdT), 10×TdT buffer, deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), dNTP, Ex Taq DNA polymerase, 10×Taq buffer, chlorine Heme, mercuric chloride, silver nitrate, magnetic beads Sera-mag SpeedBeads, magnetic beads

MyOne ^TM Streptavidin T1, nucleic acid molecular weight standards ultra-low range DNA ladder, were purchased from Saimofeike technique (Thermo Scientific Life Technologies). The experimental water was taken from the Milli-Q pure water system.

(二)超快速PCR装置的搭建(2) Construction of ultra-fast PCR device

超快速PCR装置的主要结构如图1所示，其具体的结构、连接方式和工作原理、工作过程包括：The main structure of the ultra-fast PCR device is shown in Figure 1. The specific structure, connection method, working principle and working process include:

超快速PCR装置采用Light Cycler型号的毛细管(20uL，04 929 292 001，Roche)作为PCR样品室，通过快速离心的方式，样品会分别聚集到各个毛细管一端，离心完成后带有样品的毛细管被固定在塑料支架上。塑料支架连接到步进电机(42JSF630AS-1000，Just Motioin Control)上，由该步进电机带动固定在塑料支架上的毛细管样品室在95℃的高温水浴锅和58℃的中温水浴锅之间循环转换，实现超快速PCR反应过程中的反应温度变化及控制。所述步进电机由开关电源(S-100-24，Elecall)进行供电，采用直流伺服电机驱动器(YZ-ACSD60，Moving)以及Labview(version 2014)实现步进电机的上述循环转换的频率或时间的控制。温度测量利用封装在毛细管中的热电偶来实现。热电偶信号的放大及线性化处理过程则利用温度变送器(SBWR-2260，K，Yuancheng)进行传递并采用Arduino UNO v1.0芯片进行处理。Arduino UNO芯片将接收到的温度的模拟信号转换成数字信号，然后由Arduino IDE(version 1.8.1)模块执行运算。The ultra-fast PCR device uses a Light Cycler model capillary (20uL, 04 929 292 001, Roche) as the PCR sample chamber. By rapid centrifugation, the samples are collected at one end of each capillary. After centrifugation, the capillary with the sample is fixed. On a plastic stand. The plastic bracket is connected to a stepping motor (42JSF630AS-1000, Just Motioin Control), and the capillary sample chamber fixed on the plastic bracket is driven by the stepping motor between a high temperature water bath at 95 ° C and a medium temperature water bath at 58 ° C. Cyclic conversion to achieve reaction temperature changes and control during ultra-fast PCR reactions. The stepping motor is powered by a switching power supply (S-100-24, Elecall), and the frequency or time of the above-mentioned cyclic conversion of the stepping motor is realized by a DC servo motor driver (YZ-ACSD60, Moving) and Labview (version 2014). control. Temperature measurement is achieved using a thermocouple encapsulated in a capillary. The amplification and linearization process of the thermocouple signal is transmitted by a temperature transmitter (SBWR-2260, K, Yuancheng) and processed by the Arduino UNO v1.0 chip. The Arduino UNO chip converts the received analog signal into a digital signal, which is then executed by the Arduino IDE (version 1.8.1) module.

(三)双重超快速PCR反应的建立(III) Establishment of dual ultra-fast PCR reactions

1)配制双重超快速PCR反应体系，具体见表2：1) Prepare a dual ultra-fast PCR reaction system, as shown in Table 2:

表2Table 2

进行4组反应，其中阴性对照组的反应体系中不添加Hg ²⁺和Ag ⁺；实验组一的反应体系中只添加Ag ⁺；实验组二的反应体系中只添加Hg ²⁺；实验组三的反应体系中同时添加Hg ²⁺和Ag ⁺，其中，实验组三的反应体系做两个平行实验。表中的引物Primer-1、Primer-2、Primer-3分别依次为上述表1所述的引物Primer 1、Primer 2、Primer 3。 Four groups of reactions were carried out, in which Hg ²⁺ and Ag ⁺ were not added to the reaction system of the negative control group; only Ag ⁺ was added to the reaction system of the experimental group 1; only Hg ²⁺ was added to the reaction system of the experimental group ² ; Hg ²⁺ and Ag ⁺ were simultaneously added to the reaction system, and the reaction system of the experimental group 3 was subjected to two parallel experiments. The primers Primer-1, Primer-2, and Primer-3 in the table are the primers Primer 1, Primer 2, and Primer 3 described in Table 1 above, respectively.

2)双重超快速PCR反应过程：2) Double ultra-fast PCR reaction process:

按照表2，在冰上配制10微升反应体系，迅速置于步骤(二)搭建的超快速PCR反应装置中进行温度控制，温度控制及循环数见表3：According to Table 2, 10 μl of the reaction system was prepared on ice and rapidly placed in the ultra-fast PCR reaction device constructed in the step (2) for temperature control. The temperature control and cycle number are shown in Table 3:

表3table 3

超快速PCR反应完成后，使用本实验室合成的磁珠“home-made XP beads”根据产物片段的大小对反应体系进行纯化，获得只有金属离子结合扩增的产物。After the ultra-fast PCR reaction was completed, the reaction system was purified according to the size of the product fragment using the magnetic beads "home-made XP beads" synthesized in the laboratory to obtain a product which only undergoes metal ion binding amplification.

本实验室合成磁珠“home-made XP beads”是根据“磁珠Sera-magSpeedBeads”进行PEG-8000、NaCl、Tris-HCl、EDTA的处理得到，具体制备方法见文献：Tian,Jingjing,et al."Visual Single Cell Detection of Dual-Pathogens based on Multiplex Super PCR(MS-PCR)and Asymmetric Tailing HCR(AT-HCR)."Sensors and Actuators B:Chemical(2018).Available online 3 January 2018。The synthetic magnetic beads "home-made XP beads" in this laboratory are obtained by processing PEG-8000, NaCl, Tris-HCl and EDTA according to "Magnetic beads Sera-magSpeedBeads". The specific preparation method can be found in the literature: Tian, Jingjing, et al "Visual Single Cell Detection of Dual-Pathogens based on Multiplex Super PCR (MS-PCR) and Asymmetric Tailing HCR (AT-HCR). "Sensors and Actuators B: Chemical (2018). Available online 3 January 2018.

