CN110218818B - 一种登革病毒基因片段sers检测试剂盒及其制备方法 - Google Patents

一种登革病毒基因片段sers检测试剂盒及其制备方法 Download PDF

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CN110218818B
CN110218818B CN201910512979.3A CN201910512979A CN110218818B CN 110218818 B CN110218818 B CN 110218818B CN 201910512979 A CN201910512979 A CN 201910512979A CN 110218818 B CN110218818 B CN 110218818B
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宋春元
汪联辉
刘洋
张晶晶
蒋新宇
董晨
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Abstract

本发明公开了一种登革病毒（DENV）基因片段SERS检测试剂盒及其制备方法。本发明还公开了一种登革病毒基因片段SERS检测试剂盒的检测方法。本发明通过测试基片上染料分子ROX的拉曼信号，实现对于DENV基因片段的特异性、高灵敏检测。对于DENV基因片段检测，第一试剂、第二试剂、第三试剂和第四试剂中的C1、L1、L2、C2、H1和H2为针对DENV基因片段设计的核苷酸序列不同的DNA链。本发明公开的检测试剂盒制备简单、检测灵敏度高、特异性好，在登革病毒检测等领域有广泛的应用前景。

Description

一种登革病毒基因片段SERS检测试剂盒及其制备方法
技术领域
本发明属于生物检测和光谱学检测领域，涉及一种登革病毒基因片段SERS检测试剂盒及其制备方法。
背景技术
登革病毒属于黄病毒科，四种不同的登革病毒类型(dengue 1，2，3，4)都能感染人体，这些血清型登革病毒可在初次感染时引起轻微发热，并有可能发展为更严重的登革热出血热(DHF)和登革热休克综合征(DSS)，从而导致死亡。过去40年来，登革热/登革出血热(DEN/DHF)在全球范围内急剧蔓延，流行频率和规模都在增加，迫切需要采取更有效的监测、预防和控制措施。在DEN/DHF流行的大多数国家，没有适当的监测作为评估疾病负担的手段，也没有足够的蚊虫控制或疫苗预防方案，缺乏现有的、可负担的、灵敏的和具体的诊断测试是影响DEN/DHF监测的主要障碍。对登革热病人的早期诊断也是病人管理的关键，可以避免使用昂贵和无效的抗生素，快速将发热病人分类到适当诊所，降低保健费用。随着抗病毒治疗的快速发展，早期诊断将是确定登革热患者的关键，使他们尽快得到适当的照顾，病例的及时诊断也将有助于社区的病媒控制活动，从而减少其进一步传播。
逆转录聚合酶链式反应(RT-PCR)和单克隆抗体捕获-酶联免疫吸附试验(MAC-ELISA)是目前世界范围内用于快速诊断登革病毒的实验室检测方法。然而两种方法都易产生假阳性结果。因此，生物传感检测方法因其简便、快速、灵敏等优势被应用于登革病毒的检测，表面增强拉曼散射(SERS)被称为超灵敏，功能强大且易于使用的基因检测工具，甚至能够实现单分子检测水平，且检测时只需少量样品，可提供具有非常窄特征峰的“指纹”光谱曲线，也在登革病毒检测领域取得了一定的进展。
杂交链式反应(HCR)和催化发夹组装(CHA)是两种经典的无酶核酸放大策略，这两种反应在提高无酶生物传感器的性能方面引起了人们的广泛关注。HCR是2004年Dirks和Pierce首次报道一种无酶的目标诱导信号放大策略，以单链DNA为触发链，可破坏长茎保护环的能量平衡，引发交替杂交链式反应，产生长的双链DNA。催化发夹组装(CHA)也是一种等温无酶核酸放大技术，目标DNA存在的情况下，可以促使两种发夹结构杂交形成双链结构，并释放出目标DNA循环使用。近年来，许多将反应物限制在紧密的空间中用以保持局部高浓度试剂和加速反应的模型被提出，在生物分析物检测中有广泛应用。
发明内容
