Isothermal amplification system and method based on fluorescence self-inhibition probe

An isothermal amplification and self-suppression technology, applied in the field of temperature amplification systems, can solve the problems of reducing fluorescent background signals, low detection efficiency, and poor specificity

Pending Publication Date: 2021-05-28
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to: overcome the defects of low sensitivity, poor specificity and low detection efficiency of the existing isothermal amplification technology, and provide an isothermal amplification system and amplification method based on fluorescent self-inhibiting probes, which can effectively reduce the detection rate of the reaction. The fluorescent background signal of the needle, while reducing the mismatch and non-specific extension between the probes, significantly improves the specificity and sensitivity of the isothermal amplification method for rapid detection of miRNA

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  • Isothermal amplification system and method based on fluorescence self-inhibition probe
  • Isothermal amplification system and method based on fluorescence self-inhibition probe
  • Isothermal amplification system and method based on fluorescence self-inhibition probe

Examples

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Embodiment 1

[0056] The product specificity analysis of embodiment 1 fluorescent self-inhibiting probe isothermal amplification method

[0057] This example uses the human hsa-miR-223 standard as a sample to provide an isothermal amplification system based on fluorescent self-inhibiting probes for miRNA detection.

[0058] The human hsa-miR-223 sequence was selected as the target sequence (SEQ ID NO.6); for the target sequence, a specific fluorescent self-inhibitory probe was designed, and the probe sequence was: SEQ ID NO.7-8; miR-223 The standard template was added to 20 μL of the isothermal amplification reaction system. The concentrations of the components in the reaction system are: 50mM Tris-HCl (pH 7.9@25°C), 100mM NaCl, 10mM MgCl 2 , 1mM DTT, 0.1U / μL Vent DNA polymerase, 0.2U / μL Nt.BstNBI nicking endonuclease, 0.125mMdNTPs, 0.125μM fluorescent self-inhibiting linear probe, 0.125μM fluorescent self-inhibiting hairpin probe. The isothermal amplification reaction condition is 55°C, ...

Embodiment 2

[0059] The detection sensitivity test of embodiment 2 fluorescent self-inhibiting probe isothermal amplification method

[0060] In this example, several different concentrations of human hsa-miR-223 standards were used as samples to analyze the sensitivity of the isothermal amplification detection method for fluorescent self-inhibiting probes.

[0061] The human hsa-miR-223 sequence was selected as the target sequence, and the hsa-miR-223 standard was diluted tenfold, and the miR-223 templates obtained at different concentrations were added to 20 μL of the isothermal amplification reaction system, and other The components and concentrations were the same as in Example 1, the isothermal amplification reaction lasted 90 minutes, and the fluorescent signal was collected every minute to obtain the following: Figure 4 The real-time fluorescent quantitative amplification curve and standard curve are shown. The results show that the fluorescent self-inhibiting probe isothermal amp...

Embodiment 3

[0062] The detection specificity test of embodiment 3 fluorescent self-inhibiting probe isothermal amplification method

[0063] In this embodiment, the hsa-miR-223 standard product was used as the wild-type control group, and the homologous sequences hsa-miR-223-5p and hsa-miR-599, and the mutant sequences (hsa-miR-223-Mut1, hsa-miR-223-Mut1, hsa-miR-223-Mut2, hsa-miR-223-Mut3) were detected to analyze the detection specificity of the fluorescent self-inhibiting probe isothermal amplification method.

[0064] The hsa-miR-223 sequence was used as the target sequence of the control group; its specific probe sequence was used as the detection probe; the hsa-miR-223 sequence (SEQ ID NO.6), hsa-miR-223- 5p (SEQ ID NO.9), hsa-miR-599 (SEQ ID NO.10), hsa-miR-223-Mut1 (SEQ ID NO.11), hsa-miR-223-Mut2 (SEQ ID NO.12) and the standard substance of hsa-miR-223-Mut3 (SEQ ID NO.13) were added to a 20 μL isothermal amplification reaction system, and the other components and concentrations ...

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Abstract

The invention provides an isothermal amplification system and method of a fluorescence self-inhibition probe. The isothermal amplification system comprises a combination of two fluorescence self-inhibition probes, a Vent DNA polymerase, an Nt.BstNBI nicking endonuclease, a buffer solution, a reaction raw material and miRNA target molecules. The fluorescence self-inhibition probes comprise a fluorescence self-inhibition linear probe and a fluorescence self-inhibition hairpin probe. The invention also provides an amplification method of the isothermal amplification system. According to the fluorescence self-inhibition probe isothermal amplification system and the amplification method, background fluorescence signals of the probe can be remarkably reduced, the detection signal-to-noise ratio is greatly increased, and detection of trace miRNA targets in a sample is facilitated. Meanwhile, no complementary sequence exists between the two background-free fluorescent probes, mismatching and extension between the probes can be prevented, the detection specificity is effectively improved, false positive results are avoided, and the system and method are particularly suitable for direct detection of miRNA molecular markers in clinical samples.

Description

technical field [0001] The invention relates to the technical fields of medical detection and molecular diagnosis, in particular to an isothermal amplification system and an amplification method of a fluorescent self-inhibiting probe for rapid detection of trace miRNA. Background technique [0002] MicroRNA (miRNA) is a kind of single-stranded non-coding RNA with a length of about 18-24 nucleotides, which does not code for protein, but can be widely involved in the regulation of genes. At present, circulating miRNA has been proven to be an important molecular marker for early detection of tumors and efficacy monitoring, and can be used for early screening and prognosis diagnosis and treatment of cancer. Usually, the content of circulating tumor miRNA molecular markers in human body fluids is low, and at the same time, it will be interfered by other nucleic acid molecules in body fluids. More importantly, the combined use of multiple miRNA molecular markers will greatly impr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2525/207C12Q2561/101C12Q2565/1015C12Q2545/101C12Q2521/301
Inventor 朱灵赵俊王全福朱灿灿杨柯汪磊邓国庆刘勇
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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