3)双重超快速PCR反应的扩增效果验证：3) Verification of amplification results of dual ultra-fast PCR reactions:

完成上述双重超快速PCR反应过程后，使用2％溴化乙锭预染色的琼脂糖凝胶电泳验证双重超快速PCR反应体系的扩增效果，电泳条件：120V for 25min，拍照***：Molecular Imager Gel Doc XR(Bio-Rad)。After the above double ultra-fast PCR reaction process was completed, the amplification effect of the dual ultra-fast PCR reaction system was verified by a 2% ethidium bromide pre-stained agarose gel electrophoresis. Electrophoresis conditions: 120 V for 25 min, photo system: Molecular Imager Gel Doc XR (Bio-Rad).

双重超快速PCR反应的扩增效果验证结果见图2。图2a结果表明：在Hg ²⁺、Ag ⁺分别存在或者同时存在时，本发明建立的双重超快速PCR反应体系和反应过程均实现了模板的扩增；同时由图2b的结果可以看出，磁珠起到了很好的去除杂质的作用。 The results of the amplification effect of the dual ultrafast PCR reaction are shown in Fig. 2. The results of Fig. 2a show that the double ultra-fast PCR reaction system and the reaction process established by the present invention achieve template amplification when Hg ²⁺ and Ag ⁺ are respectively present or simultaneously, and it can be seen from the results of Fig. 2b that The magnetic beads act as a good impurity removal agent.

(四)酶联核酸自组装显色模块的建立与Hg ²⁺和Ag ⁺的双重可视化检测 (IV) Establishment of enzyme-linked nucleic acid self-assembly color-developing module and dual visual detection of Hg ²⁺ and Ag ⁺

1)灵敏度实验1) Sensitivity experiment

Hg ²⁺和Ag ⁺各自的标准曲线的绘制：将上述表1所列的4条发夹探针(HairpinProbe)：Hairpin 1，Hairpin 2，Hairpin 3，Hairpin 4分别用超纯水溶解至100 μM，于95℃加热5min，后缓慢降至室温；按照上述步骤(三)中所述的双重超快速PCR反应体系和反应过程(包括磁珠纯化步骤)完成反应，不同的是：将表2所述反应体系中，只加入Hg ²⁺或只加入Ag ⁺，且使得不同反应体系中Hg ²⁺的终浓度依次为10pM、100pM、200pM、300pM、500pM、800pM、1000pM；Ag+的终浓度依次为10pM、100pM、200pM、400pM、800pM、1000pM；此外，加入Hg ²⁺的反应体系中不加表2中的Primer-3，加入Ag ⁺的反应体系中不加表2中的Primer-1。 Drawing of the respective standard curves of Hg ²⁺ and Ag ⁺ : The four hairpin probes listed in Table 1 above (HairpinProbe): Hairpin 1, Hairpin 2, Hairpin 3, Hairpin 4 were dissolved in ultrapure water to 100 μM, respectively. , heating at 95 ° C for 5 min, then slowly to room temperature; according to the double ultra-fast PCR reaction system and reaction process (including magnetic bead purification step) described in the above step (3), the difference is: Table 2 In the reaction system, only Hg ²⁺ or only Ag ⁺ is added, and the final concentration of Hg ²⁺ in different reaction systems is 10pM, 100pM, 200pM, 300pM, 500pM, 800pM, 1000pM; the final concentration of Ag+ is 10 pM, 100 pM, 200 pM, 400 pM, 800 pM, and 1000 pM; in addition, the Primer-3 in Table 2 was not added to the reaction system in which Hg ²⁺ was added, and the Primer-1 in Table 2 was not added to the reaction system in which Ag ⁺ was added.

将上述加有不同浓度的Hg ²⁺或Ag ⁺的多个反应体系完成反应后，在反应前加Hg ²⁺的反应体系中加入上述制备的Hairpin 1、Hairpin 2的超纯水溶液，在反应前加Ag+的反应体系中加入上述制备的Hairpin3、Hairpin 4的超纯水溶液，然后再分别在每个反应体系中加入自组装缓冲液(8mM Na ₂HPO ₄,2.5mM NaH ₂PO ₄,0.15M NaCl,2mM MgCl ₂,pH 7.4)，使得每个反应体系的总体积为50μL，并使每份中每一种发夹探针(HairpinProbe)的终浓度为2μM；所得所有反应体系均于37℃孵育20min，得HCR产物；然后再使用表面修饰了一层亲酶链核素的磁珠

MyOne ^TM Streptavidin T1去结合带生物素标记的HCR双链产物，去除未连接到HCR双链产物上的发夹探针等杂质，最后通过高盐洗脱去除磁珠，得HCR纯化后产物。 After the above reaction processes with different concentrations of Hg ²⁺ or Ag ⁺ are completed, the ultrapure aqueous solution of Hairpin 1 and Hairpin 2 prepared above is added to the reaction system in which Hg ²⁺ is added before the reaction, before the reaction. The ultra-pure aqueous solution of Hairpin 3 and Hairpin 4 prepared above was added to the reaction system with Ag+ addition, and then self-assembly buffer (8 mM Na ₂ HPO ₄ , 2.5 mM NaH ₂ PO ₄ , 0.15 M NaCl) was added to each reaction system. , 2 mM MgCl ₂ , pH 7.4), so that the total volume of each reaction system was 50 μL, and the final concentration of each hairpin probe (HairpinProbe) was 2 μM; each reaction system was incubated at 37 ° C. 20 min, the HCR product was obtained; then the magnetic beads modified with a layer of pro-enzyme chain were used.

MyOne ^TM Streptavidin T1 to binding with biotin-labeled double-stranded product HCR, impurity removal is not connected to the HCR hairpin probes double stranded product, and finally removing the beads by a high salt elution to yield the purified product HCR.

建立TdT酶催化的功能化核酸自组装体系，体系包括：1×TdT反应缓冲液(赛默飞科技商品),0.4mM dATP,0.6mMdGTP,20U/μL TdT酶和50μL HCR纯化后产物，于37℃反应20min后，于75℃加热10min终止酶促反应形成富G序列；取10μL酶促反应产物，加入1μL氯高铁血红素(hemin)储液(10μM)，32μL G-四链体诱导缓冲液(100mM 2-(4-morpholine)ethanesulfonic acid(MES),40mMKCl,与0.05％Triton X-100,pH 5.5)，23μL超纯水；于37℃孵育20min形成G四链体；加入8μL ABTS2-储液(20mM)与8μL过氧化氢(H ₂O ₂)储液(20mM)于室温避光孵育5min。 Establish a functionalized nucleic acid self-assembly system catalyzed by TdT enzyme, including: 1×TdT reaction buffer (Semertech), 0.4 mM dATP, 0.6 mM dGTP, 20 U/μL TdT enzyme and 50 μL HCR purified product, at 37 After reacting for 20 min at °C, the enzymatic reaction was terminated by heating at 75 °C for 10 min to form a G-rich sequence; 10 μL of the enzymatic reaction product was added, and 1 μL of hemin stock solution (10 μM) was added, and 32 μL of G-quadruplex induction buffer was added. (100 mM 2-(4-morpholine) ethanesulfonic acid (MES), 40 mM KCl, with 0.05% Triton X-100, pH 5.5), 23 μL of ultrapure water; incubated at 37 ° C for 20 min to form a G quadruplex; add 8 μL of ABTS2-storage The solution (20 mM) was incubated with 8 μL of hydrogen peroxide (H ₂ O ₂ ) stock solution (20 mM) for 5 min at room temperature in the dark.

