CN111647682A - 基于纳米酶的hpv16核酸检测试纸条 - Google Patents
基于纳米酶的hpv16核酸检测试纸条 Download PDFInfo
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- CN111647682A CN111647682A CN201910160159.2A CN201910160159A CN111647682A CN 111647682 A CN111647682 A CN 111647682A CN 201910160159 A CN201910160159 A CN 201910160159A CN 111647682 A CN111647682 A CN 111647682A
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Abstract
本发明涉及一种基于纳米酶的HPV16核酸检测试纸条。试纸条同时利用了纳米酶的磁性和过氧化物酶活性，具体如下：一次纳米酶富集后起到了核酸浓缩的作用；层析结束后，利用纳米酶的过氧化物酶活性，加入显色液进行信号放大，起到了灵敏度提高的作用。
Description
技术领域
本发明属于医药生物技术领域，具体的，涉及一种基于纳米酶的HPV16核酸检测试纸条。
背景技术
磁性纳米颗粒具有良好的生物相容性，其既具有纳米材料所特有的性质，如粒径小、比表面积大、偶联容量高，又具有磁响应性及超顺磁性，可以在恒定磁场下聚集和定位、在交变磁场下吸收电磁波产热，利用这些特性，磁性纳米颗粒被广泛应用于磁共振对比剂、磁靶向药物载体、细胞与生物分子分离、生物传感与检测以及磁感应肿瘤热疗等生物学领域。
纳米酶核酸检测试纸条的工作原理，是将能与待检核酸下游互补配对的核酸分子通过Biotin和Streptavidin固定在硝酸纤维素膜(NC膜)这一固相载体上作为检测线(T线)，将能与待检核酸上游互补配对的核酸分子通过Digoxigenin和抗Digoxigenin抗体固定在纳米酶上得到纳米酶探针，将能与纳米酶上的核酸互补配对的核酸分子通过Biotin和Streptavidin固定在NC膜上作为质控线(C线)。
当待测样品(如尿液)与纳米酶探针混合后，纳米酶探针上的核酸分子与待测样品中的目标核酸发生互补配对而形成纳米酶目标核酸复合物，然后利用纳米酶的磁性，将待测样本中的目标核酸分子进行富集，加入到96孔板的小孔中，接下来将纳米酶核酸检测试纸条插入，液体在吸水垫的作用下层析，当纳米酶目标核酸复合物层析到NC膜上的T线位置时，目标核酸的下游与T线上的核酸分子再一次互补配对而被捕获，由于纳米酶自身具有棕色因而能够在T线的位置显示条带，条带的深浅与待测液体中目标核酸的多少呈正相关。
此外，无论待测样品中是否有目标核酸，当纳米酶探针层析到NC膜上的C线位置时，此处固定的核酸都能与纳米酶探针上的核酸通过互补配对将其捕获，从而显示条带，以指示纳米酶探针和NC膜上的核酸分子均正常有效，达到了质控的目的。
发明内容
本发明涉及一种基于纳米酶的HPV16核酸检测试纸条，所述的试纸条上装有检测线、质控线、探针垫和吸收待测样品的样品垫。
(1)质控线位置装有如SEQ ID NO.2所示的核酸序列1’，
SEQ ID NO.2：5’-CAGTGATACAGGTGAAGATTTGGTAGATTT；
核酸序列1’的5’端标记Biotin，并与Streptavidin偶联；
(2)检测线位置装有如SEQ ID NO.3所示的核酸序列3，
SEQ ID NO.3：5’-TGTGTTAAATAATCATTATCATTTACTATA；
核酸序列3的5’端标记Biotin，并与Streptavidin偶联；
(3)如SEQ ID NO.1所示的核酸序列1与纳米酶探针偶联装于探针垫上，
SEQ ID NO.1：5’-AAATCTACCAAATCTTCACCTGTATCACTG；
核酸序列1的3’端标记Digoxigenin，核酸序列1；
所述的Streptavidin(from Streptomyces avidinii)氨基酸序列如SEQ ID NO.4所示：
SEQ ID NO.4：
MRKIVVAAIAVSLTTVSITASASADPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ；
所述的HPV 16的核酸序列如SEQ ID NO.5所示：
SEQ ID NO.5：
CAGTGATACAGGTGAAGATTTGGTAGATTTTATAGTAAATGATAATGATTATTTAACACAGGCAGAAACAGAGACAGCACATGCGTTGTTTACTGCACAGGAAGCAAAACAACATAGAGATGCAGTACAGGTTCTAAAACGAAAGTATT。
本发明的有益效果在于：纳米酶HPV16核酸检测试纸条同时利用了纳米酶的磁性和过氧化物酶活性，具体如下：
一次尿液50mL，纳米酶富集后用500μL缓冲液重悬，起到了核酸浓缩100倍的作用；
层析结束后，利用纳米酶的过氧化物酶活性，加入显色液进行信号放大，起到了灵敏度5倍提高的作用。
此为纳米酶首次应用于尿液检测，尿液检测有样本量大的特点，因此两者的综合效果可实现灵敏度500倍的提高。
附图说明
图1、检测尿液中目标物质成分的纳米酶检测试纸条结构示意图。
具体实施方式
实施例1，检测HPV16核酸的纳米酶试纸条
检测线，质控线的位置如图1所示，各个位置上的预装检测物如下：
1、纳米酶偶联抗Digoxigenin抗体；
2、核酸序列1能够特异地结合HPV16目标核酸序列的上游，核酸序列3能够特异地结合HPV16目标核酸序列的下游，核酸序列1和核酸序列1’互补配对；
2.1、核酸序列1；
核酸序列1的3’端标记Digoxigenin，二者孵育，则得到偶联核酸序列1的纳米酶探针；
核酸序列1如SEQ ID NO.1所示，
SEQ ID NO.1：5’-AAATCTACCAAATCTTCACCTGTATCACTG；
2.2、核酸序列1’；
核酸序列1’的5’端标记Biotin，与Streptavidin孵育，得到偶联核酸序列1’的Streptavidin，划膜固定于试纸条的质控线位置；
核酸序列1’如SEQ ID NO.2所示，
SEQ ID NO.2：5’-CAGTGATACAGGTGAAGATTTGGTAGATTT；
2.3、核酸序列3
核酸序列3的5’端标记Biotin，与Streptavidin孵育，得到偶联核酸序列3的Streptavidin，划膜固定于试纸条的检测线位置；
核酸序列3如SEQ ID NO.3所示，
SEQ ID NO.3：5’-TGTGTTAAATAATCATTATCATTTACTATA；
所述的Streptavidin(from Streptomyces avidinii)氨基酸序列如SEQ ID NO.4所示：
SEQ ID NO.4：
MRKIVVAAIAVSLTTVSITASASADPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ；
所述的HPV 16的目标核酸序列如SEQ ID NO.5所示：
SEQ ID NO.5：
CAGTGATACAGGTGAAGATTTGGTAGATTTTATAGTAAATGATAATGATTATTTAACACAGGCAGAAACAGAGACAGCACATGCGTTGTTTACTGCACAGGAAGCAAAACAACATAGAGATGCAGTACAGGTTCTAAAACGAAAGTATT。