发明目的：本发明所要解决技术问题是提供了一种登革病毒基因片段SERS检测试剂盒。
本发明还要解决的技术问题是提供了登革病毒基因片段SERS检测试剂盒的制备方法。
本发明最后要解决的技术问题是提供了登革病毒基因片段SERS检测试剂盒的检测方法。
技术方案：为了解决现有技术存在的问题，本发明采用以下技术方案：一种登革病毒基因片段SERS检测试剂盒，所述SERS检测试剂盒包括固相SERS基片、第一试剂、第二试剂、第三试剂和第四试剂，所述固相SERS基片是表面沉积有银纳米棒阵列的玻璃片，所述第一试剂包含L1-L2-C1结构，所述L1-L2-C1结构包括能与登革病毒基因片段碱基序列完全互补的发夹型结构C1、与C1部分互补的L1、以及能与L1部分碱基互补的修饰有巯基的L2，L1与L2形成L1-L2双链，L1-L2双链与C1杂交形成L1-L2-C1结构；所述第二试剂为修饰有染料分子的发夹型结构C2，所述第三试剂为修饰有染料分子的发夹型结构H1，所述第四试剂为修饰有染料分子的发夹型结构H2，其中发夹型结构H1能被C1-C2双链中的C2部分碱基打开，H2能被H1部分碱基序列打开，且H2另一部分碱基序列能打开另一发夹型结构H1，从而形成交替杂交的长的H1-H2双链。
在一些实施方式中，所述SERS检测试剂盒还包括第五试剂巯基己醇。
在一些实施方式中，针对检测DENV基因不同的片段，第一试剂中C1、L1和L2；第二试剂中的C1；第三试剂中的H1；第四试剂中的H2，它们的核苷酸序列不同。
在一些实施方式中，所述的C1、L1、L2、C2、H1、H2为人工合成。
其中，所述L1-L2-C1结构、C2、H1和H2数量多于DENV数量，向L1-L2-C1结构中加入DENV与C2，发生局域催化发夹组装(LCHA)过程，以此实现DENV链的循环放大。再加入H1和H2，发生杂交链式反应过程，以此实现染料分子信号放大。
本发明通过设计合理的核酸杂交体系，可实现目标DNA的循环使用及染料分子信号放大，进一步提高检测限。
在一些实施方式中，所述的L2的5′端修饰巯基，C2的5′端修饰染料分子，H1和H2的3′端和5′端修饰染料分子，所述染料分子为本领域常规的标记染料，包括但不限于ROX染料；
在一些实施方式中，所述固相SERS基片为银纳米棒阵列型基片(根据参考文献中物理真空蒸发沉积方法制备银纳米棒阵列，C.Y.Song，J.L.Abell，Y.P.He，S.H.Murph，Y.P.Cui，Y.P.Zhao.Gold-modified silver nanorod arrays：growth dynamics andimproved SERS properties.Journal ofMaterials Chemistry，2012，22(3)：1150-1159)，并在其表面用PDMS膜制备4×10阵列型小孔，孔径4mm，深度1mm。
在一些实施方式中，第一试剂中的C1、L1和L2为针对DENV基因片段设计的核苷酸序列不同的DNA链，L2的5′端修饰巯基，C1、L1和L2的部分核苷酸序列互补杂交形成L1-L2-C1结构；第二试剂C2为根据C1设计的5′端修饰染料分子的发夹型结构DNA。第一试剂中加入DENV与第二试剂C2，DENV打开发夹型结构形成C1-DENV双链，然后C1打开发夹型结构C2，形成C1-C2结构，并循环出DENV。
在一些实施方式中，第三试剂H1和第四试剂H2为针对C2设计的3′端和5′端修饰染料分子的核苷酸序列不同的发夹型结构DNA，C1-C2双链中的C2打开发夹型结构H1，H1打开发夹型结构H2，H2再打开另一H1，形成长链，通过L2修饰的巯基(Ag-S)将最终的带有染料分子的DNA结构固定到基片表面，实现拉曼信号的检测。
在一些实施方式中，所述C1碱基序列如SEQ ID NO：2所示，所述L1的碱基序列如SEQ ID NO：3所示，所述L2碱基序列如SEQ ID NO：4所示；所述C2碱基序列如SEQ ID NO：5所示；所述H1的碱基序列如SEQ ID NO：6所示，所述H2碱基序列如SEQ ID NO：7所示。