反应完成后利用分光光度计检测反应液在415nm的OD值，绘制Hg ²⁺和Ag ⁺各自的标准曲线，绘制结果如图3和图4所示。 After the completion of the reaction, the OD value of the reaction solution at 415 nm was measured by a spectrophotometer, and the respective standard curves of Hg ²⁺ and Ag ^{+ were} plotted, and the results are shown in FIGS. 3 and 4.

最低检测限的确定：Determination of the minimum detection limit:

根据绘制的标准曲线与3σ原则，确定Hg ²⁺的检测限分别为1.3pM，Ag ⁺的检测限为2.5pM，说明本发明所建立的检测新方法灵敏度高。 According to the drawn standard curve and the 3σ principle, the detection limit of Hg ²⁺ is determined to be 1.3 pM, and the detection limit of Ag ⁺ is 2.5 pM, indicating that the new detection method established by the invention has high sensitivity.

2)准确度实验2) Accuracy experiment

加标回收检测实验：Standardized recycling test:

按照上述步骤(三)中所述的双重超快速PCR反应体系和反应过程完成反According to the double ultra-fast PCR reaction system and the reaction process described in the above step (3)

应，不同的是：将表2所述反应体系中，只加入终浓度为400pM的Hg ²⁺，不加表2中的Primer-3和Ag ⁺，反应后按照上述灵敏度实验所述步骤进行显色，完成后，根据所制作的标准曲线，计算得出本发明所建立的方法检测出的Hg ²⁺的浓度为(400±5.33)pM，检测值接近加标量，说明本发明所建立的检测新方法检测结果可靠，准确度高。 Should be different, in the reaction system described in Table 2, only Hg ²⁺ with a final concentration of 400 pM was added, without the addition of Primer-3 and Ag ^{+ in} Table 2, and the reaction was followed by the steps described in the above sensitivity experiment. Color, after completion, according to the prepared standard curve, the concentration of Hg ²⁺ detected by the method established by the present invention is calculated to be (400±5.33) pM, and the detection value is close to the scalar quantity, indicating the detection established by the present invention. The new method has reliable detection results and high accuracy.

按照上述步骤(三)中所述的双重超快速PCR反应体系和反应过程完成反应，不同的是：将表2所述反应体系中，只加入终浓度为500pM的Ag ⁺，不加表2中的Primer-1和Hg ²⁺，反应后按照上述灵敏度实验所述步骤进行显色，完成后，根据所制作的标准曲线，计算得出本发明所建立的方法检测出的Ag ⁺的浓度，检测结果见表4。 According to the double ultra-fast PCR reaction system and the reaction process described in the above step (3), the reaction is completed, except that only the final concentration of 500 pM of Ag ⁺ is added to the reaction system described in Table 2, without adding Table 2 Primer-1 and Hg ^{2+ are} reacted and then developed according to the steps described in the above sensitivity experiment. After completion, according to the prepared standard curve, the concentration of Ag ⁺ detected by the method established by the present invention is calculated and detected. The results are shown in Table 4.

表4中本发明建立的新方法的检测值(加标回收值)接近加标量，说明本发明所建立的检测新方法检测结果可靠，准确度高。The detection value (spiked recovery value) of the new method established in the present invention in Table 4 is close to the scalar quantity, indicating that the detection method established by the invention has reliable detection results and high accuracy.

表4Table 4

3)特异性实验3) Specificity experiment

将表2所列的PCR超快速反应体系中加入终浓度均为500pM的Hg ²⁺与Cu ²⁺的实验组计为实验组a；将反应体系中加入终浓度均为500pM的Ag ⁺和Mg ²⁺的实验组计为实验组b；反应体系中加入终浓度均为500pM的Hg ²⁺和Ag+的实验组计为实验组c；按照上述步骤(三)所述的双重超快速PCR反应(除体系中金属离子的种类和浓度做相应的替换外，其它均一致)进行3组(实验组a、实验组b、实验组c)反应体系的双重超快速PCR反应，每组做4个相同的反应。 The ultra-fast PCR listed in Table 2 was added to the reaction system a final concentration of 500pM of Hg ²⁺ and Cu ²⁺ experimental group in terms of the experimental group a; was added to a final concentration of 500pM Mg and Ag ⁺ in the reaction system The experimental group of ²⁺ was counted as experimental group b; the experimental group containing Hg ²⁺ and Ag+ with a final concentration of 500 pM was counted as experimental group c; the double ultra-fast PCR reaction according to the above step (3) was Except for the type and concentration of metal ions in the system, the other ones are consistent.) Double ultra-fast PCR reactions of three groups (experimental group a, experimental group b, experimental group c), and four identical in each group. Reaction.

将上述表1所列的4条发夹探针(Hairpin Probe)：Hairpin 1，Hairpin 2，Hairpin 3，Hairpin 4分别用超纯水溶解至100μM，于95℃加热5min，后缓慢降至室温备用。The four hairpin probes listed in Table 1 above (Hairpin Probe): Hairpin 1, Hairpin 2, Hairpin 3, Hairpin 4 were dissolved in ultrapure water to 100 μM, heated at 95 ° C for 5 min, and then slowly cooled to room temperature. .