实施例2、检测目标HPV16核酸
1、加样检测
微量移液器取50μl含有梯度浓度的目标HPV16核酸溶液加入检测卡上的样品垫上，再加入50μl层析缓冲液(1％Tween 20，0.5％Triton X-100，1％NP-40，0.05％NaN3，20mM PBS，pH 7.2)，等待反应进行15min。由于磁颗粒上通过Digoxigenin抗原-抗体的相互作用偶联了与HPV16核酸配对标记的核酸序列1，它与样品溶液中的HPV16核酸结合后形成的复合物继续先前移动时，会与检测线(T线)处的HPV16核酸的另一配对的标记了Biotin的核酸序列3结合形成磁颗粒的聚集，而没有结合的BTA的磁颗粒抗体探针会继续移动到质控线(C线)处，通过与核酸序列1配对的标记了Biotin的核酸序列1’形成磁颗粒的聚集。
2、酶催化放大检测信号
在检测线T线和质控线C线处加入50μl的显色溶液(过氧化氢H2O2，浓度为530mM；过氧化物酶的生色底物二氨基联苯胺(3,3’-diaminobenzidine，DAB)浓度为816mM)，反应5min。
由于磁颗粒的过氧化物模拟酶活性，会在磁颗粒聚集的T线和C线处生成大量棕褐色的不溶性沉淀物，从而将检测信号放大，其检测灵敏度是传统胶体金试纸条检测法的500倍以上。
以上仅是本发明的具体应用范例。应理解，这些实施例仅用于说明本专利而不用于限制本发明的范围。
SEQUENCE LISTING
<110> 深圳市第二人民医院
中国科学院生物物理研究所
<120> 基于纳米酶的HPV16核酸检测试纸条
<130> CP11902123C
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213> 人工序列
<400> 1
aaatctacca aatcttcacc tgtatcactg 30
<210> 2
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<212> DNA
<213> 人工序列
<400> 2
cagtgataca ggtgaagatt tggtagattt 30
<210> 3
<211> 30
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<213> 人工序列
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tgtgttaaat aatcattatc atttactata 30
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<211> 183
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<213> Streptomyces avidinii
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Ser Ile Thr Ala Ser Ala Ser Ala Asp Pro Ser Lys Asp Ser Lys Ala
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Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln
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Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr
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Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu
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Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
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Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser
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Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile
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Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp
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Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser
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Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val Asn Asn Gly Asn
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<213> 人工序列
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ggcagaaaca gagacagcac atgcgttgtt tactgcacag gaagcaaaac aacatagaga 120
tgcagtacag gttctaaaac gaaagtatt 149
Claims (4)
1.一种基于纳米酶的HPV16核酸检测试纸条，所述的试纸条上装有检测线、质控线、探针垫和吸收待测样品的样品垫。
2.根据权利要求1所述的试纸条，其特征在于，
(1)质控线位置装有如SEQ ID NO.2所示的核酸序列1’，
核酸序列1’的5’端标记Biotin，并与Streptavidin偶联；
(2)检测线位置装有如SEQ ID NO.3所示的核酸序列3，
核酸序列3的5’端标记Biotin，并与Streptavidin偶联；
(3)如SEQ ID NO.1所示的核酸序列1与纳米酶探针偶联装于探针垫上，
核酸序列1的3’端标记Digoxigenin，核酸序列1。
3.根据权利要求1或2所述的试纸条，其特征在于，
所述的Streptavidin(from Streptomyces avidinii)氨基酸序列如SEQ ID NO.4所示：
所述的HPV 16的核酸序列如SEQ ID NO.5所示。
4.权利要求1-3任一所述的检测试纸条在制备检测疾病的医疗器械中的应用，所述的疾病为与HPV16病毒相关的疾病。
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