阅读说明：本技术 基于crispr技术的核酸检测的信号放大磁珠技术系统及其应用 (Signal amplification magnetic bead technology system for nucleic acid detection based on CRISPR technology and application thereof ) 是由 郑敦武 于 2020-08-10 设计创作，主要内容包括：本发明公开了一种报告磁珠,所述报告磁珠包括磁珠、核苷酸和高催化活性的酶相链接。本发明还公开了基于CRISPR技术的核酸检测的信号放大磁珠技术系统,本发明还公开报告磁珠的构建方法以及应用。本发明通过引入报告磁珠,将信号放大磁珠技术系统的灵敏度从aM(1000-10000 copies/mL)浓度级别提高到zM(1-10 copies/mL)浓度级别。(The invention discloses a report magnetic bead, which comprises a magnetic bead, nucleotide and enzyme with high catalytic activity. The invention also discloses a signal amplification magnetic bead technology system for nucleic acid detection based on the CRISPR technology, and a construction method and application of the report magnetic bead. The invention improves the sensitivity of a signal amplification magnetic bead technical system from aM (1000-10000copies/mL) concentration level to zM (1-10copies/mL) concentration level by introducing the report magnetic bead.)

1. The report magnetic bead is characterized by comprising a magnetic bead, nucleotides and a high-catalytic-activity enzyme which are connected.

2. The report magnetic bead of claim 1, wherein the nucleotides are one or more of single-stranded DNA, double-stranded DNA, or single-stranded RNA.

3. The reporter magnetic bead of claim 1, wherein the nucleotide is biotin-linked ssDNA.

4. The reporter magnetic bead of claim 1, wherein the high catalytic activity enzyme is one of β -Gal enzyme, horseradish peroxidase, alkaline phosphatase, carboxylic ester hydrolase, glycoside hydrolase, protease, tyrosinase, monoamine oxidase, nitroreductase, thioredoxin reductase, and γ -glutamyltransferase.

5. The method for constructing a report magnetic bead according to any one of claims 1 to 4, comprising the steps of: and linking the magnetic beads and nucleotides in a covalent bond mode to obtain nucleotide-magnetic beads, and linking the enzyme with high catalytic activity and the nucleotide-magnetic beads through streptavidin and biotin.

6. Signal amplification magnetic bead technology system for nucleic acid detection based on CRISPR technology, characterized in that the signal amplification magnetic bead technology system comprises the report magnetic bead of any one of claims 1 to 4.

7. Use of the reporter bead of any one of claims 1 to 4 or the signal amplifying bead technology system of claim 6 for preparing a nucleic acid detection reagent or kit.

8. A nucleic acid detection kit comprising the reporter magnetic bead according to any one of claims 1 to 4 or the signal amplifying magnetic bead technology system according to claim 6.

9. An ultra-high sensitivity nucleic acid detection method based on CRISPR technology is characterized by comprising the following steps:

1) performing thermal inactivation and cracking on virus liquid to release RNA molecules of viruses, extracting the RNA molecules in virus suspension by using nano nucleic acid magnetic beads, adding the extracted magnetic beads into a reaction system of RT-PRA isothermal amplification, and performing RT-RPA amplification on target molecules to obtain first-stage signal amplification;

2) performing secondary extraction on DNA molecules obtained by amplifying the primary signals by using original nano magnetic beads, adding the DNA molecules into CRISPR protein with side-cleavage activity to specifically recognize and cleave virus target DNA molecules, activating the non-specific cleavage activity of the CRISPR protein with the side-cleavage activity, and starting the amplification of the secondary signals;

3) adding the report magnetic beads of any one of claims 1 to 4 into a reaction system, cutting ssDNA to release free enzymes with high catalytic activity into system supernatant by using non-specific cutting activity of CRISPR protein with side cutting activity, adsorbing all the magnetic beads by using a magnet, taking out supernatant containing the free enzymes with high catalytic activity, adding a fluorescent substrate for reaction, and performing reading detection by using an enzyme labeling instrument to obtain a detection result.

10. The nucleic acid detection method of claim 9, wherein the CRISPR protein with side-cleavage activity is one or more of Cas13a, Cas12a, Cas14, Cas12b, Cas13b and Csm 6.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a signal amplification magnetic bead technical system for nucleic acid detection based on a CRISPR (clustered regularly interspaced short palindromic repeats) technology and application thereof.

