CN112400016A

CN112400016A - Kit and method for detecting DNA

Info

Publication number: CN112400016A
Application number: CN201980042833.5A
Authority: CN
Inventors: 周飞; 冯先文; 陈龙胜; 付瑜
Original assignee: TDK Corp
Current assignee: TDK Corp
Priority date: 2018-06-26
Filing date: 2019-06-24
Publication date: 2021-02-23
Also published as: WO2020003087A1

Abstract

The present disclosure provides a kit and method for detecting DNA. In particular, the present disclosure relates to gene chip-based kits for detecting DNA samples, signal amplification systems for the kits, and methods of use of the kits. When the DNA fragment to be detected is determined, the object to be detected is unified through a biotransformation process outside a test system, so that the substrate of the chip only corresponds to a unified standard substance. Therefore, the unified chip and the substrate can detect a plurality of target objects, the preparation of the chip is simplified, and the cross contamination of different substrates is avoided. Meanwhile, the kit contains a signal amplification system, and when the signal amplification system is used for detecting DNA, the detection sensitivity is higher.

Description

Kit and method for detecting DNA

Technical Field

The present disclosure relates to a detection kit for a DNA sample, a signal amplification system for the detection kit, and a detection method for a DNA sample. In particular, the present disclosure relates to gene chip-based kits for DNA sample detection, signal amplification systems for the kits, and methods of use of the kits.

Background

DNA chips, also known as gene chips or gene microarrays, are oligonucleotide chips or DNA microarrays, which are fabricated by attaching high density arrays of DNA fragments to glass, nylon, etc. in an array arrangement by microarray technology. The DNA chip technology is that oligonucleotide is synthesized in situ on a solid support or a large number of DNA probes are directly immobilized on the surface of the support in a microscopic printing manner, and then hybridized with a labeled sample, and the genetic information of the sample can be obtained by detecting and analyzing the hybridization signal. Generally speaking, a gene chip is a two-dimensional DNA probe array formed by fixing tens of thousands or even millions of DNA fragments (gene probes) of a specific sequence on a support such as a silicon wafer or a glass slide having a square meter of 2c by a micro-processing technique, and is called a gene chip because it is very similar to an electronic chip on an electronic computer.

The capture and detection of disease markers using immobilized supports is a common method for modern diagnostic testing. However, the existing solutions mainly detect various disease targets by means of a probe chip corresponding to specific binding, and the chip and the substrate must correspond to the target detection object. This results in the simultaneous detection of multiple targets, which requires the simultaneous planting of multiple substrates on a single chip. Such disadvantages are that the preparation process of the chip is complicated and difficult to control, and the substrates of adjacent chips may be contaminated with each other, which may eventually lead to inaccurate or unstable detection results.

In the process of DNA detection, the prior art mainly adopts the following two approaches: (1) template amplification techniques, which increase sensitivity by amplifying a target sequence; (2) the signal amplification system can improve the sensitivity by amplifying the signal intensity, and has the advantages of quick response, simplicity, convenience and no interference factor of template amplification.

However, many disease-related living substances (such as specific proteins, enzymes, molecules or molecular groups such as sugars, which are called markers in disease detection) are low in content in living bodies and thus cannot be easily detected.

Meanwhile, it is considered that almost all drug metabolizing enzymes have genetic polymorphisms, and most of the polymorphisms of drug metabolizing enzymes are a Single Nucleotide Polymorphism (SNP) caused by having a plurality of alleles at the same genetic locus. Alleles encode metabolic enzymes with different drug metabolizing abilities, which have important effects on both the individual response to treatment and drug toxicity and side effects. Therefore, the method can predict the treatment effect of the drug by detecting whether the drug metabolizing enzyme has mutation or not, and has very important effect on personalized medicine.

Therefore, many amplification methods have been proposed to increase the sensitivity of DNA mutation or SNP detection. However, the existing method mainly aims at the amplification effect in the test system, and a preposed signal transfer system does not exist, and meanwhile, the amplification efficiency is relatively low.