在一些优选实施方式中，第五试剂的浓度不同，巯基己醇的浓度为10μM-10mM，最佳浓度为1mM；当巯基己醇浓度过低时，表面封闭不完全，会有大量的非特异性吸附，使背景信号过强；当巯基己醇浓度过高时，当巯基己醇浓度过高时，在替换掉非特异性吸附的DNA后，会接着替换固定在基片上的DNA结构，使SERS强度降低。
本发明内容还包括所述的登革病毒基因片段SERS检测试剂盒的制备方法，包括以下步骤：
1)固相SERS基片的制备；
2)第一试剂的获得：根据登革病毒基因片段设计并合成对应的发夹型结构C1，再根据所述C1设计并合成对应的L1，再根据L1设计并合成L2；
3)第二试剂的获得：根据C1设计并合成发夹型结构C2；
4)第三试剂的获得：根据所述C2设计并合成对应的发夹型结构H1；
5)第四试剂的获得：根据H1设计并合成发夹型结构H2。
其中，所述C1、L1、L2、C2、H1和H2浓度相同，浓度均为1-10μM。在一些优选实施方式中，浓度为1-2μM。实验设计的C1、L1、C2、H1、H2与L2浓度对应相同，只要保证Cl、L1、C2、H1、H2的浓度足够即可。
本发明内容还包括所述的登革病毒基因片段SERS检测试剂盒的检测方法，包括以下步骤：(所有试剂用前需加热至95℃后退火至室温)
1)用超纯水多次清洗试剂盒中的固相SERS基片；并在其表面用PDMS膜制备4×10阵列型小孔，孔径4mm，深度1mm；
2)向试剂盒的第一试剂中加入检测样品与第二试剂共培养；
3)向步骤2)中加入第三试剂与第四试剂得到溶液；
4)将步骤1)得到的固相SERS基片与步骤3)的溶液共培育；
5)缓冲液冲洗步骤4)得到的基片后，用第五试剂封闭基片表面；
6)纯水冲洗步骤5)得到的基片后，进行SERS检测。
其中，所述步骤2)中的共培养条件为24-28℃，培养1.5-2h。
其中，所述步骤3)培养条件为24-28℃，1.5-2h。
其中，所述步骤4)的培养条件为25-30℃，60％-80％湿度环境，培养3-4h。
其中，所述步骤5)的培养条件为25-30℃，60％-80％湿度环境，静置培养10-20min。
作为进一步优选，步骤2)中所述第一试剂中加入待检测样品和第二试剂培养条件为25℃，培养1.5h。
作为进一步优选，步骤3)中加入第三试剂和第四试剂后培养条件为25℃，培养1.5h。
作为进一步优选，步骤4)中所述固相SERS基片与步骤3)溶液共培育为取20μL溶液滴于基片表面小孔中，培养条件为25℃，80％湿度环境，培养3h。
作为进一步优选，步骤5)中所述加入巯基己醇浓度范围为10μM-10mM，加入巯基己醇最优浓度为1mM，体积为20μL，培养条件为25℃，80％湿度环境，静置培养10min。
作为进一步优选，步骤6)所述拉曼检测为检测C2、H1和H2上标记的染料分子的拉曼信号。
其中，本发明所述的登革病毒基因片段SERS检测试剂盒的检测方法，分别加入不同浓度DENV基因片段的缓冲液，得到不同浓度基因片段对应的SERS信号强度，以DENV基因片段浓度的对数为横坐标，以SERS信号强度为纵坐标做出工作曲线。最后，通过加入待测样品溶液共培养后，最后测试得到SERS信号，对照工作曲线计算得出待测样品中流感病毒基因片段的浓度。
本发明的该试剂盒的检测浓度范围为1fM-10nM。
本发明的检测原理：向第一试剂中加入待检测样品和第二试剂，样品中的待检测DENV基因片段打开第一试剂中L1-L2-C1中的发夹型结构C1互补杂交形成C1-DENV双链，C1-DENV双链中的C1打开第二试剂发夹型结构C2形成C1-C2双链，并释放出DENV，发生LCHA反应，实现DENV循环。之后加入第三试剂发夹型结构H1和第四试剂发夹型结构H2，C1-C2双链中的C2打开发夹型结构H1，形成C2-H1双链，C2-H1双链结构中的H1打开发夹型结构H2，H2又打开另一个H1，形成长的DNA链，发生HCR反应，实现染料分子信号放大。随后，将固相SERS基片与上述试剂混合培育，溶液中的L2通过S-银键固定到基片表面，将染料分子捕获到基片上。然后将第五试剂滴加到SERS基片表面进行封闭，减少基片非特异性吸附。最后，检测拉曼信号，通过测试基片上染料分子的拉曼信号，实现对于DENV基因片段的特异性、高灵敏检测。
