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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (11): 28-34    DOI: 10.13523/j.cb.2008124
技术与方法     
双链探针实时荧光PCR核酸检测新技术研究*
刘丽艳1**,刘琪琦1**,张影2,王升启1***()
1 军事医学研究院辐射医学研究所 北京 100850
2 郑州美灵生物技术有限公司 郑州 450000
The Study of a Novel Nucleic Acid Detection Technology by Double-stranded Probe Real-time PCR
LIU Li-yan1**,LIU Qi-qi1**,ZHANG Ying2,WANG Sheng-qi1***()
1 Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China
2 Zhengzhou Maylink Biotechnology Co. Ltd, Zhengzhou 450000, China
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摘要：

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。
关键词： 双链探针;  TaqMan探针;  实时荧光PCR;  乙肝病毒;  CYP2C19*2    
Abstract:

Objective: Using a “double-stranded probe” real-time fluorescent PCR technology to improve the sensitivity of HBV nucleic acid detection, complete the genotype detection of metabolic enzyme CYP2C19 *2 in a tube. Methods: The double-stranded probe and the TaqMan probe was used to simultaneously detect different concentrations of HBV in serum samples by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Then, according to the Ct value of nucleic acid detection by instrument to statistical analysis of results; the double-stranded probe was used to detect samples of different genotypes of metabolic enzyme CYP2C19*2 in a tube, and the detection of nucleic acid Ct value and genotype analysis were performed by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Results: In the detection of HBV serum samples at different concentrations, the fluorescence background of the double-stranded probe was low and the detection sensitivity was higher than the TaqMan probe. And significant differences were noted between the two probes (P<0.05);The metabolic enzyme CYP2C19*2 genotypes of 36 samples were detected using the double-stranded probe, the results were consistent with those of Sanger sequencing. Conclusion: The double-stranded probe real-time fluorescent PCR detection technology can complete the highly sensitive nucleic acid detection of the target gene and also the genotype analysis.
Key words: Double-stranded probe    TaqMan probe    Real-time PCR    HBV    CYP2C19*2
收稿日期: 2020-08-15 出版日期: 2020-12-11
ZTFLH:  Q819  
基金资助: * 国家科技重大专项(2018ZX10711001-003-003);国家自然科学基金重点项目(81830101)
通讯作者: 王升启     E-mail: sqwang@bmi.ac.cn
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引用本文:

刘丽艳,刘琪琦,张影,王升启. 双链探针实时荧光PCR核酸检测新技术研究*[J]. 中国生物工程杂志, 2020, 40(11): 28-34.

LIU Li-yan,LIU Qi-qi,ZHANG Ying,WANG Sheng-qi. The Study of a Novel Nucleic Acid Detection Technology by Double-stranded Probe Real-time PCR. China Biotechnology, 2020, 40(11): 28-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2008124    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I11/28