Disclosure of Invention

Problems to be solved by the invention

It is an object of the present disclosure to overcome the above-mentioned disadvantages of the prior art and to provide a novel DNA sample detection kit which can detect a plurality of targets using a unified chip and substrate, thereby avoiding cross-contamination of different substrates.

In one embodiment, the present disclosure also provides a method for detecting a DNA sample using the above kit.

It is another object of the present disclosure to overcome the above-mentioned disadvantages of the prior art by providing a kit and a method for DNA sample detection, wherein the kit comprises a signal amplification system, and when the signal amplification system is used for detecting DNA, the detection sensitivity is higher.

Means for solving the problems

In order to achieve the purpose, the technical scheme adopted by the disclosure is as follows.

In one embodiment, the present disclosure relates to a DNA sample detection kit, which includes a capture probe, a transfer substance, a terminal signal substance, an unmodified solid support, and a magnetic bead modified with an affinity substance M5; wherein the content of the first and second substances,

the capture probe is a DNA fragment, and the capture probe contains a partial nucleotide sequence B of the DNA fragment to be detected in the DNA sample₁Complementary paired DNA fragments, said partial nucleotide sequence B₁Containing a site to be detected;

the intermediate and the terminal signal substance are both DNA fragments, and the intermediate is prepared fromNucleotide sequence L at one end₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pair, nucleotide sequence L at the other end of the said intermediate₂Partial nucleotide sequence C of terminal signaler₁Complementary pairing; and

the kit also comprises a chip with a substrate, wherein the substrate is a DNA fragment, and part or all of the fragment of the substrate and part of the nucleotide sequence C of the terminal signal substance₂Complementary pairing; and/or the kit also comprises a signal converter, the signal converter is a DNA fragment, and part or all of the fragment of the signal converter and part of the nucleotide sequence C of the terminal signal converter₃Complementary pairing;

wherein, the terminal signal substance or the signal conversion substance is labeled with an affinity substance M6, and the affinity substance M6 and the affinity substance M5 have affinity.

In a specific embodiment of the above scheme, Tm1 is a partial nucleotide sequence B of the capture probe and the DNA fragment to be detected₁The temperature at which bound base pairs separate; tm2 is the nucleotide sequence L of the transfer product₁And partial nucleotide sequence B of the DNA fragment to be detected₂The temperature at which bound base pairs separate; tm3 is the nucleotide sequence L of the transfer product₂Partial nucleotide sequence C of terminal signaler₁The temperature at which bound base pairs separate; tm4 is partial nucleotide sequence C of the terminal signal substance₃The temperature at which base pair separation occurs with the signal transducer; the numerical value of Tm3 is the lowest among Tm1, Tm2, Tm3 and Tm 4.

In a specific embodiment of the above protocol, where the kit comprises a chip with a substrate, the partial nucleotide sequence C of the terminal signaler₂A partial nucleotide sequence C longer than the terminal signaler₁Length of (d); when the kit comprises a signal converter, a partial nucleotide sequence C of the terminal signal substance₃A partial nucleotide sequence C longer than the terminal signaler₁Length of (d).

In a particular aspect of the aboveIn one embodiment, the capture probe is a DNA fragment, and the capture probe has a partial nucleotide sequence B₁The length of the complementary paired DNA fragments is 6-20 bp.

In a specific embodiment of the above protocol, the capture probe has a partial nucleotide sequence B₁The length of the complementary paired DNA fragments is 10-15 bp.

In a specific embodiment of the above protocol, the capture probe has a modification that enhances binding for base pairing.

In a specific embodiment of the above scheme, the modification is at least one of PNA, LNA, MNA, ANA, TNA, CeNA, GNA, XNA, HNA, INA, BNA.

In a specific embodiment of the above embodiment, the unmodified solid phase carrier is a magnetic bead or a microparticle made of glass or nylon.