Background

The CRISPR nucleic acid detection technology can be used for detecting DNA or RNA molecules from plants, animals, microorganisms, viruses and the like. The current proteins for CRISPR nucleic acid detection include Cas proteins with alternative nucleic acid cleavage activity, such as Cas13a, Cas12a, Cas14, Cas12b, Cas13b and Csm 6.

Cas13a protein molecule: recognizes a specific single-stranded RNA molecule, activates alternative nucleic acid cleavage activity, and nonspecifically cleaves any single-stranded RNA molecule.

The crRNA nucleic acid molecule can bind to the Cas13a protein molecule to form a crRNA-Cas13a complex. When crRNA nucleic acidWhen the molecule is matched with a target RNA molecule, Cas13a specifically cleaves the target RNA, thereby activating the alternative nucleic acid cleavage activity of Cas13a and performing efficient non-specific cleavage of any single-stranded RNA molecule encountered. One target RNA molecule can activate one Cas13a protein, and the activated Cas13a protein can cleave a large number of arbitrary single-stranded RNA molecules. The characteristic of the Cas13a protein can be used for specifically detecting a certain RNA sequence. Specifically binding crRNA is designed for the target RNA molecule. Adding RNA molecules to be detected, specific crRNA molecules, Cas13a protein molecules and reporter RNA molecules into the reaction system. One commonly used reporter RNA molecule is an oligonucleotide: one end is attached with a fluorescent group (HEX), and the other end is provided with a quenching group (BHQ 1). An intact reporter RNA molecule will not fluoresce due to quenching. When the RNA molecule to be detected is matched with the crRNA-Cas13a, the crRNA-Cas13a specifically cuts the RNA molecule to be detected and activates the activity of nonspecific RNA hydrolase of Cas13a (bypass nucleic acid cutting activity), so that the reporter RNA molecule is cut, the fluorescent group is free from the action of the quenching group, and fluorescence is released. Thus, the presence of the target RNA molecule can be detected by a fluorescent signal. If the target RNA molecule is not present in the system, the fluorescent gene of the reporter molecule will not be illuminated. The fluorescence signal can be emitted only when the target RNA molecule is present in the system, the activity of the nonspecific RNA hydrolase of Cas13a (alternative nucleic acid cleavage activity) is activated, and fluorescein is free from the action of the quenching group. The detection sensitivity can reach nM (10)^-9M) concentration level.

In order to improve the detection sensitivity, the molecule to be detected needs to be subjected to nucleic acid amplification before the Cas13a reaction is performed, and the commonly used nucleic acid amplification methods include: RPA amplification, LAMP amplification, PCR amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA, and SMAP2, and the like. The amplified and enriched molecules to be detected are subjected to Cas13a reaction, and the detection sensitivity can reach aM (10)^-18M) concentration level, i.e., 1000-.

Cas13a eggWhite blood can be used to detect RNA molecules as well as DNA molecules. If the molecule to be detected is DNA, the DNA molecules are amplified and enriched by RPA to obtain a large amount of double-stranded DNA molecules. If the molecule to be detected is RNA, the RNA is subjected to reverse transcription to form cDNA, and then is subjected to RPA amplification (RT-RPA) enrichment to obtain a large amount of double-stranded DNA molecules. After RPA amplification or RT-RPA amplification, molecules to be detected are enriched in a large amount and become double-stranded DNA molecules, which is the first round of signal amplification. Double-stranded DNA molecules are converted into single-stranded RNA molecules by in vitro transcription (e.g., T7 transcription). When the single-stranded RNA molecule is matched with the crRNA-Cas13a, the crRNA-Cas13a specifically cuts the RNA molecule, activates the activity of nonspecific RNA hydrolase of Cas13a (bypass nucleic acid cutting activity), further cuts the reporter RNA molecule, enables the fluorescent group to get rid of the action of the quenching group, and releases fluorescence. Thus, the presence of the target molecule can be detected by the fluorescent signal. One Cas13a molecule can cleave a large number of reporter RNA molecules, which is the second round of signal amplification. Through two rounds of signal amplification (nucleic acid amplification + Cas13a cleavage), the detection sensitivity can reach aM (10)^-18M) concentration level, i.e., 1000-.