分别将完成双重超快速PCR反应的每个反应体系(10ul体系)加入到显色反应体系中。每组(实验组a、实验组b、实验组c)反应体系的第一个反应体系分别加入到3个编号均为1的微孔1中(Hairpin 1与Hairpin 2的超纯水溶液提前溶解在微孔1中)，每组反应体系的第二个反应体系分别加入到3个编号均为2的微孔2中(Hairpin 3与Hairpin 4的超纯水溶液提前溶解在微孔2中)，每组反应体系的其余两个反应体系分别添加到3个微孔3与3个微孔4中(微孔3与微孔4中不放置任何Hairpin作为阴性对照)，再分别在每个微孔中加入自组装缓冲液(8mM Na ₂HPO _4,2.5mM NaH ₂PO ₄,0.15M NaCl,2mM MgCl ₂,pH 7.4)，并使每一种发夹探针(Hairpin Probe)的终浓度为2μM，每个微孔均为50微升，所有微孔于37℃孵育20min，经磁珠分别纯化后得HCR纯化产物。 Each reaction system (10 ul system) which completed the double ultra-fast PCR reaction was separately added to the color reaction system. The first reaction system of each group (experimental group a, experimental group b, experimental group c) was added to three microwells numbered 1 (Hairpin 1 and Hairpin 2 ultra-pure aqueous solution were dissolved in advance). In microwell 1), the second reaction system of each reaction system is added to three micropores 2 of number 2 (Hairpin 3 and Hairpin 4 ultra-pure aqueous solution are dissolved in micropores 2 in advance), each The remaining two reaction systems of the group reaction system were added to 3

micropores

3 and 3 micropores 4 respectively (no Hairpin was placed in microwell 3 and microwell 4 as a negative control), and then in each microwell. Self-assembly buffer (8 mM Na ₂ HPO _4, 2.5 mM NaH ₂ PO ₄ , 0.15 M NaCl, 2 mM MgCl ₂ , pH 7.4) was added, and the final concentration of each hairpin probe (Hairpin Probe) was 2 μM. Each microwell was 50 microliters, and all the microwells were incubated at 37 ° C for 20 min, and purified by magnetic beads to obtain HCR purified product.

将上述HCR纯化产物分别加到含有1×TdT反应缓冲液(赛默飞科技商品),0.4mM dATP,0.6mM dGTP,20U/μL TdT酶的微孔中，于37℃反应20min后，于75℃加热10min终止酶促反应形成富G序列；在酶促反应产物中加入1μLhemin储液(10μM)，32μL G-四链体诱导缓冲液(100mM 2-(4-morpholine)ethanesulfonic acid(MES),40mMKCl,与0.05％Triton X-100,pH 5.5)，23μL超纯水；于37℃孵育20min形成G四链体；加入8μL ABTS2-储液(20mM)与8μL过氧化氢(H ₂O ₂)储液(20mM)于室温避光孵育5min。 The above HCR purified product was separately added to a microwell containing 1×TdT reaction buffer (Semertech), 0.4 mM dATP, 0.6 mM dGTP, 20 U/μL TdT enzyme, and reacted at 37 ° C for 20 min, at 75 The enzymatic reaction was terminated by heating at °C for 10 min to form a G-rich sequence; 1 μL of hemin stock solution (10 μM) and 32 μL of G-quadruplex induction buffer (100 mM 2-(4-morpholine) ethanesulfonic acid (MES) were added to the enzymatic reaction product. 40 mM KCl, with 0.05% Triton X-100, pH 5.5), 23 μL of ultrapure water; incubation at 37 ° C for 20 min to form a G quadruplex; add 8 μL of ABTS 2 stock solution (20 mM) and 8 μL of hydrogen peroxide (H ₂ O ₂ ) Stock solutions (20 mM) were incubated for 5 min at room temperature in the dark.

实验结果如图5所示，图5结果表明本发明所建立的检测方法和生物传感器对Cu ²⁺、Mg ²⁺无交叉反应，可同时实现Hg ²⁺和Ag ⁺的双重特异性检测。 The experimental results are shown in Fig. 5. The results of Fig. 5 show that the detection method and biosensor established by the invention have no cross-reaction with Cu ²⁺ and Mg ²⁺ , and can simultaneously achieve dual specific detection of Hg ²⁺ and Ag ⁺ .

以上所述实施例仅表达了本发明的实施方式，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，但凡采用等同替换或等效变换的形式所获得的技术方案，均应落在本发明的保护范围之内。The embodiments described above are only illustrative of the embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention, but the technical solutions obtained in the form of equivalent substitution or equivalent transformation. All should fall within the scope of protection of the present invention.

Claims

一种金属的检测方法，其特征在于，所述方法包括体外核酸扩增技术,所述体外核酸扩增技术的反应体系包括下游引物、模板和至少两种上游引物；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；所述下游引物包括可特异性扩增所述模板的核苷酸序列。A method for detecting a metal, comprising: an in vitro nucleic acid amplification technique, wherein the reaction system of the in vitro nucleic acid amplification technique comprises a downstream primer, a template, and at least two upstream primers; the at least two upstream primers The base of at least one nucleotide of each of the respective nucleotide sequences is different; each of the upstream primers of the at least two upstream primers comprises: a complementary sequence A, a tether, a complementary sequence B, and a specific expansion Increasing the nucleotide sequence of the template and a nucleotide that can bind to the metal to be tested; the linking arm is located between the complementary sequence A and the complementary sequence B, the core capable of specifically amplifying the template a nucleotide sequence and a nucleotide which can bind to the metal to be detected are located at the 5' or 3' end of the upstream primer; the nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inverted Complementary; the tether comprises a structure that inhibits polymerase binding and/or a structure that inhibits extension of a new strand during nucleic acid amplification in vitro; the downstream primer comprises a nucleotide sequence that specifically amplifies the template.
根据权利要求1所述的方法，其特征在于，所述方法还包括下述1)-3)中的至少一种：The method of claim 1 wherein said method further comprises at least one of the following 1) - 3):

1)所述体外核酸扩增技术包括超快速PCR反应，所述超快速PCR反应的反应过程包括：90-98℃，2-6s；50-60℃，2-8s；共20-40个循环；1) The in vitro nucleic acid amplification technique comprises an ultra-fast PCR reaction comprising: 90-98 ° C, 2-6 s; 50-60 ° C, 2-8 s; a total of 20-40 cycles ;

2)所述连接臂包括具长链结构的化合物；2) the connecting arm comprises a compound having a long chain structure;

3)所述可与待测金属结合的核苷酸具体包括含胸腺嘧啶或胞嘧啶的核苷酸。3) The nucleotide which can bind to the metal to be tested specifically includes a thymine- or cytosine-containing nucleotide.
根据权利要求1和/或2所述的方法，其特征在于，所述方法还包括下述1)-8)中的至少一种：The method according to claim 1 and/or 2, characterized in that the method further comprises at least one of the following 1)-8):