本发明面向登革病毒快速检测需求，结合SERS技术与信号放大技术，构建了用于登革病毒基因片段检测的SERS检测试剂盒，并公开了其检测方法。为登革病毒高灵敏、特异性检测提供了方法，也拓展SERS技术在登革病毒检测中的应用。
有益效果：与现有技术相比，本发明的优点是：本发明利用银纳米棒阵列型SERS基片作为检测基片，具有优异的SERS性能，并结合核酸信号放大技术，运用局域催化发夹组装反应(LCHA)将加入的DENV进行循环使用，并应用杂交链式反应(HCR)实现染料分子信号放大，进一步提高检测灵敏度，该传感器检测限达到几百阿摩尔级(～100aM)。同时对各种病毒基因片段检测具有的普适性。本发明通过测试基片上染料分子的拉曼信号，实现对于登革病毒基因片段的特异性、高灵敏检测。对于DENV基因片段检测，第一试剂、第二试剂、第三试剂和第四试剂中的C1、L1、L2、C2、H1、H2为针对DENV基因片段设计的核苷酸序列不同的DNA链。本发明公开的检测试剂盒制备简单、检测灵敏度高、特异性好，在登革病毒检测等领域有广泛的应用前景。
附图说明
图1是登革病毒基因片段SERS检测试剂盒工作原理图。
图2是实施例1中SERS检测试剂盒DENV基因片段巯基己醇浓度优化实验。(A)是试剂盒优化不同浓度巯基己醇对应的SERS谱图；(B)是(A)中SERS谱线1503cm-1拉曼位移处峰值统计。
图3是实施例2中SERS检测试剂盒用于缓冲液中DENV基因片段检测的工作曲线。(A)是试剂盒检测不同浓度DENV基因片段对应的SERS谱图；(B)是(A)中SERS谱线1503cm-1拉曼位移处峰值统计。
图4是实施例2中SERS检测试剂盒DENV基因片段特异性检测，(A)是试剂盒检测DENV基因片段、单碱基错配序列、双碱基错配序列、三碱基错配序列缓冲液样品的SERS谱图；(B)是(A)中SERS谱线1503cm-1拉曼位移处峰值统计。
图5是实施例2中SERS检测试剂盒均一性验证。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。以下实施例是为了对本发明作进一步详细说明，并非对发明的限制。
本发明所使用的10种DNA链片段均为人工合成得到，均由生工生物工程(上海)股份有限公司合成。第四试剂巯基己醇由Sigma-Aldrich合成。
DENV碱基序列为：
5′-TGGTGCTGTTGAATCAACAGGTTCT-3′
对应的C1、L1、L2、C2、H1、H2碱基序列为：
C1：5′-AGAACCTGTTGATTCAACAGCACCAACATAGGAGTGCTGTTGAATCAACATTTTTTTTTTTTTTTTTTTT-3′；
L1：5′-TCGTTACTTAAATGGTCAGAAATATGGGATTAACCATGGTGTTTATGATATGAAGTGTTGGAAGCIAAAAAAAAAAAAAAAAAAAA-3′；
L2：5′-SH-TTAATCCCATATTTCTGACCATTTAACGAACGAAGCTTCCAACACTTCATATCATAAACACCATGG-3′；
C2：5′-ROX-CAACAGCACTCCTATGTTGGTGCTGTTGAATCAACATAGGAGTGTT
CTCGGCAGTATCAT-3′
H1：5′-ROX-GTTCTCGGCAGTATCATTTGACACATGATACTGCCGAGAACACTCCTA-ROX-3′
H2：5′-ROX-GTGTCAAATGATACTGCCGAGAACTAGGAGTGTTCTCGGCAGTATCAT-ROX-3′
DENV基因片段特异性实验对应的单碱基错配序列(M1)、双碱基错配序列(M2)，三碱基错配(M3)碱基序列为：
单碱基错配序列(M1)：5′-TGGTGCTGTTGAATCAACAGGCTCT-3′；
双碱基错配序列(M2)：5′-TGGTGCTGTTGAGTCAACAGGCTCT-3′；
三碱基错配序列(M3)：5′-TGGAGCTGTTGAGTCAACAGGCTCT-3′；
