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Hairpin probe group based on enzyme assisted cascade cycle amplification, preparation method and use

A technology of hairpin probes and cascading cycles, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as complex operating procedures and expensive equipment

Inactive Publication Date: 2020-10-20
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The HPLC method has good sensitivity and high selectivity, but the equipment required is expensive and the operation procedures are complicated, requiring professionals to operate

Method used

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  • Hairpin probe group based on enzyme assisted cascade cycle amplification, preparation method and use
  • Hairpin probe group based on enzyme assisted cascade cycle amplification, preparation method and use
  • Hairpin probe group based on enzyme assisted cascade cycle amplification, preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] This embodiment provides a hairpin probe set based on enzyme-assisted cascade cyclic amplification, including the following probes:

[0068] The first hairpin probe, from the 5' end to the 3' end, sequentially includes an aptamer sequence that binds to a biological target and a first binding sequence, and part of the base pairing between the aptamer sequence and the first binding sequence makes the first hairpin probe Form a hairpin structure. Wherein, the aptamer sequence includes sequence a, sequence b and sequence c sequentially from the 5' end to the 3' end, and the first binding sequence includes sequence d sequentially from the 5' end to the 3' end, and sequence b complementary to sequence b * . Sequence b of the aptamer sequence and sequence b of the first binding sequence * The pairing forms the stem region of the hairpin probe, and the sequence d of the aptamer sequence and the sequence c of the first binding sequence form the stem loop region of the hairpin ...

Embodiment 2

[0079] This embodiment provides a method for preparing a hairpin probe set based on enzyme-assisted cascade cycle amplification, comprising the following steps:

[0080] Centrifuge the dry powder of hairpin probe sequences H1, H2 and H3 at 12000rpm for 5 minutes, and then dissolve them in 20mM Tris-HNO 3 Buffer (20mM NaNO 3 ,10mM NH4 NO 3 ,2mM Mg(NO 3 ) 2 , pH 7.4), a 100 μM stock solution was obtained. Next, stock solutions (100 μM) of hairpin probe sequences H1, H2 and H3 were heated to 95° C. for 5 minutes, and then slowly cooled to room temperature to form hairpin structures.

Embodiment 3

[0082] This embodiment provides a fluorescent detection method for a biological target, specifically aflatoxin B1 (AFB1), comprising the following steps:

[0083] S1, mix the sample to be tested, the first hairpin probe, the second hairpin probe, the third hairpin probe and T7 exonuclease in the first buffer solution, heat the mixture and incubate, then cool to room temperature , to obtain the first reaction solution; specifically:

[0084] Mix 1 μL of the sample to be tested that may contain the target AFB1, 2 μL of the first hairpin probe H1 (1 μM), 2 μL of the second hairpin probe H2 (1 μM), 3 μL of the third hairpin probe H3 (1 μM) and 1 μL of T7 Exo (10U / μL) was sequentially added to 42μL of 1×NEBuffer 4 buffer (20mM Tris-HNO 3 ,50mM KNO 3 ,10mM Mg(NO 3 ) 2 , 1mM DTT, pH 7.9). The mixture was incubated at 37° C. for 90 minutes, and then cooled to room temperature to obtain the first reaction solution.

[0085] S2, taking the first reaction solution and adding it to ...

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Abstract

The invention relates to the field of biochemical analysis and detection, and in particular relates to a hairpin probe group based on enzyme assisted cascade cycle amplification, a preparation methodand use. According to the hairpin probe group provided by the invention, a biological target to be detected is used as a first-level target to trigger first target cycle amplification, so that enrichment amplification of a secondary target sequence is realized; simultaneously, the secondary target sequence also can trigger second target cycle amplification; fluorescent signal sequences are continuously released; and thus, cascade cycle amplification of the fluorescent signal sequences is realized. By adoption of the hairpin probe group, a lot of fluorescent signal sequences can be triggered and generated only through trace biological targets; the very high fluorescence signal intensity is generated; the detection sensitivity of the biological target is obviously increased; the detection limit is increased; simultaneously, the specificity of the cascade cycle amplification of the probe in combination with the biological target is high; and a detection result is accurate.

Description

technical field [0001] The invention relates to the field of biochemical analysis and detection, in particular to a hairpin probe set based on enzyme-assisted cascade cycle amplification, a detection system, a biosensor, a preparation method, and an application. Background technique [0002] At present, biochemical analysis research mainly focuses on the analysis and detection of bioactive substances, heavy metals, biomacromolecules (nucleic acids, proteins, enzymes and polypeptides, etc.), biopharmaceuticals and microorganisms in organisms, food or the environment. Therefore, biochemical analysis and detection are of great significance to the preventive diagnosis of diseases, environmental monitoring, and food monitoring. Usually, only trace amounts of target substances exist in the actual biochemical analysis samples used for biochemical analysis detection, so high sensitivity and high accuracy have always been the improvement goals of biochemical analysis detection method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6844
CPCC12Q1/6844C12Q2525/205C12Q2525/301C12Q2563/107C12Q2565/607C12Q2521/319
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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