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Biological probe and detection method for detecting miRNA and application

A biological probe and probe technology, applied in the field of biosensors, can solve problems such as complex experimental design, increased experimental cost, and a large number of molecular beacons, achieving high analytical performance, high sensitivity and specificity, and improved sensitivity

Active Publication Date: 2019-10-22
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the inventors have found that there are still some deficiencies in the above-mentioned literature, such as in scheme 1, a large number of molecular beacons and primers are required, which increases the cost of the experiment, and the experimental design is relatively complicated, and in the two cycle processes, only constant Turn on the molecular beacon to achieve fluorescence emission. The molecular beacon itself is not amplified, and is limited by the concentration of the molecular beacon, which affects the sensitivity of the detection.

Method used

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  • Biological probe and detection method for detecting miRNA and application
  • Biological probe and detection method for detecting miRNA and application
  • Biological probe and detection method for detecting miRNA and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Biological probes for detecting miRNA

[0060] This embodiment provides a biological probe for detecting miRNA, including a first arch probe and a second arch probe; the first arch probe and the second arch probe are both single-stranded Complementary hybridization of the terminal bases of linear molecules;

[0061] The first arch probe includes a target miRNA binding sequence region, an endonuclease recognition sequence region and a DNA polymerase extension sequence region; the first arch probe binds to the target miRNA and undergoes a chain extension and shearing reaction. The released oligonucleotides are secondary primers;

[0062] The second arch probe includes a secondary primer binding sequence region, an endonuclease recognition sequence region and a DNA polymerase extension reporter sequence region; the second arch probe is combined with the secondary primer and undergoes chain extension and The cleavage reaction releases the reporter sequence.

[0063] Fur...

Embodiment 2

[0069] Example 2 Biosensor for detecting miRNA

[0070] This embodiment provides a biosensor for detecting miRNA, including the biological probe for detecting miRNA prepared in Example 1.

[0071] Further, it includes A reaction reagent and B reaction reagent, and the A reaction reagent includes: a first arch probe, 1000 nM (nmol / L), 5 μL;

[0072] MiRNA to be tested, 5μL;

[0073] Endonuclease buffer, 15μL; the endonuclease buffer is 0.5×Nt.BstNBI buffer, 0.5×Nt.BstNBI buffer contains 25mM (mmol / L) Tris-HNO 3 , 50mM NaNO 3 , 5mM Mg(NO 3 ) 2 , And 0.5mM dithiothreitol (dithiothreitol) aqueous solution, pH 7.9, 25℃;

[0074] The B reaction reagent includes:

[0075] The second arch probe, 1500nM, 5μL;

[0076] DNA polymerase, 1U / μL, 2.5μL; the DNA polymerase is Vent (exo-) DNA polymerase

[0077] Endonuclease, 4U / μL, 2.5μL; said endonuclease is Nt.BstNBI endonuclease;

[0078] dNTPs, 5000μM, 5μL;

[0079] RNase inhibitor, 3U / μL, 5μL;

[0080] 1×ThermoPol buffer solution, 5μL; the 1×ThermoPol b...

Embodiment 3

[0083] Example 3 Biosensor for detecting miRNA

[0084] This embodiment provides a biosensor for detecting miRNA, including the biological probe for detecting miRNA prepared in Example 1.

[0085] Further, it includes A reaction reagent and B reaction reagent, and the A reaction reagent includes: a first arch probe, 1500 nM, 5 μL;

[0086] MiRNA to be tested, 3μL;

[0087] Endonuclease buffer, 17μL; the endonuclease buffer is 0.5×Nt.BstNBI buffer, 0.5×Nt.BstNBI buffer is 25mM Tris-HNO 3 , 50mM NaNO 3 , 5mM Mg(NO 3 ) 2 , And 0.5mM dithiothreitol (dithiothreitol) aqueous solution, pH 7.9, 25℃;

[0088] The B reaction reagent includes:

[0089] The second arch probe, 1500nM, 5μL;

[0090] DNA polymerase, 1U / μL, 3μL; the DNA polymerase is Vent (exo-) DNA polymerase

[0091] Endonuclease, 6U / μL, 2μL; The endonuclease is Nt.BstNBI endonuclease;

[0092] dNTPs, 6000μM, 5μL;

[0093] RNase inhibitor, 4U / μL, 3μL;

[0094] 1×ThermoPol buffer solution, 7μL; the 1×ThermoPol buffer solution contains 20mM ...

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Abstract

The invention belongs to the technical field of biosensors and relates to a biological probe and detection method for detecting miRNA and application. The biological probe comprises a first arch probebody and a second arch probe body; the first arch probe body and the second arch probe body are both formed by complementary hybridization of terminal bases of two single-stranded linear molecules; the first arched probe body comprises a target miRNA binding sequence region, an endonuclease recognition sequence region and a DNA polymerase extension sequence region; oligonucleotide which is released through chain extension and a shear reaction of the first arch probe body and the target miRNA after binding is adopted as a secondary primer; the second arch probe body comprises a secondary primer binding sequence region, an endonuclease recognition sequence region and a DNA polymerase extension report sequence region; the second arch probe body is bonded with the secondary primer and then subjected to chain extension and a shear reaction to release a report sequence. The miRNA detection method based on mediation of the biological probe of the miRNA has the advantages of being high in sensitivity and specificity, simple in design, rapid in detection and low in cost.

Description

Technical field [0001] The invention belongs to the technical field of biosensors, and specifically relates to a biological probe for detecting miRNA and a fluorescent biosensor for detecting miRNA, its use and a method for detecting miRNA. Background technique [0002] MicroRNA (miRNA) is a type of endogenous short non-coding RNA molecule with a length of 17-25 nucleotides. It is produced by two RNase III enzymes (Drosha and Dicer) continuously cutting long primary transcripts. miRNAs have regulatory functions and are widely present in eukaryotes. Most miRNAs are also highly conservative, sequential and tissue specific. So far, more than 1,000 miRNAs have been found in the human genome. Because they can inhibit the transcription and translation of target mRNA, or can cut target mRNA and promote its degradation, they play a vital role in a variety of human biological processes. The role of cell growth, differentiation, apoptosis and proliferation. More and more evidences show t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2533/10C12Q2521/301C12Q2565/607C12Q2525/207C12Q2563/137Y02A50/30
Inventor 吴昊邹霈刘娅灵王洪勇吴军韩国庆
Owner JIANGSU INST OF NUCLEAR MEDICINE
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