基于双酶切级联信号放大的核酸检测方法

doi:10.7503/cjcu20180002

高等学校化学学报 ›› 2018, Vol. 39 ›› Issue (7): 1427.doi: 10.7503/cjcu20180002

基于双酶切级联信号放大的核酸检测方法

廉翔^1,², 吴望华², 范宏亮³, 张勇¹(), 张涛²()

1. 山西大学化学化工学院, 太原 030006
2. 浙江大学工业控制技术国家重点实验室, 智能系统与控制研究所, 分析仪器研究中心, 杭州 310027
3. 浙江省医学科学院卫生学研究所, 杭州 310013

收稿日期:2018-01-02 出版日期:2018-07-10 发布日期:2018-06-21
作者简介:联系人简介: 张涛, 男, 博士, 副教授, 主要从事生化传感与分析仪器方面的研究. E-mail: zhtao@zju.edu.cn; 张勇, 男, 博士, 教授, 主要从事光谱分析方面的研究. E-mail: zhangyong@sxu.edu.cn
基金资助:
国家自然科学基金(批准号: 21275129)、工业控制技术国家重点实验室自主课题(批准号: ICT1805)、国家重大科学仪器设备开发专项(批准号: 2013YQ470781)和浙江省医药卫生科技计划项目(批准号: 2015KYA061, 2016KYB070)资助.

Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection^†

LIAN Xiang^1,², WU Wanghua², FAN Hongliang³, ZHANG Yong^1,^*(), ZHANG Tao^2,^*()

1. College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China
2. Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control,State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310027, China
3. Department of Environmental Medicine,Institute of Hygiene, Zhejiang Academy of Medical Sciences,Hangzhou 310013, China

Received:2018-01-02 Online:2018-07-10 Published:2018-06-21
Contact: ZHANG Yong,ZHANG Tao E-mail:zhangyong@sxu.edu.cn;zhtao@zju.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.21275129), the Autonomous Research Project of the State Key Laboratory of Industrial Control Technology, China(No.ICT1805), the National Key Foundation for Exploring Scientific Instruments, China(No.2013YQ470781) and the Medical and Health Technology Project of Zhejiang Province, China(Nos.2015KYA061, 2016KYB070).

摘要/Abstract

摘要：

利用茎环结构定位探针构建了一个基于双酶切反应的级联信号放大体系, 并将其用于核酸的检测. 在该体系中, 茎环结构定位探针首先是内切酶Tth Endonuclease Ⅳ的作用底物, 被剪切后又作为定位探针介导切口酶Nt.BstNBI对分子信标实施剪切, 将这2步剪切反应结合起来可有效克服切口酶对于目标核酸中特定识别序列的依赖, 同时进一步提高了检测灵敏度. 实验结果表明, 荧光信号与目标DNA浓度的对数值呈线性相关, 响应范围为1 pmol/L~1 nmol/L, 并且具有良好的识别单碱基变异的能力. 此外, 本方法序列设计简单, 通用性强, 仅改变定位探针的部分序列即可实现对不同目标DNA的检测. 对掺杂于血清中的目标DNA的检测结果验证了本方法在实际样品检测中的应用潜力.

关键词: 级联信号放大, 分子信标, 切口酶, 定位探针, 核酸检测

Abstract:

Dual enzyme cleavage-based cascade signal amplification for nucleic acids detection was developed. In this system, a modified DNA aligner(MDA) that contains an abasic site was first cleaved by Tth Endonuclease Ⅳ in the presence of target DNA, and then served as an aligner to mediate the cleavage of molecular beacon by nicking endonuclease Nt.BstNBI. These cascade reactions not only overcame the sequence dependence of Nt.BstNBI, but also improved the detection sensitivity. The results showed a good linear correlation between the fluorescence intensity and the logarithm of target DNA concentration(lgc) ranging from 1 pmol/L to 1 nmol/L. Moreover, the proposed method also showed a good capability of identifying single base mutation in target DNA. In addition, this method also features simple probe design and excellent universality. By modifying a small fragment on MDA’s loop, it can be used to sense various target DNAs. Experiments with target DNA spiked in human serum showed the potential of applying this method to real samples.

Key words: Cascade signal amplification, Molecular beacon, Nicking endonuclease, DNA aligner, Nucleic acid detection

中图分类号:

O655.9

TrendMD:

廉翔, 吴望华, 范宏亮, 张勇, 张涛. 基于双酶切级联信号放大的核酸检测方法. 高等学校化学学报, 2018, 39(7): 1427.

LIAN Xiang, WU Wanghua, FAN Hongliang, ZHANG Yong, ZHANG Tao. Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection^†. Chem. J. Chinese Universities, 2018, 39(7): 1427.