1)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列通过连接臂连接后得到的引物；1) one of the at least two upstream primers comprising: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing through a tether;

2)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；2) One of the at least two upstream primers comprises: substituting a nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing by one or several nucleotides and/ Or a primer obtained by deleting and/or adding a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing, which are ligated by a tether;

3)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物；3) one of the at least two upstream primers comprises: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether;

4)所述至少两种上游引物中的一种包括：将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；4) One of the at least two upstream primers comprises: substituting a nucleotide sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing by one or several nucleotides and/ Or a primer obtained by deleting and/or adding a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing;

5)所述下游引物包括序列表中SEQ ID NO：3所示的核苷酸序列；5) the downstream primer comprises the nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing;

6)所述下游引物包括将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；6) The downstream primer comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing and SEQ ID NO: 3 in the Sequence Listing a nucleotide sequence having the same function as the nucleotide sequence shown;

7)所述模板包括序列表中SEQ ID NO：6所示的核苷酸序列；7) the template comprises the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;

8)所述模板包括将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列。8) The template comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing and SEQ ID NO: 6 in the Sequence Listing. A nucleotide sequence having the same function as a nucleotide sequence.
根据权利要求1、2和/或3所述的方法，其特征在于，所述方法还包括核酸自组装显色反应,所述核酸自组装显色反应的反应体系包括发卡序列，所述发卡序列包括发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补和/或反向互补。The method according to claim 1, 2 and/or 3, characterized in that the method further comprises a nucleic acid self-assembly color reaction, the reaction system of the nucleic acid self-assembly color reaction comprising a hairpin sequence, the hairpin sequence Including a card issue sequence 1 and/or a card issue sequence 2, the card issue sequence 1 includes: a complementary sequence C, a complementary sequence D and more than 3 arbitrary nucleotides, wherein the three or more arbitrary nucleotides are located in the card issuance sequence 5' and/or 3'; the cardinal sequence 2 comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Complementary sequence A and/or complementary sequence B is complementary and/or inversely complementary, and said complementary sequence C is complementary and/or inversely complementary to said complementary sequence E.
根据权利要求4所述的方法，其特征在于，所述发卡序列包括下述1)-4)中的至少一种：The method of claim 4 wherein said card issuance sequence comprises at least one of the following 1)-4):

1)所述发卡序列一包括序列表中SEQ ID NO：7所示的核苷酸序列和/或SEQ ID NO：7所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：7所示核苷酸序列具有相同功能的核苷酸序列；1) The hairpin sequence 1 comprises the nucleotide sequence shown by SEQ ID NO: 7 in the sequence listing and/or the nucleotide sequence shown in SEQ ID NO: 7 is substituted by one or several nucleotides and/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing;

2)所述发卡序列二包括序列表中SEQ ID NO：8所示的核苷酸序列和/或SEQ ID NO：8所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：8所示核苷酸序列具有相同功能的核苷酸序列；2) The hairpin sequence 2 comprises a nucleotide sequence represented by SEQ ID NO: 8 in the sequence listing and/or a nucleotide sequence represented by SEQ ID NO: 8 substituted by one or several nucleotides and/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing;

3)所述发卡序列一包括将序列表中SEQ ID NO：9所示的核苷酸序列和/或SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；3) The hairpin sequence 1 comprises a nucleotide sequence represented by SEQ ID NO: 9 in the sequence listing and/or a nucleotide sequence represented by SEQ ID NO: 9 by one or several nucleotide substitutions and/or Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;

4)所述发卡序列二包括将序列表中SEQ ID NO：10所示的核苷酸序列和/或SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列。4) The hairpin sequence two comprises substituting one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 10. Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing.
根据权利要求4和/或5所述的方法，其特征在于，所述核酸自组装显色反应还包括下述1)-2)中所述的至少一种：The method according to claim 4 and/or 5, wherein the nucleic acid self-assembly color reaction further comprises at least one of the following 1) to 2):

1)将权利要求1、2和/或3所述方法制备得到的至少一种反应体系与权利要求4和/或5所述方法中的至少一种发卡序列混合，于37℃孵育20min；再加入TdT酶反应体系，37℃反应20min后，于75℃加热10min；然后再加入氯高铁血红素和G-四链体诱导缓冲液于37℃孵育20min；最后加入ABTS2-和过氧化氢进行显色反应；1) mixing at least one reaction system prepared by the method of claim 1, 2 and/or 3 with at least one of the hairpin sequences of the methods of claims 4 and/or 5, incubating at 37 ° C for 20 min; Add TdT enzyme reaction system, react at 37 ° C for 20 min, then heat at 75 ° C for 10 min; then add hemin and G-quadruplex induction buffer for 20 min at 37 ° C; finally add ABTS2- and hydrogen peroxide to display Color reaction

2)所述核酸自组装显色反应的反应体系中，所述发卡序列一或发卡序列二的终浓度均为2μM。2) In the reaction system of the nucleic acid self-assembly color reaction, the final concentration of the hairpin sequence or the hairpin sequence 2 is 2 μM.
一种用于检测金属的试剂盒和/或生物传感器，其特征在于，所述试剂盒和/或生物传感器包括下述1)-6)所述中的至少一种：A kit and/or biosensor for detecting metal, characterized in that the kit and/or biosensor comprises at least one of the following 1) to 6):

1)包括至少两种上游引物，所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；1) comprising at least two upstream primers, the bases of at least one of the nucleotide sequences of the at least two upstream primers being different; each of the at least two upstream primers being upstream The primer comprises: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence which can specifically amplify the template, and a nucleotide which can bind to the metal to be tested; the tether is located in the complementary sequence A and Between the complementary sequences B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be detected are located at the 5' end or the 3' end of the upstream primer; the complementary sequence A and the nucleotide sequence of the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or a structure that inhibits new strand extension during nucleic acid amplification in vitro;

2)包括模板，所述模板至少包括互补序列G、互补序列H和序列I；其中，所述互补序列G、互补序列H分别与至少两种上游引物中的可特异性扩增所述模板的核苷酸序列互补和/或反向互补；所述序列I的核苷酸序列与下游引物中的可特异性扩增所述模板的核苷酸序列相同；2) comprising a template comprising at least a complementary sequence G, a complementary sequence H and a sequence I; wherein the complementary sequence G, the complementary sequence H and the at least two upstream primers respectively specifically amplify the template The nucleotide sequence is complementary and/or reverse complementary; the nucleotide sequence of the sequence I is identical to the nucleotide sequence of the downstream primer that specifically amplifies the template;

