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Fluorescence detection kit based on simultaneous detection on DNA and miRNA

A fluorescence detection and kit technology, applied in the field of detection kits and fluorescence detection kits, can solve the problems of high detection cost, long detection time, complicated detection process, etc., and achieve good response, simplified detection process, high sensitivity and high sensitivity. repeatable effect

Active Publication Date: 2018-08-21
XIANGTAN UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the shortcomings in the prior art and improve the existing photochemical analysis technology in the detection of multiple disease markers, the process is complicated, the detection cost is high, and the detection time is long. The purpose of the present invention is to provide a low-cost, high-efficiency Sensitive, high-selective detection, designed based on the simultaneous detection of DNA and miRNA fluorescence detection kits, can be efficient, fast and highly sensitive simultaneous detection of DNA and miRNA

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  • Fluorescence detection kit based on simultaneous detection on DNA and miRNA
  • Fluorescence detection kit based on simultaneous detection on DNA and miRNA
  • Fluorescence detection kit based on simultaneous detection on DNA and miRNA

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Effect test

Embodiment 1

[0025] Design of a detection probe system in a fluorescent detection kit for simultaneous detection of viral DNA and miRNA:

[0026] The system consists of three parts: the locked G-quadruplex is connected to the 5' of the DNA end, and the weak light AgNCs and cDNA complementary sequences are connected to the 3' of the other end. Region 1 and region 2 on the probe are compatible with the target 1 and target 2 complementary sequence

Embodiment 2

[0028] Feasibility determination of fluorescence detection kit in simultaneous detection of DNA and miRNA (1) Fluorescence spectrum test

[0029] Such as image 3 As shown, the probe only responds to a specific target and emits at the corresponding wavelength. In the presence of T1 (Target 1), only the G-quadruplex / ThT dimer-specific fluorescence signal with a characteristic peak at 487 nm was recorded, but no obvious AgNC fluorescence signal was observed at the emission wavelength of 607 nm ( image 3 a). In contrast, no fluorescence signal of G-quadruplex / ThT dimer and strong AgNC fluorescence signal were observed in the presence of T2 (Target 2) ( image 3 b). The co-existence of T1 and T2 simultaneously enhanced the fluorescence signal of G-quadruplex / ThT dimer and AgNCs ( image 3 c). The results demonstrate the effectiveness of simultaneous detection of multiple viral genes.

[0030] (2) Circular dichroism (CD) spectrometry

[0031] Circular dichroism (CD) was use...

Embodiment 3

[0033] Sensitivity Determination of Fluorescent Detection Kit in Simultaneous Detection of DNA and miRNA

[0034] Such as Figure 5 As shown in a, the fluorescence intensity at the emission wavelength of 487 nm increases with the increase of H5N1 gene concentration. In addition, the fluorescence change value (F-F0) showed a clear linear dependence (R 2=0.9954)( Figure 5 b). The regression equation is F-F0=1.6493C+11.5513, wherein F0 and F represent the fluorescence intensity without and with the target object respectively, and C represents the concentration of T1 respectively. The estimated detection limit was 0.45 nM (3σ / slope, σ represents the standard deviation of the blank solution, n=11). Then, the change in the fluorescence emission spectrum at the T2 concentration was measured. As the concentration of H1N1 genes increased, Figure 5 c shows the enhanced fluorescence intensity at an emission wavelength of 607 nm. The F-F0 value of the calibration chart has a line...

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Abstract

The invention discloses a fluorescence detection kit based on simultaneous detection on DNA and miRNA. The kit comprises a fluorescence probe, metal ions, a fluorescence dye, and a reaction buffer. The fluorescence probe is used to carry out specific identification on a molecular recognition component; the terminal 5' of DNA of the probe is connected to a locked G-quadruplex; the 3' terminal of the probe and the locked area thereof are completely complementary with the hybridization sequence of cDNA / AgNCs (silver nano cluster), and the area 1 and the area 2 of the probe are complementarily paired sequences of a target 1 and a target 2. When a target exists, the G-quadruplex and AgNCs can generate a fluorescence signal at the same time; a universal platform is provided for detection of different targets; at the same time, the detection system has very good specificity, common analogs will not influence the detection results; during the whole detection process, separation or purificationis not needed; detection can be carried out after simple mixing, the operation is simple, the cost is low, and the responding is quick. The selectivity on similar oligonucleotides is high; the responding on targets in a serum sample is good, and the detection kit can further be used in biomedicine and clinical diagnosis.

Description

technical field [0001] The invention relates to a detection kit, in particular to a fluorescence detection kit based on simultaneous detection of DNA and miRNA, belonging to the field of molecular detection. Background technique [0002] Viral infectious diseases or cancers have always threatened human health and have had a huge impact on human society. The emergence of diseases may be accompanied by changes in various diagnostic indicators, which cause difficulties in early and accurate diagnosis during disease treatment. Simultaneous detection of multiple biomarkers, such as DNAs, miRNAs, small molecules, and proteins, has been attractive to researchers in recent years because of its higher detection efficiency and more precise diagnostic capabilities compared with single detection . However, due to the complexity of the detection system and the need for special signals that are not interfered, it is difficult to detect targets at the same time. Therefore, it is also ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816
CPCC12Q1/6816C12Q2525/207C12Q2563/107
Inventor 蔡昌群韩云鹏李诗雨
Owner XIANGTAN UNIV
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