The process comprises the following steps:

the DNA molecule to be detected is amplified > nucleic acid amplification > in vitro transcription > Cas13a cleavage of the reporter RNA molecule > release fluorescence; or the RNA molecule to be detected is > reverse transcription > nucleic acid amplification > in vitro transcription > Cas13a cleavage of the reporter RNA molecule > release fluorescence

Cas12a protein molecule: recognizing specific single-stranded DNA molecules or double-stranded DNA, activating the alternative nucleic acid cleavage activity, and non-specifically cleaving any single-stranded DNA molecules;

the Cas12a system differs from the Cas13a system in that: cas12a specifically recognizes either a single-stranded DNA molecule or a double-stranded DNA molecule; non-specifically cleaved is a single-stranded DNA molecule. Thus the reporter in the Cas12a system needs to be replaced with a single stranded DNA molecule.

The crRNA nucleic acid molecule can bind to a Cas12a protein molecule to form a crRNA-Cas12a complex. When the crRNA nucleic acid molecule is matched with the target DNA molecule (single-stranded DNA or double-stranded DNA), Cas12a specifically cleaves the target DNA (single-stranded DNA or double-stranded DNA), thereby activating the alternative nucleic acid cleavage activity of Cas12a to effect highly efficient non-specific cleavage of any single-stranded DNA molecule encountered. One target DNA molecule can activate one Cas12a protein, and the activated Cas12a protein can cleave a large number of arbitrary single-stranded DNA molecules. Therefore, the reporter molecule in the Cas12a system needs to be replaced by a single-stranded DNA molecule, one end of the reporter DNA molecule is attached with a fluorescein, and the other end is attached with a quencher, and the action principle is similar to Cas13 a.

Since Cas12a can recognize double-stranded DNA molecules, RPA amplified can be used directly in Cas12a cleavage reaction without in vitro transcription.

The process comprises the following steps: the DNA molecule to be detected > nucleic acid amplification (e.g., RPA amplification) > Cas12a cleavage of the reporter DNA molecule > releases fluorescence; alternatively, the RNA molecule to be detected is > reverse transcription > nucleic acid amplification (e.g., RPA amplification) > Cas12a cleavage of the reporter DNA molecule > releases fluorescence.

Cas14 protein molecule: recognizing specific single-stranded DNA, activating the alternative nucleic acid cleavage activity, and non-specifically cleaving any single-stranded DNA molecule. The Cas14 system differs from the Cas13a system in that: cas14 specifically recognizes a single-stranded DNA molecule; non-specifically cleaved is a single-stranded DNA molecule. The reporter molecule in the Cas14 system is a single stranded DNA molecule.

The crRNA nucleic acid molecule can bind to a Cas14 protein molecule to form a crRNA-Cas14 complex. When the crRNA nucleic acid molecule matches the target single-stranded DNA molecule, Cas14 specifically cleaves the target single-stranded DNA molecule and thus activates the alternative nucleic acid cleavage activity of Cas14, resulting in efficient non-specific cleavage of any single-stranded DNA molecule encountered. A target single-stranded DNA molecule can activate a Cas14 protein, and the activated Cas14 protein can cleave a large number of arbitrary single-stranded DNA molecules. The reporter molecule in the Cas14 system is a single-stranded DNA molecule, one end of the reporter DNA molecule is attached with a fluorescein, and the other end is provided with a quencher, and the action principle is similar to Cas13 a.

Since Cas14 recognizes a single-stranded DNA molecule, the double-stranded DNA after RPA amplification needs to be single-stranded (for example, single-stranded DNA can be obtained by T7 exonuclease degradation), and then Cas14 cleavage reaction is performed.

The process comprises the following steps: the DNA molecule to be detected is > nucleic acid amplification (e.g., RPA amplification) > single-stranded processing > Cas14 cleavage of the reporter DNA molecule > release fluorescence; alternatively, the RNA molecule to be detected is > reverse transcription > nucleic acid amplification (e.g., RPA amplification) > single stranded treatment > Cas14 cleavage of the reporter DNA molecule > release fluorescence.