3)包括下游引物、模板和至少两种上游引物；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；所述下游引物包括可特异性扩增所述模板的核苷酸序列；3) comprising a downstream primer, a template and at least two upstream primers; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; the at least two upstream primers Each of the upstream primers includes: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; Between the complementary sequence A and the complementary sequence B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be tested are located at the 5' end or the 3' end of the upstream primer. The nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits new strands during nucleic acid amplification in vitro; An extended structure; the downstream primer comprising a nucleotide sequence that specifically amplifies the template;

4)包括至少两种上游引物和发卡序列；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；4) comprising at least two upstream primers and a hairpin sequence; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; of the at least two upstream primers Each upstream primer comprises: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; the tether is located at the complementary Between sequence A and complementary sequence B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be detected are located at the 5' end or the 3' end of the upstream primer; The nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits new strand extension during nucleic acid amplification in vitro; structure;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；The card-issuing sequence includes: a card-issuing sequence one and/or a card-issuing sequence two, the card-issuing sequence one comprising: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, and the three or more arbitrary nucleotides Located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, said complementary sequence C being complementary or oppositely complementary to said complementary sequence E;

5)包括至少两种上游引物、模板和发卡序列；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；5) comprising at least two upstream primers, a template and a hairpin sequence; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; the at least two upstream primers Each of the upstream primers includes: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; Between the complementary sequence A and the complementary sequence B, the nucleotide sequence which can specifically amplify the template and the nucleotide which can bind to the metal to be tested are located at the 5' end or the 3' end of the upstream primer. The nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits new strands during nucleic acid amplification in vitro; Extended structure

所述模板至少包括互补序列G、互补序列H和序列I；其中，所述互补序列G、互补序列H分别与至少两种上游引物中的可特异性扩增所述模板的核苷酸序列互补和/或反向互补；所述序列I的核苷酸序列与下游引物中的可特异性扩增所述模板的核苷酸序列相同；The template comprises at least a complementary sequence G, a complementary sequence H and a sequence I; wherein the complementary sequence G and the complementary sequence H are complementary to nucleotide sequences of at least two upstream primers that specifically amplify the template, respectively And/or reverse complement; the nucleotide sequence of the sequence I is identical to the nucleotide sequence of the downstream primer that specifically amplifies the template;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；The card-issuing sequence includes: a card-issuing sequence one and/or a card-issuing sequence two, the card-issuing sequence one comprising: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, and the three or more arbitrary nucleotides Located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, said complementary sequence C being complementary or oppositely complementary to said complementary sequence E;

6)包括下游引物、模板、至少两种上游引物和发卡序列；所述至少两种上游引物的各自的核苷酸序列中至少有一个核苷酸的碱基是不同的；所述至少两种上游引物的中的每种上游引物包括：互补序列A、连接臂、互补序列B、可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增所述模板的核苷酸序列和可与待测金属结合的核苷酸位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；6) comprising a downstream primer, a template, at least two upstream primers, and a hairpin sequence; the bases of at least one of the nucleotide sequences of the at least two upstream primers are different; the at least two Each of the upstream primers includes: a complementary sequence A, a tether, a complementary sequence B, a nucleotide sequence that can specifically amplify the template, and a nucleotide that can bind to the metal to be tested; An arm is located between the complementary sequence A and the complementary sequence B, and the nucleotide sequence capable of specifically amplifying the template and the nucleotide which can bind to the metal to be detected are located at the 5' end of the upstream primer or a 3' end; the nucleotide sequence of the complementary sequence A and the complementary sequence B are complementary and/or inversely complementary; the linker comprises a structure that inhibits polymerase binding and/or inhibits nucleic acid amplification in vitro The structure of the extension of the new chain;

所述下游引物包括可特异性扩增所述模板的核苷酸序列；The downstream primer includes a nucleotide sequence that specifically amplifies the template;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；The card-issuing sequence includes: a card-issuing sequence one and/or a card-issuing sequence two, the card-issuing sequence one comprising: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, and the three or more arbitrary nucleotides Located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, said complementary sequence C being complementary or oppositely complementary to said complementary sequence E;

具体的，所述可与待测金属结合的核苷酸具体包括含胸腺嘧啶或胞嘧啶的核苷酸；Specifically, the nucleotide that can bind to the metal to be tested specifically includes a thymine- or cytosine-containing nucleotide;

具体的，所述至少两种上游引物中的一种包括下述1)-4)中的至少一种：Specifically, one of the at least two upstream primers includes at least one of the following 1)-4):

1)将序列表中SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列通过连接臂连接后得到的引物；1) a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing via a tether;

2)将序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；2) subjecting the nucleotide sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing to one or several nucleotide substitutions and/or deletions and/or additions and to the SEQ ID in the Sequence Listing a primer obtained by linking a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 2 to the nucleotide sequence shown by SEQ ID NO: 2;

3)将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物；3) a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether;

4)将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；4) subjecting the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing to one or several nucleotide substitutions and/or deletions and/or additions and to the SEQ ID in the Sequence Listing a primer obtained by linking a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 5 to the nucleotide sequence shown by SEQ ID NO: 5;

具体的，所述下游引物包括序列表中SEQ ID NO：3所示的核苷酸序列，和/或将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；Specifically, the downstream primer comprises the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, and/or the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing is subjected to one or several nucleotides. a nucleotide sequence having a substitution and/or deletion and/or addition and having the same function as the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

具体的，所述模板包括序列表中SEQ ID NO：6所示的核苷酸序列,和/或将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列；Specifically, the template includes the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing, and/or the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing is subjected to one or several nucleotides. a nucleotide sequence substituted and/or deleted and/or added and having the same function as the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing;

具体的，所述连接臂包括具长链结构的化合物；Specifically, the connecting arm comprises a compound having a long chain structure;

具体的，所述连接臂为oxyethyleneglycol bridge，oxyethyleneglycol的化学结构为：Specifically, the connecting arm is an oxyethyleneglycol bridge, and the chemical structure of the oxyethyleneglycol is:

具体的，所述至少两种上游引物中的一种包括：AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT或TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；Specifically, one of the at least two upstream primers comprises: AGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT or TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTGCACATAACACCCC;

