Portable nucleic acid detection method based on glucose signal

A detection method and technology for glucose, applied in the fields of biology and chemistry, to achieve the effect of low price, no need for professional equipment, and high sensitivity

Pending Publication Date: 2022-07-26
ZHEJIANG UNIV
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Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects of the existing nucleic acid detection technology, with the help of the CRISPR Cas system and the functionalized magnetic bead sensor, to construct a new platform that is portable, does not require the assistance of large instruments, is low in cost, has good selectivity and high sensitivity

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  • Portable nucleic acid detection method based on glucose signal
  • Portable nucleic acid detection method based on glucose signal
  • Portable nucleic acid detection method based on glucose signal

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Embodiment Construction

[0061] The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings:

[0062] 1. Preparation of functionalized magnetic bead sensors

[0063] 1.1 Preparation of conjugated invertase functional magnetic beads

[0064] The preparation of functionalized magnetic beads is mainly divided into four steps: (1) Activation of the 3'-terminal thiol group in Bio-Linker-SH: 5 μL of 1 mM MBs-Linker, 1 μL of 30 mM tris(2-carboxyethyl) phosphine (TCEP) and 1 μL of buffer A (1M phosphate buffer, pH 5.5), mixed well, and incubated at room temperature for 1 h. The activated Bio-Linker-SH (ie, SEQ ID No. 18 of the sequence listing) was isolated by Amicon-3K, and washed with buffer B (0.1M NaCl, 0.1M phosphate buffer, pH 7.3), which was repeated 8 times. (2) Activation of invertase: prepare 2.0-60.0 mg / mL invertase aqueous solution, and add 1 mg of 4-(N-maleimidomethyl)cyclohexane-1-ca...

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Abstract

The invention provides a portable nucleic acid detection method based on a glucose signal. According to the design, by means of a chemical coupling mode, enzyme production is modified to magnetic beads by utilizing ssDNA, so that the functionalized magnetic bead sensor is prepared. The specific detection process comprises the following steps: (1) amplifying a nucleic acid detection fragment by virtue of an isothermal amplification technology; (2) identifying the amplified fragment through a CRISPR / Cas12a system, and releasing glucoproducing enzymes on the functionalized magnetic beads by using nuclease activity of the amplified fragment; and (3) hydrolyzing the free enzyme catalytic substrate through magnetic separation to form glucose. And (4) the glucose concentration can be measured by virtue of a portable glucometer, so that quantitative detection of nucleic acid is realized. The method is convenient to operate, free of assistance of large instruments, low in cost, good in selectivity and high in sensitivity, and has a certain application prospect.

Description

technical field [0001] The invention belongs to the fields of biology and chemistry, and relates to a portable nucleic acid detection method based on glucose signals. Background technique [0002] Nucleic acid detection is to use the theory and technology of molecular biology and biochemistry to directly or indirectly probe the existence or integrity of nucleic acid, and analyze the structure and function of nucleic acid from the level of nucleic acid structure, replication, transcription or translation. Studies have shown that known organisms other than prions contain nucleic acid molecules, so nucleic acid detection has important application value in many fields such as life genetic research, infectious disease detection, genetic disease diagnosis, and tumor research. [0003] The most widely used nucleic acid detection methods include sequencing technology and nucleic acid amplification technology. Taking the nucleic acid detection of SARS-CoV-2 as an example, Wu et al. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N11/14C12R1/93
CPCC12Q1/701C12Q1/6844C12N11/14C12N9/2431C12N9/2411C12N9/2454C12N9/2465C12N9/2471C12Y302/01022C12Y302/01023C12Y302/01011C12Y302/01026C12Q2521/327C12Q2525/161C12Q2563/125C12Q2563/143C12Q2563/149
Inventor 徐志南黄迪石朱伟方蒙君
Owner ZHEJIANG UNIV
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