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Fluorescent biosensor to detect BRCA1 gene and preparation method thereof

A biotin and gene detection technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of poor specificity, long detection time and cumbersome process of BRCA1 gene detection, and achieve simple and convenient operation and detection speed. Fast and reproducible results

Active Publication Date: 2019-07-05
CHONGQING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a fluorescent biosensor for detecting BRCA1 gene and its preparation method, which is used to solve the problem of poor detection specificity, cumbersome process, and time-consuming detection of BRCA1 gene in the prior art. long wait question

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  • Fluorescent biosensor to detect BRCA1 gene and preparation method thereof
  • Fluorescent biosensor to detect BRCA1 gene and preparation method thereof
  • Fluorescent biosensor to detect BRCA1 gene and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Preparation of fluorescent BRCA1 biosensor

[0061] 1. Materials and methods

[0062] 1.1 Materials

[0063] Streptavidin-modified magnetic beads (SMBs, product number: S1420s) and Nb.BbvCI (product number: R0631L) were purchased from NEB Company in the United States, and the HPLC purified oligonucleotide sequences (including SEQ ID NO.1-8 Sequences shown) were synthesized by Shanghai Sangon Bioengineering Co., Ltd. TE buffer (10mM TrisHCl, 1mM EDTA, pH 8.0, catalog number: B548106-0500), PBS buffer (136.89mM NaCl; 2.67mM KCl; 8.1mM NaCl 2 HPO 4 ; 1.76mM KH 2 PO 4 , Cat. No.: E607008-0500) was purchased from Shanghai Sangon Bioengineering Co., Ltd., 20bp DNA marker (Cat. No.: SD0524) was purchased from Dalian Takara Company, MgCl 2 (Product No.: MB0328-500G) was purchased from Chongqing Maoye Chemical Reagent Co., Ltd.

[0064] 1.2 Testing instruments

[0065] The Cary Eclipse fluorescence spectrometer is a product of Agilent, California.

[0066] 1....

Embodiment 2

[0088] Example 2 Verifies the feasibility of the fluorescent BRCA1 detection sensor

[0089] 1. We verified the feasibility of the constructed double amplification system by fluorescence method and non-denaturing polyacrylamide gel electrophoresis.

[0090] Such as figure 2 As shown, curve a: represents the fluorescence signal of the triplex L-P-S, showing a low signal, indicating a small background.

[0091] Curve b: Indicates the fluorescent signal generated when there is only a lack of target substance in the system. Due to the lack of target substance, PWTs still maintain a double-stranded state, so DWTs and SDs cannot form the recognition site of Nb.BbvCI, and DNA Walker cannot trigger it. No intermediate chain will be generated. Although L-P-S and A exist at this time, the entropy drive cannot be triggered, so the signal is relatively low.

[0092] Curve c: Indicates the fluorescent signal generated in the absence of Nb.BbvCI. In this case, although the target substan...

Embodiment 3

[0097] Example 3 Fluorescent BRCA1 detection sensor and optimization of its use conditions

[0098] We also further optimized several important conditions during the experiment, namely the ratio of auxiliary single-chain PWTs and SDs, the concentration of Nb.BbvCI, the digestion time of Nb.BbvCI and the reaction time of ESDRs. For each condition, five points were selected from low concentration to high concentration to carry out a series of experiments.

[0099] 1. In order to investigate the effect of the ratio of single-chain PWTs and SDs on the fluorescent BRCA1 detection sensor, this experiment adopts a fixed SDs concentration of 1500nM, using different concentrations of single-chain PWTs (0, 25, 50, 75, 100nM), the results It shows that when the concentration of single-chain PWTs reaches or exceeds 75nM, that is, when the molar ratio of PWTs to SDs reaches or exceeds 1:20, the generated fluorescence response signal reaches the maximum value. Such as Figure 4 It can be ...

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Abstract

The invention provides a fluorescent biosensor to detect BRCA1 gene and a preparation method thereof. A composite for detecting BRCA1 gene comprises biotin-modified DNA Walker chains (DWTs), a protective probe (PP), biotin-modified substrate chain DNA (SDs), streptavidin-modified magnetic beads (SMBs), a FAM-modified fluorescent signal chain P, a Dabcyl-modified L chain, an S chain and an auxiliary chain A. The fluorescent biosensor and detection system to detect BRCA1 are constructed successfully herein; BRCA1 measurement with the fluorescent biosensor applied herein shows high sensitivity, good stability and good reproducibility. Compared with the prior art, the fluorescent biosensor has high detection speed, good sensitivity, good operational simplicity and convenience, short detectioncycle, good specificity, low false positive rate and low false negative rate. The fluorescent biosensor is applicable to the measurement of actual samples and the like and is expectedly applicable asa sensor having actual application value.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a fluorescent biosensor for detecting BRCA1 gene and a preparation method thereof. Background technique [0002] China used to be considered a low-risk breast cancer country, but in recent years, with the continuous improvement of people's living standards, changes in lifestyles and environmental pollution caused by industrialization, the incidence of breast cancer in China is 3% per year- 4% growth rate, much higher than the international average annual growth rate. China has become the main country of breast cancer incidence. According to the World Population Standardized Incidence and Mortality Rankings, the incidence of breast cancer ranks fourth among all malignant tumors, and the mortality rate ranks sixth among all malignancies. In China's coastal developed provinces, the incidence of breast cancer is much higher than in other regions. Breast cancer has bec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806
CPCC12Q1/6886C12Q1/6806C12Q2563/131
Inventor 颜玉蓉丁世家程伟马洪敏晏小玉王通阙海英刘萍甘秀峰
Owner CHONGQING MEDICAL UNIVERSITY
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