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Probe and fluorescence sensor for quantitatively detecting hepatitis B virus DNA and method

A fluorescent sensor, hepatitis B virus technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, fluorescence / phosphorescence, etc., can solve the problems of high cost, poor stability, cumbersome operation of HBVDNA detection methods, etc., to achieve low cost and expand Effects of detection versatility and low synthesis cost

Pending Publication Date: 2021-08-13
CHONGQING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above-mentioned shortcoming of prior art, the object of the present invention is to provide a kind of probe, fluorescent sensor and method for quantitative detection of hepatitis B virus DNA, for solving the complicated operation and high cost of HBV DNA detection method in the prior art , poor stability and other issues

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  • Probe and fluorescence sensor for quantitatively detecting hepatitis B virus DNA and method
  • Probe and fluorescence sensor for quantitatively detecting hepatitis B virus DNA and method
  • Probe and fluorescence sensor for quantitatively detecting hepatitis B virus DNA and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Fabrication of fluorescent sensors and detection of DNA

[0053] 1. Materials and methods

[0054] 1.1 Materials

[0055] HPLC-purified oligonucleotides were synthesized by Shanghai Sangong. AgNO 3 and NaBH 4 Purchased from SIGMA-ALDRICH Co., Ltd. (St. Louis, USA). PBS buffer (20mM, pH 7.0) was purchased from Shanghai Sangong Company.

[0056] 1.2 Testing instruments

[0057] The Cary Eclipse fluorescence spectrophotometer is a product of Agilent.

[0058] 1.3 Detection principle

[0059] figure 1 The detection principle of the present invention is shown: in a homogeneous system, the target substance, the first probe enhanced sequence G-rich and the second probe DNA silver nano-cluster sequence AgNCs-HBV form a three-stranded structure, and G-rich can Enhanced fluorescent signal of AgNCS-HBV. The content of the target substance can be known by detecting the fluorescent signal.

[0060] The nucleotide sequence of G-rich is:

[0061] 5'-TCCATATAACTTCGCATGGGTGG...

Embodiment 2

[0079] Validation of the feasibility of a fluorescent sensor for the detection of HBV DNA

[0080] In embodiment 1, the overall detection process is verified with page electrophoresis fluorescence detection, and the results are as follows figure 2 shown.

[0081] figure 2 Shown are the comparison graphs of fluorescence signals and electrophoresis graphs (insert graphs) to verify the feasibility, in which the red curve is the experimental group, the black curve is the control group lacking the target substance, and the blue curve is the control group with only AgNCs-HBV; figure 2 The insert figure in is the electrophoresis verification figure, in which lane 1 is the target substance, and lane 2 is AgNCs-HBV and G-rich.

[0082] Depend on figure 2 It can be seen that when only DNA silver nanoclusters exist, the fluorescence signal is extremely low; when there is no target substance, the nanocluster probes will not self-combine to generate fluorescent signals; when the tar...

Embodiment 3

[0085] Fluorescence sensor for detection of HBV DNA and optimization of its application conditions

[0086] In this embodiment, several important conditions in the experimental process are: the number of stem bases combined with triple strands; the reaction temperature of G-rich and AgNCs-HBV hybrid system, the reaction time of G-rich and AgNCs-HBV hybrid system; DNA, The configuration ratio of silver nitrate and sodium borohydride; the reaction pH of the fluorescent reaction system (ie buffer pH); High values ​​select at least five points respectively for a series of experiments.

[0087] In order to investigate the influence of the number of bases in the stem structure on the stability and reaction speed of the developed fluorescent sensor, this embodiment further studies the optimal number of foothold bases (2 to 10), and the results are as follows image 3 shown. Depend on image 3 It can be seen that when the number of stem bases is 4, 5, and 6, the signal-to-noise rat...

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Abstract

The invention discloses a probe and a fluorescence sensor for quantitatively detecting hepatitis B virus DNA and a method. The fluorescence sensor comprises a first probe enhanced sequence G-rich and a second probe DNA silver nanocluster sequence AgNCs-HBV, the G-rich and the AgNCs-HBV dissolve in a buffer solution, a target substance to be detected is added, mixed incubation is performed, a fluorescence reaction system is constructed, fluorescence detection is performed, and a fluorescence signal can be obtained. The fluorescence sensor provided by the invention realizes high-sensitivity detection of HBV based on a silver nano-cluster beacon, can improve the detection speed by tuning DNA silver nano-cluster sequence AgNCs-HBV fluorescence through the proximity enhancement sequence G-rich, is low in synthesis cost, can overcome the limitation of the traditional PCR method for detecting HBV on site and personnel requirements, is good in universality, stability and reproducibility, and is suitable for large-scale screening or quantitative detection of the hepatitis B HBV in communities or hospitals.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, a fluorescent sensor and a method for quantitatively detecting hepatitis B virus DNA. Background technique [0002] Hepatitis B virus (HBV) is an important human infectious agent that has caused serious health problems of public concern worldwide. The virus is extremely harmful and spreads widely through blood and bodily fluids. HBV infection can lead to liver-related diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Generally, the concentration level of HBV DNA in serum is considered as an important index to evaluate the therapeutic effect of chronic hepatitis B. Therefore, accurate quantitative detection of serum HBV DNA is of great significance for infection diagnosis. [0003] At present, the most commonly used clinical HBV gene detection methods are quantitative polymerase chain reaction (qPCR) and molecular beacon (MB) technology. qPCR ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/682G01N21/64C12N15/11
CPCC12Q1/706C12Q1/682G01N21/6428C12Q2563/137C12Q2565/607C12Q2563/107
Inventor 丁世家苟小龙伊明
Owner CHONGQING MEDICAL UNIVERSITY
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