Cas12b, Cas13b, Csm6 are similar in principle to the Cas protein described above and will not be described; however, the detection sensitivity of the probe to nucleic acid can only reach aM (10) no matter Cas13a, Cas12a, Cas14, Cas12b, Cas13b or Csm6^-18M) concentration level (i.e., 1000-. Many clinical tests require achievement of zM (10)^-21M) concentration level (i.e., 1-10copies/mL) can be met.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to improve the sensitivity of specific nucleic acid detection based on CRISPR technology in the prior art, and improve the sensitivity from aM (1000-10000copies/mL) concentration level to zM (1-10copies/mL) concentration level. In order to achieve the purpose of the invention, the invention introduces catalytic enzyme as a third-stage signal amplification system on the basis of two rounds of signal amplification of original nucleic acid amplification and nonspecific arbitrary cleavage of Cas protein, so that the invention provides a signal amplification magnetic bead technical system for nucleic acid detection based on CRISPR technology. The invention also provides a method for identifying target nucleotide molecules by using the system, detects target nucleotide sequence molecules from various source samples, and constructs various detection kits and methods. The cell detection technology of the invention realizes high-sensitivity detection, can detect a large amount of samples and nucleic acid molecules with extremely low content, and has high sensitivity.

The technical scheme is as follows: in order to solve the above problems, the technical solution of the present invention is to provide a reporter magnetic bead, wherein the reporter magnetic bead comprises a magnetic bead, nucleotides and a high catalytic activity enzyme linked together.

Wherein, the nucleotide is one or more of single-stranded DNA, double-stranded DNA or single-stranded RNA.

Wherein the nucleotide is biotin-linked ssDNA.

Wherein, two ends of the ssDNA molecule are respectively marked with digoxin and biotin.

The enzyme with high catalytic activity comprises but not limited to beta-Gal enzyme, and the beta-Gal enzyme can also be replaced by HRP (horse radish peroxidase), so that whether a sample contains a target molecule can be judged by observing color change by naked eyes, the dependence on a fluorescence measuring instrument is eliminated, and the method is used for quick screening of communities. The β -Gal enzyme may be replaced with other phosphatases such as AP (alkaline phosphatase), carboxylic ester hydrolase, glycoside hydrolase, or protease; the β -Gal enzyme may be substituted with an oxidoreductase such as tyrosinase, monoamine oxidase, Nitroreductase (NTR) and thioredoxin reductase; the β -Gal enzyme may be replaced with a transferase such as γ -glutamyltransferase (GGT).

Wherein beta-Gal enzyme (beta-galactosidase) is labeled with streptavidin and coupled to the other, free end of a single-stranded DNA (or RNA) molecule via streptavidin-biotin interaction.

The invention also comprises a construction method of the report magnetic bead, which comprises the following steps: and linking the magnetic beads and nucleotides in a covalent bond mode to obtain nucleotide-magnetic beads, and linking the enzyme with high catalytic activity and the nucleotide-magnetic beads through streptavidin and biotin.

The invention further provides a signal amplification magnetic bead technology system for nucleic acid detection based on CRISPR technology, and the signal amplification magnetic bead technology system comprises the report magnetic bead.

The invention also comprises the application of the report magnetic bead or the signal amplification magnetic bead technical system in the preparation of a nucleic acid detection reagent or a kit.

The invention also comprises a nucleic acid detection kit, wherein the kit comprises the report magnetic bead or the signal amplification magnetic bead technical system.

The invention also discloses an ultrahigh-sensitivity nucleic acid detection method based on the CRISPR technology, which comprises the following steps:

1) performing thermal inactivation and cracking on virus liquid to release RNA molecules of viruses, extracting the RNA molecules in virus suspension by using nano nucleic acid magnetic beads, adding the extracted magnetic beads into a reaction system of RT-PRA isothermal amplification, and performing RT-RPA amplification on target molecules to obtain first-stage signal amplification;

2) performing secondary extraction on DNA molecules obtained by amplifying the primary signals by using original nano magnetic beads, adding the DNA molecules into CRISPR protein with side-cleavage activity to specifically recognize and cleave virus target DNA molecules, activating the non-specific cleavage activity of the CRISPR protein with the side-cleavage activity, and starting the amplification of the secondary signals;

3) adding the report magnetic beads into a reaction system, cutting the ssDNA to release free enzymes with high catalytic activity into system supernatant by the non-specific cutting activity of the CRISPR protein with the side cutting activity, adsorbing all the magnetic beads by using a magnet, taking out the supernatant containing the free enzymes with high catalytic activity, adding a fluorescent substrate for reaction, and performing reading detection by using an enzyme labeling instrument to obtain a detection result.

The CRISPR protein with the paracleaving activity is one or more of Cas13a, Cas12a, Cas14, Cas12b, Cas13b and Csm 6.

The invention is not limited to the use in Cas12a, and Cas proteins with alternative nucleic acid cleavage activity, such as Cas13a, Cas14, Cas12b, Cas13b and Csm6, are also applicable; the method is also suitable for nucleic acid detection of animals, plants, microorganisms and the like.