图/表 9

参考文献 24

[1]	Tan W. H., Donovan M. J., Jiang J. H., Chem.Rev., 2013, 113(4), 2842—2862
[2]	Zhao Y. X., Chen F., Li Q., Wang L. H., Fan C. H., Chem.Rev., 2015, 115(22), 12491—12545
[3]	Sonkar S. C., Sachdev D., Mishra P. K., Kumar A., Mittal P., Saluja D., Biosens. Bioelectron., 2016, 86, 41—47
[4]	Sanders R., Mason D. J., Foy C. A., Huggett J. F., Anal. Bioanal.Chem., 2014, 406(26), 6471—6483
[5]	Chen G. H., Tang X. Y., Sun Y., Zhou L., Li D., Bai Y., Mai K. J., Li Y. Y., Wu Q. W., Ma J. Y., J. Virol. Methods, 2018, 251, 129—132
[6]	Chen P. P., Peng W., Zhang Y. X., Chen J. B., Jiang X. M., Zheng C. B., Hou X. D., Anal.Chem., 2016, 88(24), 12386—12392
[7]	Xu W. T., Wang C. G., Zhu P. Y., Guo T. X., Xu Y. C., Huang K. L., Luo Y. B., Analyst, 2016, 141(8), 2542—2552
[8]	Xu Y. Z., Niu C., Xiao X. F., Zhu W. Y., Dai Z., Zou X. Y., Anal.Chem., 2015, 87(5) 2945—2951
[9]	Kong X. J., Wu S., Cen Y., Chen T. T., Yu R. Q., Chu X., Analyst, 2016, 141(14), 4373—4380
[10]	Li J., Macdonald J., Biosens. Bioelectron., 2015, 64, 196—211
[11]	Deng H. M., Gao Z. Q., Anal. Chim. Acta, 2015, 853(1), 30—45
[12]	Tan E., Erwin B., Dames S., Ferguson T., Buechel M., Irvine B., Voelkerding K., Niemz A., Biochem., 2008, 47(38), 9987—9999
[13]	Shinde D. N., Ramírezborrero A. J., Hintze A., Adami C., Niemz A., Nucleic Acids Res., 2012, 40(11), e87
[14]	Tyagi S., Kramer F. R., Nat.Biotechnol., 1996, 14(3), 303—308
[15]	Dong H. F., Meng X. D., Dai W. H.,Cao Y., Lu H. T., Zhou S. F., Zhang X. J., Anal.Chem., 2015, 87(8), 4334—4340
[16]	Zhang W. C., Hu F., Zhang Q., Zhang J. Q., Mao Y. B., Wang P., Gu Y. Q., Talanta, 2018, 179, 685—692
[17]	Zhang L. L., Wong J. X. H., Li X. C., Li Y. C., Yu H. Z., Anal.Chem., 2015, 87(10), 5062—5067
[18]	Zhang P., Beck T., Tan W. H., Angew. Chem. Int.Ed., 2001, 40(2), 402—405
[19]	Xu H., Zhang R. B., Li F., Zhou Y. Y., Peng T., Wang X. D., Shen Z. F., Anal. Bioanal.Chem., 2016, 408(22), 6181—6188
[20]	Li J.W. J.,Chu Y. Z., Lee B. Y. H., Xie X. L. S., Nucleic Acids Res., 2008, 36(6), e36
[21]	Zhao Y. X., Chen F., Wu Y. Y., Dong Y. H., Fan C. H., Biosens.Bioelectron., 2013, 42(1), 56—61
[22]	Xu W., Xue X. J., Li T. H., Zeng H. Q., Liu X. G., Angew.Chem., 2009, 48(37), 6849—6852
[23]	Kong R. M., Zhang X. B., Zhang L. L., Huang Y., Lu D. Q., Tan W. H., Shen G. L., Ru R. Q., Anal.Chem., 2011, 83(1), 14—17
[24]	Wu W. H., Zhang T., Han D., Fan H. L., Zhu G. Z., Ding X., Wu C. C., You M. X., Qiu L. P., Li J., Zhang L. Q., Lian X., Hu R., Mu Y., Zhou J. G., Tan W. H., Chem.Sci., 2018, 9(11), 3050—3055

Name	Sequence(5'-3')
Molecular Beacon-1(MB-1)	FAM-GCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGC-Dabcyl
Molecular Beacon-2(MB-2)	FAM-CTGCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGCAG-Dabcyl
DNA Aligner(DA)	TCGGTATCTCCTGACTCACGTTTTTCGTGAGTCAACGTCCAGTAC
Modified DNA Aligner(MDA)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCACAT- GAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-FAM (MDA-FAM)	FAM-GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCA- CATGAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-1(MDA-1)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACACCACTCAACCTTCTAGACT- GACTCTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-2(MDA-2)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACTGCTCACATCTGATTCGACACTGT- CACTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Target DNA	ACTCATGTGAGCAAGGTGACACTTG
Target DNA-1(T-1)	GAGTCAGTCTAGAAGGTTGAGTGGT
Target DNA-2(T-2)	GTGACAGTGTCGAATCAGATGTGAGCA
Mutant target DNA-1(MT-1)	ACTCATGTGAGCAAGcTGACACTTG
Mutant target DNA-2(MT-2)	ACTCAcGTGAGCAAGcTGACACTTG
Mutant target DNA-3(MT-3)	ACTgATGTGAGCAAGcTGACACaTG