以下实施例中银纳米棒阵列型SERS基片采用真空电子束蒸发镀膜技术制备，具体方法按文献(C.Y.Song，J.L.Abell，Y.P.He，S.H.Murph，Y.P.Cui，Y.P.Zhao.Gold-modifiedsilver nanorod arrays：growth dynamics and improved SERS properties.JournalofMaterials Chemistry，2012，22(3)：1150-1159.)中报道的方法制备。基片表面覆盖一层具有阵列型(4×10)小孔的PDMS膜，孔径4mm，深度1mm。
实施例1 DENV基因片段检测的SERS试剂盒制备及检测方法：
(1)银纳米棒阵列型SERS基片用超纯水多次冲洗；
(2)向PCR管中加入第一试剂L1和L2单链和发夹型结构C1，25℃，杂交120min；
(3)向步骤(2)得到的混合液中加入DENV和第二试剂C2，25℃，杂交90min；
(4)向步骤(3)得到的混合液中加入第三试剂H1和第四试剂H2，25℃，杂交90min；
以上过程每种DNA用量保证最终PCR管内L1、L2、C1、C2、H1和H2浓度为1μM。
(5)取步骤(4)得到的混合液(1μM)，20μL滴于SERS基片上的小孔中，在25℃，80％湿度环境中培育3h，然后用TM缓冲液(10mM磷酸盐，100mM氯化钠，pH 7.4)冲洗干净；
(6)向步骤(5)处理所得基片小孔中分别加入20μL，浓度10μM、100μM、1mM、10mM巯基己醇，培养10min后用缓冲液轻轻冲洗干净，随后进行SERS检测，测试得到染料分子的SERS谱线，参见图2。
实施例2DENV基因片段检测的SERS试剂盒制备及检测方法：
(1)银纳米棒阵列型SERS基片用超纯水多次冲洗；
(2)向PCR管中加入第一试剂L1和L2单链和发夹型结构C1，25℃，杂交120min；
(3)向步骤(2)得到的混合液中加入DENV和第二试剂C2，25℃，杂交90min；
(4)向步骤(3)得到的混合液中加入第三试剂H1和第四试剂H2，25℃，杂交90min；
以上过程每种DNA用量保证最终PCR管内L1、L2、C1、C2、H1和H2浓度为1μM。
(5)取步骤(4)得到的混合液(1μM)，20μL滴于SERS基片上的小孔中，在25℃，80％湿度环境中培育3h，然后用TM缓冲液(10mM磷酸盐，100mM氯化钠，pH 7.4)冲洗干净；
(6)向步骤(5)处理所得基片小孔中加入20μL浓度1mM巯基己醇，培养10min后用缓冲液轻轻冲洗干净，随后进行SERS检测，测试得到染料分子的SERS谱线。
(7)工作曲线：分别加入浓度为1fM-10nM的DENV，获得DENV检测的校准曲线。图3A为不同DENV浓度下所得的SERS光谱图，图3B为其各谱线在1503cm-1拉曼位移处所对应的的SERS峰强度。(拉曼测试条件：扫描时间1s，激光功率1％，物镜放大倍率20x，累计次数1次，激发光波长633nm)通过对SERS强度与DENV浓度对数关系的拟合，得到：
工作曲线：IDENV＝511×1g CDENV+8808(R2＝0.9955)，通过计算检测限为490aM。
(8)特异性验证：分别加入1nM DENV，10nM单碱基错配序列(M1)，10nM双碱基错配序列(M2)，10nM三碱基错配序列(M3)和blank(TM缓冲液)，测得SERS信号强度，来研究传感检测策略的特异性。见图4，图4A为不同实验条件下所得SERS光谱图，图4B为其1503cm-1拉曼位移处所对应的SERS峰强度，可以看出，加入1nM DENV时SERS强度明显强于其他实验条件，说明该检测策略具有较好的特异性。
(9)均一性验证：取检测1nM DENV时的50个随机点，记录其SERS强度，用来验证该检测策略的均一性，如图5所示，相对标准偏差(RSD)为8.78％，说明所提出的AgNRs基底上的传感策略具有良好的均一性。
(10)回收率验证实验：分别向TM缓冲液中加入4个不同浓度的DENV基因特征片段配制得到4个待检测样品，对4个待检测样品进行回收率验证实验。实验结果如表1。
表1 DENV检测回收率验证
Figure BDA0002093886790000091
从表1中可以看出1、2、3和4样品中DENV的检出的浓度与配制值接近，回收率在93.10-114.00％之间，样品检出准确性高。
对比例