图1  双链探针结构示意图
图2  双链探针与TaqMan探针荧光本底信号值检测结果图
名称 序列(5'-3') 5' 荧光标记 3'荧光标记
HBV-F GYTATCGCTGGATGTGTCTGC
HBV-R GACAAACGGGCAACATACCTT
HBV-P CCTCTKCATCCTGCTGCTATGCCTCAT FAM BHQ1
GCATAGCAGCAGGATGM FAM BHQ1
CYP2C19*2-F ATTATTGTTTTCTCTTAGATAT
CYP2C19*2-R AAGTCCCGAGGGTTGTTGAT
CYP2C19*2-P TATTTCCCAGGAACCCA FAM BHQ1
TATGGGTTCCCGGGAAATAAT HEX BHQ1
表1  HBV、CYP2C19*2引物及探针序列
样本浓度(IU/ml) Ct值
双链探针 TaqMan探针
2.66×108 13.39 16.60
2.45×108 13.52 16.62
1.93×108 13.90 16.55
1.33×107 18.08 21.92
1.08×107 18.34 22.10
1.03×107 18.55 22.29
1.49×106 22.00 25.74
1.45×106 22.20 26.19
1.04×106 21.57 25.36
2.10×105 24.58 28.28
1.97×105 24.68 28.33
1.35×105 25.27 29.13
1.13×104 29.16 32.96
1.02×104 29.32 32.72
1.01×104 29.35 32.88
1.12×103 32.88 36.54
1.09×103 33.71 37.07
1.05×103 34.05 37.84
9.81×101 36.71 No Ct
8.58×101 37.00 No Ct
7.10×101 38.03 No Ct
1.81×101 38.89 No Ct
1.12×101 39.32 No Ct
1.17×101 39.37 No Ct
表2  双链探针与TaqMan探针检测24份定量HBV样本Ct值结果
编号 Ct值
野生链 突变链
G/A G/G A/A G/A G/G A/A
1 32.44 31.90 No Ct 30.66 No Ct 29.49
2 34.31 32.45 No Ct 32.59 No Ct 32.53
3 32.66 36.97 No Ct 30.65 No Ct 36.32
4 37.66 30.77 No Ct 36.08 No Ct 31.11
5 34.07 32.10 No Ct 32.94 No Ct 24.50
6 29.14 36.04 No Ct 28.45 No Ct 32.80
7 29.09 35.12 No Ct 27.61 No Ct 28.68
8 31.78 37.14 No Ct 30.29 No Ct 28.83
9 28.48 28.64 No Ct 26.95 No Ct 29.97
10 31.46 32.21 No Ct 29.74 No Ct 30.01
11 36.60 32.33 No Ct 34.92 No Ct 31.19
12 32.27 33.63 No Ct 31.50 No Ct 31.23
表3  双链探针检测3种CYP2C19* 2基因型样本结果
图3  双链探针检测36份不同CYP2C19* 2基因型样本结果图
[1] Heid C A, Stevens J, Livak K J, et al. Real time quantitative PCR. Genome Research, 1996,6(10):986-994.
[2] Shi S R, Ni B, Guo Y, et al. Detection of 2019 novel coronavirus in various biological specimens of novel coronavirus pneumonia. West China Medical Journal, 2020,35(2):132-136.
[3] Liu R, Han H, Liu F, et al. Positive rate of RT-PCR detection of SARS-CoV-2 infection in 4880 cases from one hospital in Wuhan, China, from Jan to Feb 2020. Clinica Chimica Acta, 2020,505:172-175.
[4] Henritzi D, Hoffmann B, Wacheck S, et al. A newly developed tetraplex real time RT-PCR for simultaneous screening of influenza virus types A, B, C and D. Influenza and Other Respiratory Viruses, 2019; 13(1):71-82.
doi: 10.1111/irv.12613 pmid: 30264926
[5] Feng W N, Gu W Q, Zhao N, et al. Comparison of the superARMS and droplet digital PCR for detecting EGFR mutation in ctDNA from NSCLC patients. Translational Oncology, 2018,11(2):542-545.
[6] Wilson H L, Tran T, Druce J, et al. Neutralization assay for zika and dengue viruses by use of real-time-PCR-based endpoint assessment. Journal of Clinical Microbiology, 2017,55(10):3104-3112.
[7] Li-Wan-Po A, Girard T, Farndon P, et al. Pharmacogenetics of CYP2C19: functional and clinical implications of a new variant CYP2C19*17. British Journal of Clinical Pharmacology, 2010,69(3):222-230.
pmid: 20233192
[8] Günther S, Asper M, R?ser C, et al. Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro. Antiviral Research, 2004,63(3):209-215.
[9] Zhang M, Gong Y, Osiowy C, et al. Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis. Hepatology, 2002,36(3):723-728.
pmid: 12198666
[10] Sz?llosi J, Damjanovich S, Mátyus L. Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research. Cytometry, 1998,34(4):159-179.
pmid: 9725457
[11] Ozaki H, McLaughlin L W. The estimation of distances between specific backbone-labeled sites in DNA using fluorescence resonance energy transfer. Nucleic Acids Research, 1992,20(19):5205-5214.
pmid: 1408835
[12] Arya M, Shergill I S, Williamson M, et al. Basic principles of real-time quantitative PCR. Expert Review of Molecular Diagnostics, 2005,5(2):209-219.
[13] Wang S Q, Wang X H, Chen S H, et al. A new fluorescent quantitative polymerase chain reaction technique. Analytical Biochemistry, 2002,309(2):206-211.
[14] Tyagi S, Kramer F R. Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology, 1996,14(3):303-308.
[15] Livak K J, Flood S J, Marmaro J, et al. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications, 1995,4(6):357-362.
[16] Murray J L, Hu P, Shafer D A. Seven novel probe systems for real-time PCR provide absolute single-base discrimination, higher signaling, and generic components. Journal of Molecular Diagnostics, 2014,16(6):627-638.
[17] Li Q, Luan G, Guo Q, et al. A new class of homogeneous nucleic acid probes based on specific displacement hybridization. Nucleic Acids Research, 2002,30(2):E5.
[18] Song Y, Dou F, Zhou Z, et al. Microarray-based detection and clinical evaluation for helicobacter pylori resistance to clarithromycin or levofloxacin and the genotype of CYP2C19 in 1083 patients. Biomed Research International, 2018,2018:2684836.
[19] Do H, Krypuy M, Mitchell P L, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies. BMC Cancer, 2008,8:142.
pmid: 18495026
[20] Chen A, Kao Y F, Brown C M. Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons. Nucleic Acids Research, 2005,33(4):1169-1181.
[21] Wang S Q, Liu Q Q, Zhang Y, et al. The structure and application of a double-stranded oligonucleotide nucleic acid probe: China, ZL201811643407.0. 2019-04-19[2020-08-08]. http://pss-system.cnipa.gov.cn/sipopublicsearch/patentsearch/showViewList-jumpToView.shtml.
[22] 刘颖梅. 甲型H1N1流感的病原学诊断//中国药理学会, 第十届全国化疗药理暨抗感染药理高峰论坛资料汇编. 北京:中国药理学会, 2010.
Liu Y M. The etiological diagnosis of influenza A H1N1//Chinese Pharmacological Society,Data Collection of The 10th National Chemotherapy Pharmacology and Anti-infective Pharmacology Summit Forum. Beijing: Chinese Pharmacological Society, 2010.
[23] McAnulty J M. Emerging infectious diseases. Public Health Research & Practice, 2016,26(5):2651653.
[24] Morens D M, Folkers G K, Fauci A S. The challenge of emerging and re-emerging infectious diseases. Nature, 2004,430(6996):242-249.
pmid: 15241422
[25] Zhang W J, Liao P. Analysis of the causes of similarities and differences in multiple nucleic acid tests for a case of Corona Virus Disease 2019. Chongqing Medicine:1-5.[2020-08-08]. http://kns.cnki.net/kcms/detail/50.1097.R.20200316.0821.002.html.
[26] Phan T. Novel coronavirus: From discovery to clinical diagnostics. Infection Genetics and Evolution, 2020,79:104211.
[27] Zhang J, Zhong J, Ding J, et al. Simultaneous detection of human CYP2C19 polymorphisms and antibiotic resistance of Helicobacter pylori using a personalised diagnosis kit. Journal of Global Antimicrobial Resistance, 2018,13:174-179.
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