Wherein the enzyme catalytic substrate includes but is not limited to FDG [ fluoroscein di-beta-D-galactopyranoside ] substrate]CAS #: 17817-20-8, FDG is more sensitive than radioactive label by 100-fold and 1000-fold, can be used for detecting single enzyme molecules, and is a highly sensitive fluorescent substrate of beta-galactosidase. The substrate reaction is: conversion of FDG to FMG first (reaction efficiency 1.9. mu. mole min)^-1mg^-1) In the presence of FMG conversion to fluorescent signal (reaction efficiency 22.7. mu. mole min)^-1mg^-1)。

When the Cas protein cleaves a single-stranded DNA (or RNA) molecule, the β -Gal enzyme is detached from the magnetic beads. Collecting the beta-Gal enzyme in the supernatant and transferring the beta-Gal enzyme into a reaction solution containing a beta-Gal fluorescent probe, wherein one beta-Gal enzyme molecule can catalyze and generate 2000-3000 fluorescent molecules. So that the detection signal is amplified in the third stage, and the detection sensitivity of the concentration level of zM (1-10copies/mL) is realized. (first amplification of nucleic acids; second amplification of cleavage of the DNA (or RNA) molecule by the Cas protein; third amplification by enzymatic action of the β -Gal enzyme molecule on the molecular substrate.

In the signal amplification magnetic bead technical system, nucleotides are fixed on the surface of a magnetic bead through immobilization, and one end of each nucleotide is fixed on one end of the surface of the magnetic bead and is fixed with an enzyme with high catalytic activity, so that the CRISPR protein with the side-cleavage activity is combined with a gRNA molecule or a crRNA molecule to form a protein nucleic acid compound which has a specific recognition specific sequence and can be activated by specific nucleotide molecules to have the side-cleavage activity. The protein nucleic acid compound can perform nonspecific cutting on a nucleotide sequence fixed on the surface of magnetic beads, release high-activity enzyme into a solution system, remove unreleased enzyme through the magnetic beads, and finally detect the enzyme released into the solution by adding a fluorescent substrate, thereby realizing the detection of the target nucleotide sequence.

Preferably, the "three-stage signal amplification" of the present invention means: first-stage amplification RPA isothermal amplification; the second stage amplifies the cleavage of ssDNA molecules by Cas12 a; the third amplification is by enzymatic action of the beta-galactosidase molecule on the fluorescent probe molecule. The invention uses Cas12a, can recognize and non-specifically cut single-stranded DNA molecules, one is that the DNA molecules are more stable, and simultaneously can design different crRNAs for different viruses or target molecules to solve the problem of specificity of different detections. The CRISPR molecular diagnosis technology of the invention has high specificity, can not cause the cleavage reaction of Cas protein as long as the mismatch of one or more bases exists, and the solution of high sensitivity is the innovative introduced 'three-stage signal amplification' method of the invention: RT-RPA isothermal amplification, Cas12a non-specific cleavage activity, "β -galactosidase: FDG fluorescent probes ", which are 100-fold and 1000-fold more sensitive than radioactive labeling.

Compared with the fluorescence-DNA-quenching group method used by other CRISPR nucleic acid detection units, the beta-galactosidase signal detection of the last stage of the method has the advantages that the molecular signal amplification of the first-stage enzymatic reaction is increased, and the beta-galactosidase substrate catalyzing capability is higher than the Cas12a cleavage efficiency.

The extraction technology of the nanometer magnetic bead nucleic acid involved in the whole reaction is mature, the automatic operation is easy, the related magnetic bead extraction automatic equipment is low in price, the manual operation can be carried out if the batch quantity is small, the operation is very simple, and only a magnetic rod is needed. The RNA molecules of the viruses extracted for the first time are used, so that all the viral nucleic acids can be fully utilized; the RT-RPA isothermal amplification DNA molecules extracted for the second time can ensure that all the specific activating molecules are completely collected, and the detection sensitivity is improved.

Followed by the use of reporter beads: magnetic bead-nucleic acid DNA-beta-galactosidase can release a large amount of free beta-galactosidase due to non-specific cleavage of Cas12a, and meanwhile, due to the help of magnetic beads, a reaction system can be controlled to be 20-200 mu L, so that the concentration of the beta-galactosidase released by cleaving DNA on the magnetic beads is greatly increased, a reaction substrate FDG is added, and a microplate reader can be directly used for reading after incubation, so that the sensitivity of the magnetic bead-nucleic acid DNA-beta-galactosidase is improved to the level of zM (5-50 copies/mL).