收集已报道的登革病毒基因检测其它方法的灵敏度。
表2 DENV基因不同检测方法总结
Figure BDA0002093886790000092
Figure BDA0002093886790000101
本发明的所提出的SERS策略的灵敏度检测限为490aM，远远优于表1中所列出已报道的登革病毒基因检测其它方法的灵敏度。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
序列表
<110> 南京邮电大学
<120> 一种登革病毒基因片段SERS检测试剂盒及其制备方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> DENV基因片段(DENV)
<400> 1
tggtgctgtt gaatcaacag gttct 25
<210> 2
<211> 70
<212> DNA
<213> C1( C1)
<400> 2
agaacctgtt gattcaacag caccaacata ggagtgctgt tgaatcaaca tttttttttt 60
tttttttttt 70
<210> 3
<211> 86
<212> DNA
<213> L1(L1)
<400> 3
tcgttactta aatggtcaga aatatgggat taaccatggt gtttatgata tgaagtgttg 60
gaagctaaaa aaaaaaaaaa aaaaaa 86
<210> 4
<211> 66
<212> DNA
<213> L2( L2)
<400> 4
ttaatcccat atttctgacc atttaacgaa cgaagcttcc aacacttcat atcataaaca 60
ccatgg 66
<210> 5
<211> 60
<212> DNA
<213> C2(C2)
<400> 5
caacagcact cctatgttgg tgctgttgaa tcaacatagg agtgttctcg gcagtatcat 60
<210> 6
<211> 48
<212> DNA
<213> H1(H1)
<400> 6
gttctcggca gtatcatttg acacatgata ctgccgagaa cactccta 48
<210> 7
<211> 48
<212> DNA
<213> H2(H2)
<400> 7
gtgtcaaatg atactgccga gaactaggag tgttctcggc agtatcat 48
<210> 8
<211> 25
<212> DNA
<213> 单碱基错配(M1)
<400> 8
tggtgctgtt gaatcaacag gctct 25
<210> 9
<211> 25
<212> DNA
<213> 双碱基错配(M2)
<400> 9
tggtgctgtt gagtcaacag gctct 25
<210> 10
<211> 25
<212> DNA
<213> 三碱基错配(M3)
<400> 10
tggagctgtt gagtcaacag gctct 25

Claims (9)

1.一种登革病毒基因片段SERS检测试剂盒，其特征在于，所述SERS检测试剂盒包括固相SERS基片、第一试剂、第二试剂、第三试剂和第四试剂，所述固相SERS基片是表面沉积有银纳米棒阵列的玻璃片，所述第一试剂包含L1-L2-C1结构，所述L1-L2-C1结构包括能与登革病毒基因片段碱基序列完全互补的发夹型结构C1、与C1部分互补的L1、以及能与L1部分碱基互补的修饰有巯基的L2，L1与L2形成L1-L2双链，L1-L2双链与C1杂交形成L1-L2-C1结构；所述第二试剂为修饰有染料分子的发夹型结构C2，所述第三试剂为修饰有染料分子的发夹型结构H1，所述第四试剂为修饰有染料分子的发夹型结构H2，其中发夹型结构H1能被C1-C2双链中的C2部分碱基打开，H2能被H1部分碱基序列打开，且H2另一部分碱基序列能打开另一发夹型结构H1，从而形成交替杂交的长的H1-H2双链，所述C1碱基序列如SEQ ID NO：2所示，所述L1的碱基序列如SEQ ID NO：3所示，所述L2碱基序列如SEQ ID NO：4所示；所述C2碱基序列如SEQ ID NO：5所示；所述H1的碱基序列如SEQ ID NO：6所示，所述H2碱基序列如SEQ ID NO：7所示。