具体的，所述下游引物还包括在所述下游引物上标记免疫标记；所述免疫标记包括生物素；所述生物素标记在所述下游引物的5’端第一个核苷酸上；所述标记方法属于现有技术；Specifically, the downstream primer further comprises marking an immunolabel on the downstream primer; the immunolabel comprises biotin; the biotin label is on the first nucleotide of the 5' end of the downstream primer; The marking method belongs to the prior art;

具体的，所述发卡序列包括下述1)-4)中的至少一种：Specifically, the card issue sequence includes at least one of the following 1)-4):

1)所述发卡序列一包括序列表中SEQ ID NO：7所示的核苷酸序列，和/或将SEQ ID NO：7所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：7所示核苷酸序列具有相同功能的核苷酸序列；1) the hairpin sequence 1 comprises the nucleotide sequence shown in SEQ ID NO: 7 in the Sequence Listing, and/or the nucleotide sequence shown in SEQ ID NO: 7 is substituted by one or several nucleotides and a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing;

2)所述发卡序列二包括序列表中SEQ ID NO：8所示的核苷酸序列，和/或将SEQ ID NO：8所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：8所示核苷酸序列具有相同功能的核苷酸序列；2) The card-issuing sequence II comprises the nucleotide sequence shown in SEQ ID NO: 8 in the Sequence Listing, and/or the nucleotide sequence shown in SEQ ID NO: 8 is substituted by one or several nucleotides and a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing;

3)所述发卡序列一包括将序列表中SEQ ID NO：9所示的核苷酸序列，和/或将SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；3) The card-issuing sequence one comprises the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, and/or the nucleotide sequence shown in SEQ ID NO: 9 is substituted by one or several nucleotides. And/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;

4)所述发卡序列二包括将序列表中SEQ ID NO：10所示的核苷酸序列，和/或将SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列；4) The hairpin sequence 2 comprises the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing, and/or the nucleotide sequence shown by SEQ ID NO: 10 is substituted by one or several nucleotides. And/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing;

具体的，所述试剂盒和/或生物传感器包括下述1)-5)：Specifically, the kit and/or biosensor includes the following 1)-5):

1)TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；1) TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC;

2)AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT；2) AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT;

3)序列表中SEQ ID NO：6所示的核苷酸序列；3) the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;

4)Biotin-TCATCGCACCGTCAAAGGAACC；4) Biotin-TCATCGCACCGTCAAAGGAACC;

5)序列表中SEQ ID NO：7所示的核苷酸序列、序列表中SEQ ID NO：8所示的核苷酸序列、序列表中SEQ ID NO：9所示的核苷酸序列和序列表中SEQ ID NO：10所示的核苷酸序列。5) the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing, and The nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing.
一种银的检测方法，其特征在于，所述方法包括体外核酸扩增技术,所述体外核酸扩增技术的反应体系包括上游引物、下游引物和模板，A method for detecting silver, characterized in that the method comprises an in vitro nucleic acid amplification technology, and the reaction system of the in vitro nucleic acid amplification technology comprises an upstream primer, a downstream primer and a template.

所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；The upstream primer comprises: a primer obtained by ligating the nucleotide sequence shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing via a tether, or SEQ ID NO: 4 and/or in the sequence listing. The nucleotide sequence set forth in SEQ ID NO: 5 is substituted and/or deleted and/or added by one or several nucleotides and is nucleotide represented by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing. a primer obtained by ligating a nucleotide sequence having the same function by a ligation arm; the linker comprises a structure which inhibits polymerase binding and/or a structure which inhibits elongation of a new strand during nucleic acid amplification in vitro;

所述下游引物包括：序列表中SEQ ID NO：3所示的核苷酸序列，或将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；The downstream primer comprises: the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing is substituted by one or several nucleotides and / Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

所述模板包括：序列表中SEQ ID NO：6所示的核苷酸序列，或将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列；The template includes: the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing is substituted by one or several nucleotides and/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing;

具体的，所述方法还包括下述1)-2)中的至少一种：Specifically, the method further includes at least one of the following 1)-2):

1)所述体外核酸扩增技术包括超快速PCR反应，所述超快速PCR反应的反应过程包括：90-98℃，2-6s；50-60℃，2-8s；共20-40个循环；1) The in vitro nucleic acid amplification technique comprises an ultra-fast PCR reaction comprising: 90-98 ° C, 2-6 s; 50-60 ° C, 2-8 s; a total of 20-40 cycles ;

2)所述方法还包括核酸自组装显色反应,所述核酸自组装显色反应的反应体系包括发卡序列，所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；2) The method further comprises a nucleic acid self-assembly color reaction, the nucleic acid self-assembly color reaction reaction system comprising a hairpin sequence, the card issue sequence comprising: a card issue sequence one and/or a card issue sequence two, the card issue sequence The invention comprises: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, wherein the three or more arbitrary nucleotides are located at the 5' end and/or the 3' end of the card issue sequence; Included is a complementary sequence E and a complementary sequence F; said complementary sequence D is complementary and/or inversely complementary to a complementary sequence F; said complementary sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B And the complementary sequence C is complementary or inversely complementary to the complementary sequence E;

具体的，所述发卡序列包括下述1)-2)中至少一种：Specifically, the card issuance sequence includes at least one of the following 1)-2):

1)所述发卡序列一包括将序列表中SEQ ID NO：9所示的核苷酸序列和/或SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；1) The hairpin sequence 1 comprises a nucleotide sequence represented by SEQ ID NO: 9 in the sequence listing and/or a nucleotide sequence represented by SEQ ID NO: 9 by one or several nucleotide substitutions and/or Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;

2)所述发卡序列二包括将序列表中SEQ ID NO：10所示的核苷酸序列和/或SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列。2) The hairpin sequence two comprises substituting one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 10. Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing.
一种用于检测银的试剂盒和/或生物传感器，其特征在于，所述试剂盒和/或生物传感器包括下述1)-4)所述中的至少一种：A kit and/or biosensor for detecting silver, characterized in that the kit and/or biosensor comprises at least one of the following 1) to 4):

1)包括上游引物，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；1) comprising an upstream primer comprising: a primer obtained by ligating the nucleotide sequence shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing via a tether, or SEQ ID in the Sequence Listing The nucleotide sequence shown by NO: 4 and/or SEQ ID NO: 5 is substituted and/or deleted and/or added by one or several nucleotides and is SEQ ID NO: 4 and/or SEQ ID in the Sequence Listing. NO: a primer obtained by ligating a nucleotide sequence having the same function as a nucleotide sequence of 5, which is linked by a tether; the linker comprises a structure which inhibits polymerase binding and/or inhibits an in vitro nucleic acid amplification process Chain extended structure;

