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CRISPR-Cas12a-based MOF-DNA hydrogel colorimetric detection kit and method

A MOF-DNA, detection method technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., to achieve the effect of intuitive results

Active Publication Date: 2021-02-09
INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of Cas12a combined with nanomaterials for highly sensitive detection of non-nucleic acid targets in the fields of disease diagnosis, food quality, and environmental control has rarely been reported.

Method used

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  • CRISPR-Cas12a-based MOF-DNA hydrogel colorimetric detection kit and method
  • CRISPR-Cas12a-based MOF-DNA hydrogel colorimetric detection kit and method
  • CRISPR-Cas12a-based MOF-DNA hydrogel colorimetric detection kit and method

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Embodiment 1

[0053]This example is used to illustrate the method for colorimetric detection of estradiol based on the CRISPR-Cas12a MOF-DNA hydrogel of the present invention. The specific implementation steps are as follows:

[0054]1) Synthesis of precursor Ps-X / Y: primer X / Y (50μL, 100μmol / L), 30% AA (6.2μL) and 5×TAE / Mg2+(18.5μL) After mixing, place it under vacuum for 15 minutes. Subsequently, 1% APS (13.3 μL) and 3.5% TEMED were added and placed under vacuum for 20 minutes to obtain Ps-X / Y. The sequence of primer X is 5'-Acrydite-TTATTCTTGTCTCCCGAGAT-3' (SEQ ID NO: 1); the sequence of primer Y is 5'-Acrydite-TTATTTCACAGATGAGTATC-3' (SEQ ID NO: 2).

[0055]2) Synthesis of hydrogel: Ps-X (30μL), Ps-Y (30μL), Linker (20μL, 50μmol / L) and MOF (20μL, 1mg / mL) are mixed and reacted together at 65℃ for 5min, and then cooled to Keep it at 25°C for 20 minutes to form a gel.figure 2 TEM images of MOF and hydrogel MOF are shown. The sequence of the linker is 5'-GATACTCATCTGTGATTATTTTATTTTATTATCTCGGGAGACAAG-3'...

Embodiment 2

[0064]This example is used to illustrate the method for colorimetric detection of estradiol in milk based on the CRISPR-Cas12a MOF-DNA hydrogel of the present invention.

[0065]1) Pre-treatment of milk samples: Centrifuge the milk purchased from the supermarket for 20 minutes to remove the upper fat. Then use phosphate buffer to dilute at a ratio of 1:10 for use.

[0066]2) Magnetic bead recognition: Hybridize a solution of MMPs-APT (500μL, 1mg / mL) and excess complementary oligonucleotide (cDNA) at 37°C for 30min to obtain MMPs-apt-cDNA complex. Remove unreacted cDNA by magnetic separation and resuspend to 50 μL. Then, 5μL of the target substance (E2 / PSA) standard solution and 5μL of MMPs-apt-cDNA were incubated at 37°C for 1h. Subsequently, the dissociated cDNA (8 μL) was collected by magnetic separation.

[0067]3) CHA isothermal amplification: all DNA is denatured at 95℃ for 5 minutes and then slowly cooled, and the cooling and annealing process lasts for 1 hour; H1 (1μL, 100μM) and H2 (...

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Abstract

The invention belongs to the field of biological detection, and relates to a CRISPR-Cas12a-based MOF-DNA hydrogel colorimetric detection kit and method. The method comprises the following steps: S1, preparing DNA hydrogel-MOF; S2, hybridizing MMPs-APT and a solution of complementary oligonucleotide cDNA to obtain MMPs-apt-cDNA, performing magnetic separation to remove unreacted cDNA and performingre-suspension, incubating a sample solution and a MMPs-apt-cDNA re-suspension solution, and collecting dissociated cDNA through magnetic separation; S3, performing CHA isothermal amplification on thecollected cDNA; S4, incubating Cas12a, crRNA and a Cas12a matching buffer solution, and adding the DNA hydrogel-MOF and an isothermal amplification product to obtain cut hydrogel; and S5, performingcolor development and detection on the cut hydrogel. According to the method, the limitation that a CRISPR-Cas12a-based sensor is only used for nucleic acid detection is broken through, and the CRISPR-Cas12a-based sensor is combined with DNA hydrogel to be successfully applied to the detection of an estradiol small-molecule target object.

Description

Technical field[0001]The invention belongs to the field of biological detection, and specifically relates to a CRISPR-Cas12a-based MOF-DNA hydrogel colorimetric detection kit for small molecules, and a CRISPR-Cas12a-based MOF-DNA hydrogel colorimetric detection kit Small molecule approach.Background technique[0002]Estradiol (estradiol, E2), also known as "estrus" and "coupling hormone". Secreted by granular cells of follicles in the ovary. Its metabolites are estrone and estriol. The target organs are the uterus, vagina, fallopian tube and pituitary. As a common estrogen, it can interfere with human body secretion and endanger human health. Due to the abuse of steroid growth hormone, E2 is widely present in commodities such as meat and dairy products. Hormones in animals are excreted into the environment as a typical environmental endocrine disruptor. E2 has the characteristics of non-degradability, long residual period and lipophilicity, and can be absorbed into the body through th...

Claims

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Application Information

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IPC IPC(8): C12Q1/682C12Q1/6825
CPCC12Q1/682C12Q1/6825C12Q2525/205C12Q2531/119C12Q2521/327C12Q2545/114C12Q2565/607C12Q2563/103Y02A50/30
Inventor 高志贤王瑜彭媛李双韩殿鹏任舒悦秦康
Owner INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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