Preferably, the invention also discloses an ultrahigh-sensitivity nucleic acid detection method based on the CRISPR technology, which comprises the following steps:

1) performing thermal inactivation and cracking on virus liquid to release RNA molecules of viruses, extracting the RNA molecules in virus suspension by using nano nucleic acid magnetic beads, adding the extracted magnetic beads into a reaction system of RT-PRA isothermal amplification, and performing RT-RPA amplification on target molecules to obtain first-stage signal amplification;

2) amplifying the first-stage signal to obtain DNA fractionPerforming secondary extraction on the molecules by using original nano magnetic beads, adding DNA molecules into the Cas12a specific recognition and cleavage virus target DNA molecules, activating the non-specific cleavage activity of Cas12a, and starting secondary signal amplification; a Cas12a molecule activated by specific cleavage can be non-specifically cleaved 10^8-9A nucleic acid molecule. After two rounds of signal amplification, the detection sensitivity at this stage can reach 1-10aM level;

3) adding reporter magnetic beads carrying reporter 'ssDNA-beta-galactosidase' into a reaction system, cutting the ssDNA at the non-specific cleavage activity of Cas12a to release free beta-galactosidase to a system supernatant, adsorbing all the magnetic beads by using a magnet, taking out the supernatant containing the free beta-galactosidase, adding a substrate for reaction, and performing reading detection by using an enzyme labeling instrument to obtain a detection result.

Due to non-specific cleavage of Cas12a, beta-galactosidase can be released, and due to the help of magnetic beads, a reaction system can be controlled to be 20-200 muL, so that the concentration of the beta-galactosidase released by DNA cleavage on the magnetic beads is greatly increased, and then a reaction substrate FDG fluorescent probe is added, wherein one beta-galactosidase can catalyze 10^3～4The FDG releases fluorescence, the reading can be directly carried out by using a microplate reader after incubation, the signal is amplified by the third stage, and at the moment, the amplified detectable fluorescence signal molecules of one RNA virus molecule are as follows: 10^19～21The sensitivity of the sensor can reach 1-10 zM level. At this time an RNA virus molecule has amplified the detectable fluorescent signal molecule: 10^19-21The sensitivity of the sensor can reach 1-10 zM level.

In the first amplification stage, in addition to the RT-RPA isothermal amplification, other nucleic acid amplification methods can be used, including but not limited to: LAMP amplification, PCR amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA, and SMAP2, and the like.

Has the advantages that: compared with the prior art, the invention has the following advantages: according to the invention, a third-level signal amplification system (report magnetic bead) is introduced on the basis of two rounds of signal amplification of nucleic acid amplification and non-specific arbitrary cleavage of Cas protein, so that the sensitivity is improved from aM (1000-10000copies/mL) concentration level to zM (1-10copies/mL) concentration level.

Drawings

FIG. 1, structure of a conventional reporter;

FIG. 2 is a schematic diagram of a coronavirus RNA detection method constructed based on Cas12a system;

FIG. 3 is a schematic diagram of the working principle of the present invention;

FIG. 4 is a schematic diagram of the operating principle of the amplification system of the present invention;

FIG. 5 is a schematic diagram of the principle of action of the reporter beads of the present invention;

FIG. 6 is a schematic diagram of a detection process of the signal amplifying magnetic bead technology system of the present invention;

FIG. 7 is a graph showing the results of an experiment in example 1 of the present invention;

FIG. 8 is a graph showing the results of an experiment in example 2 of the present invention;

FIG. 9 is a graph showing the results of the experiment in example 3 of the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings.

Pseudovirus: pseudoviruses (pseudoviruses), also known as "pseudoviruses", are a class of chimeric virus particles that express recombinant glycoproteins of one replication-defective virus (viral vector) on the surface of another virus and are useful as control samples for nucleic acid detection. For example, the pseudo virus used in COVID-19 research is to pack SARS-CoV-2 specific nucleic acid sequence into retrovirus or bacteriophage to construct a structure of protein-coated RNA, and the RNA extraction effect is theoretically consistent with that of original virus. The synthesis of all nucleotide sequences of the invention is completed by Nanjing Kingsrei Biotech Co. The synthesis of report magnetic beads was performed by nutria biotechnology limited.