2)包括上游引物、下游引物和模板，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；2) comprising an upstream primer, a downstream primer, and a template, the upstream primer comprising: a primer obtained by ligating the nucleotide sequence represented by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether, or The nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing is substituted and/or deleted and/or added by one or several nucleotides and is SEQ ID NO: 4 in the Sequence Listing. And/or a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown in SEQ ID NO: 5 by a ligation arm; the linker comprises a structure capable of inhibiting polymerase binding and/or inhibiting nucleic acid in vitro a structure in which a new chain extends during amplification;

所述下游引物包括：序列表中SEQ ID NO：3所示的核苷酸序列，或将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；The downstream primer comprises: the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing is substituted by one or several nucleotides and / Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

所述模板包括：序列表中SEQ ID NO：6所示的核苷酸序列，或将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列；The template includes: the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing is substituted by one or several nucleotides and/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing;

3)包括上游引物和发卡序列，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；3) comprising an upstream primer and a hairpin sequence, the upstream primer comprising: a primer obtained by ligating the nucleotide sequence represented by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether, or a sequence listing The nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 is substituted and/or deleted and/or added by one or several nucleotides and is SEQ ID NO: 4 and / in the Sequence Listing. Or a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown in SEQ ID NO: 5 by a ligation arm; the linker comprises a structure which inhibits polymerase binding and/or inhibits nucleic acid amplification in vitro The structure of the new chain extension in the process;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；The card-issuing sequence includes: a card-issuing sequence one and/or a card-issuing sequence two, the card-issuing sequence one comprising: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, and the three or more arbitrary nucleotides Located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, said complementary sequence C being complementary or oppositely complementary to said complementary sequence E;

4)包括上游引物、下游引物、模板和发卡序列，所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物，或将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；4) comprising an upstream primer, a downstream primer, a template and a hairpin sequence, the upstream primer comprising: a primer obtained by ligating the nucleotide sequence shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the sequence listing through a tether Or the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing is subjected to one or several nucleotide substitutions and/or deletions and/or additions and to the SEQ ID in the Sequence Listing. a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 5 by a ligation arm; the linker comprises a structure capable of inhibiting polymerase binding and/or Inhibiting the structure of new strand extension during in vitro nucleic acid amplification;

所述下游引物包括：序列表中SEQ ID NO：3所示的核苷酸序列，或将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；The downstream primer comprises: the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing is substituted by one or several nucleotides and / Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing;

所述模板包括：序列表中SEQ ID NO：6所示的核苷酸序列，或将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列；The template includes: the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, or the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing is substituted by one or several nucleotides and/or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing;

所述发卡序列包括：发卡序列一和/或发卡序列二，所述发卡序列一包括：互补序列C、互补序列D和3个以上的任意核苷酸，所述3个以上的任意核苷酸位于所述发卡序列的5’端和/或3’端；所述发卡序列二包括互补序列E和互补序列F；所述互补序列D与互补序列F互补和/或反向互补；所述互补序列C与所述互补序列A和/或互补序列B互补和/或反向互补，所述互补序列C与所述互补序列E互补或反向互补；The card-issuing sequence includes: a card-issuing sequence one and/or a card-issuing sequence two, the card-issuing sequence one comprising: a complementary sequence C, a complementary sequence D and three or more arbitrary nucleotides, and the three or more arbitrary nucleotides Located at the 5' end and/or the 3' end of the hairpin sequence; the card issue sequence two comprises a complementary sequence E and a complementary sequence F; the complementary sequence D is complementary and/or inversely complementary to the complementary sequence F; Sequence C is complementary and/or inversely complementary to said complementary sequence A and/or complementary sequence B, said complementary sequence C being complementary or oppositely complementary to said complementary sequence E;

具体的，所述连接臂包括具长链结构的化合物；Specifically, the connecting arm comprises a compound having a long chain structure;

再具体的，所述连接臂为oxyethyleneglycol bridge，oxyethyleneglycol的化学结构为：More specifically, the connecting arm is an oxyethyleneglycol bridge, and the chemical structure of the oxyethyleneglycol is:

具体的，所述下游引物还包括在所述下游引物上标记免疫标记；所述免疫标记包括生物素；所述生物素标记在所述下游引物的5’端第一个核苷酸上；所述标记方法属于现有技术；Specifically, the downstream primer further comprises marking an immunolabel on the downstream primer; the immunolabel comprises biotin; the biotin label is on the first nucleotide of the 5' end of the downstream primer; The marking method belongs to the prior art;

具体的，所述发卡序列包括下述1)-2)中至少一种：Specifically, the card issuance sequence includes at least one of the following 1)-2):

1)所述发卡序列一包括将序列表中SEQ ID NO：9所示的核苷酸序列和/或SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；1) The hairpin sequence 1 comprises a nucleotide sequence represented by SEQ ID NO: 9 in the sequence listing and/or a nucleotide sequence represented by SEQ ID NO: 9 by one or several nucleotide substitutions and/or Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;

2)所述发卡序列二包括将序列表中SEQ ID NO：10所示的核苷酸序列和/或 SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列；2) The hairpin sequence two comprises substituting one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 10. Or a nucleotide sequence which is deleted and/or added and which has the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing;

具体的，所述试剂盒和/或生物传感器包括下述1)-4)：Specifically, the kit and/or biosensor includes the following 1)-4):

1)TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC；1) TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge-AACTATACAACCTACTACCTCATTTTTTTTTTTGCACATAACACCCC;

2)序列表中SEQ ID NO：6所示的核苷酸序列；2) the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;

3)Biotin-TCATCGCACCGTCAAAGGAACC；3) Biotin-TCATCGCACCGTCAAAGGAACC;

4)序列表中SEQ ID NO：7所示的核苷酸序列、序列表中SEQ ID NO：8所示的核苷酸序列、序列表中SEQ ID NO：9所示的核苷酸序列和序列表中SEQ ID NO：10所示的核苷酸序列；4) the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 8 in the Sequence Listing, the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing, and a nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing;

其中，oxyethyleneglycol的化学结构为：Among them, the chemical structure of oxyethyleneglycol is:
权利要求1、2、3、4、5、6和/或8所述方法、权利要求7和/或9所述试剂盒和/或生物传感器的应用。The method of claim 1, 2, 3, 4, 5, 6 and/or 8, the use of the kit and/or the biosensor of claim 7 and/or 9.