Name	Sequence(5'-3')
Molecular Beacon-1(MB-1)	FAM-GCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGC-Dabcyl
Molecular Beacon-2(MB-2)	FAM-CTGCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGCAG-Dabcyl
DNA Aligner(DA)	TCGGTATCTCCTGACTCACGTTTTTCGTGAGTCAACGTCCAGTAC
Modified DNA Aligner(MDA)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCACAT- GAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-FAM (MDA-FAM)	FAM-GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCA- CATGAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-1(MDA-1)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACACCACTCAACCTTCTAGACT- GACTCTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-2(MDA-2)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACTGCTCACATCTGATTCGACACTGT- CACTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Target DNA	ACTCATGTGAGCAAGGTGACACTTG
Target DNA-1(T-1)	GAGTCAGTCTAGAAGGTTGAGTGGT
Target DNA-2(T-2)	GTGACAGTGTCGAATCAGATGTGAGCA
Mutant target DNA-1(MT-1)	ACTCATGTGAGCAAGcTGACACTTG
Mutant target DNA-2(MT-2)	ACTCAcGTGAGCAAGcTGACACTTG
Mutant target DNA-3(MT-3)	ACTgATGTGAGCAAGcTGACACaTG

Target sample	1	2	3	4	5	6	7	8	9
Added/(pmol·L^-1)	10.0	10.0	10.0	100.0	100.0	100.0	500.0	500.0	500.0
Found/(pmol·L^-1)	12.1	9.9	8.7	91.3	110.8	95.4	589.0	461.5	522.2
Recovery(%)	117.1	95.1	83.1	90.9	110.4	95.0	117.7	92.2	104.3

Target sample	1	2	3	4	5	6	7	8	9
Added/(pmol·L^-1)	10.0	10.0	10.0	100.0	100.0	100.0	500.0	500.0	500.0
Found/(pmol·L^-1)	12.1	9.9	8.7	91.3	110.8	95.4	589.0	461.5	522.2
Recovery(%)	117.1	95.1	83.1	90.9	110.4	95.0	117.7	92.2	104.3

基于双酶切级联信号放大的核酸检测方法

Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection^†

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 24

相关文章 8

编辑推荐

Metrics

本文评价

[1]	张志庆,汪珊珊,张子辰,马杰,王秀凤,周亭,王芳,张国栋. 基于滚环合成的聚分子信标用于凝血酶的靶向检测[J]. 高等学校化学学报, 2019, 40(12): 2465.
[2]	袁浩钧, 郜晚蕾, 景奉香, 刘松生, 周洪波, 贾春平, 金庆辉, 赵建龙. 一种用于核酸高灵敏检测的液滴式数字聚合酶链式反应芯片[J]. 高等学校化学学报, 2017, 38(7): 1140.
[3]	刘斌, 羊小海, 王柯敏, 谭蔚泓. 基于分子信标对hOGG1活性的定量分析[J]. 高等学校化学学报, 2012, 33(03): 486.
[4]	王青, 崔亮, 羊小海, 王柯敏, 何磊良. 基于锁核酸的高选择性新型分子信标[J]. 高等学校化学学报, 2010, 31(7): 1322.
[5]	孟祥贤, 羊小海, 王柯敏, 郭秋平, 李军, 唐志文. 分子信标用于核酸连续复制过程的体外实时监测[J]. 高等学校化学学报, 2009, 30(8): 1538.
[6]	王炜刘斌李军王柯敏谭蔚泓唐红星. 分子信标研究5-氟尿嘧啶对鼻咽癌HNE1细胞p33^ING1基因转录水平的影响[J]. 高等学校化学学报, 2006, 27(2): 268.
[7]	李军, 王柯敏, 谭蔚泓, 刘斌, 郭秋平, 唐志文, 刘凌风. 分子信标荧光探针用于抑癌基因ING1表达产物的定量测定[J]. 高等学校化学学报, 2004, 25(3): 421.
[8]	刘凌凤, 唐志文, 王柯敏, 谭蔚泓, 李军, 郭秋平, 孟祥贤, 黄杉生, 李杜. 基于分子信标实时监测大肠杆菌DNA连接酶催化的DNA连接过程[J]. 高等学校化学学报, 2003, 24(10): 1761.

基于双酶切级联信号放大的核酸检测方法

Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection†

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 24

相关文章 8

编辑推荐

Metrics

本文评价

Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection^†