2.根据权利要求1所述的登革病毒基因片段SERS检测试剂盒，其特征在于，所述SERS检测试剂盒还包括第五试剂巯基己醇。
3.根据权利要求1~2任一项所述的登革病毒基因片段SERS检测试剂盒的制备方法，其特征在于，包括以下步骤：
1）固相SERS基片的制备；
2）第一试剂的获得：根据登革病毒基因片段设计并合成对应的发夹型结构C1，再根据所述C1设计并合成对应的L1，再根据L1设计并合成L2；
3）第二试剂的获得：根据C1设计并合成发夹型结构C2；
4）第三试剂的获得：根据所述C2设计并合成对应的发夹型结构H1；
5）第四试剂的获得：根据H1设计并合成发夹型结构H2。
4.根据权利要求3所述的登革病毒基因片段SERS检测试剂盒的制备方法，其特征在于，所述C1、L1、L2、C2、H1和H2浓度相同，浓度均为1-10 µM。
5.权利要求1~2任一项所述的登革病毒基因片段SERS检测试剂盒的检测方法，其不用于疾病的诊断和治疗，其特征在于，包括以下步骤：
1）用超纯水多次清洗试剂盒中的固相SERS基片；
2）向试剂盒的第一试剂中加入检测样品与第二试剂共培养；
3）向步骤2）中加入第三试剂与第四试剂得到溶液；
4）将步骤1）得到的固相SERS基片与步骤3）的溶液共培育；
5）缓冲液冲洗步骤4）得到的基片后，用第五试剂封闭基片表面；
6）纯水冲洗步骤5）得到的基片后，进行SERS检测。
6.根据权利要求5所述的一种登革病毒基因片段SERS检测试剂盒的检测方法，其特征在于，所述步骤2）中的共培养条件为24-28℃，培养1.5-2h。
7.根据权利要求5所述的一种登革病毒基因片段SERS检测试剂盒的检测方法，其特征在于，所述步骤3）培养条件为24-28℃，1.5-2h。
8.根据权利要求5所述的一种登革病毒基因片段SERS检测试剂盒的检测方法，其特征在于，所述步骤4）的培育条件为25-30℃，60%-80%湿度环境，培养3-4h。
9.根据权利要求5所述的一种登革病毒基因片段SERS检测试剂盒的检测方法，其特征在于，所述步骤5）的培养条件为25-30℃，60%-80%湿度环境，静置培养10-20 min。
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CN109811089A (zh) * 2019-01-02 2019-05-28 南京邮电大学 一种流感病毒基因片段sers检测试剂盒及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A cascade amplification strategy of catalytic hairpin assembly and hybridization chain reaction for the sensitive fluorescent assay of the model protein carcinoembryonic antigen;Wenting Yang et al.;《Mikrochim Acta》;20180110;100 *
Highly sensitive SERS assay of DENV gene via a cascade signal amplification strategy of localized catalytic hairpin assembly and hybridization chain reaction;Chunyuan Song et al.;《Sens Actuators B Chem》;20201215;128970 *
结合信号放大技术的病毒基因SERS传感器的构建及应用研究;刘洋;《中国优秀硕士学位论文全文数据库（基础科学辑）》;20200215;A006-686 *

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