In a specific embodiment of the above protocol, the affinity substance M5 is amino, polylysine, thiol, bovine serum albumin, avidin, sepharose or polyacrylamide gel.

In a preferred embodiment, the affinity substance M5 is streptavidin and the affinity substance M6 is biotin.

In a specific embodiment of the above scheme, the solid phase carrier in the chip comprises Al₂O₃Glass, polymer and nylon.

In a specific embodiment of the above scheme, the partial nucleotide sequence C of the terminal signaler₁The length of (a) is 10-15 bp; partial nucleotide sequence C of the terminal signaler₂The length of the probe is 16-60 bp; partial nucleotide sequence C of the terminal signaler₃The length of the probe is 16-60 bp.

In a specific embodiment of the above scheme, the partial nucleotide sequence C of the terminal signaler₂The length of the probe is 25-60 bp; partial nucleotide sequence C of the terminal signaler₃The length of the DNA fragment is 25-60 bp.

In a specific embodiment of the above protocol, where the kit comprises a chip with a substrate, the nucleotide sequence C is homologous to the substrate₂The length of the complementary pairing nucleotide sequence is longer than the length of the remaining nucleotide sequence in the substrate.

In a specific embodiment of the above protocol, the DNA sample is from human blood.

In a specific embodiment of the above protocol, the DNA fragment to be tested in the DNA sample is a DNA fragment with a mutation site.

In a specific embodiment of the above protocol, the DNA fragment to be tested in the DNA sample is an FGFR3 gene fragment containing the G380R mutation site.

In a specific embodiment of the above scheme, the nucleotide sequence of the DNA fragment to be detected in the DNA sample is shown as SEQ ID NO. 1; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 2; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 3; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 4; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 5; the nucleotide sequence of the substrate is shown as SEQ ID NO. 6.

In a specific embodiment of the above scheme, the DNA fragment to be detected in the DNA sample is a CYP2C19 gene fragment containing G681A site.

In a specific embodiment of the above scheme, the nucleotide sequence of the DNA fragment to be detected in the DNA sample is shown as SEQ ID NO. 7; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 8; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 9; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 10; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 11; the nucleotide sequence of the substrate is shown as SEQ ID NO. 12.

In a specific embodiment of the above protocol, a standard solution of the DNA fragment to be tested is also included.

In a preferred embodiment, the detection kit further comprises a hybridization solution and a washing solution.

In a specific embodiment of the above protocol, the eluent comprises deionized formamide, 2 XSSC, 5 XDenhard's, SDS, and deionized water; the washing solution contains Tris-Citric, NaCl and Tween 20.

In another embodiment, the present disclosure also relates to a method of detecting a DNA sample, the method comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

(2) adding the solid phase carrier coupled with the capture probe and the DNA fragment to be detected in the DNA sample into the hybridization solution, so that the capture probe and the partial nucleotide sequence B of the DNA fragment to be detected in the DNA sample₁Hybridizing; after the reaction is finished, separating the solid phase carrier coupled with the DNA fragment to be detected and the capture probe;

(3) adding the solid phase carrier separated in the step (2) and the transfer substance into a hybridization solution to enable the nucleotide sequence L at one end of the transfer substance₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with the transfer product, the DNA fragment to be detected and the capture probe;

(4) adding the solid phase carrier separated in the step (3) and a terminal signal substance into a hybridization solution to enable the nucleotide sequence L at the other end of the transit substance₂Partial nucleotide sequence C of terminal signaler₁Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with a terminal signal substance, a transfer substance, a DNA fragment to be detected and a capture probe;

(5) adding the solid phase carrier and the signal converter separated in the step (4) into the hybridization solution to ensure that the signal converter and the partial nucleotide sequence C of the terminal signal converter₃Hybridizing to form a complex of a terminal signal substance and a signal converter, and separating a solid phase carrier coupled with the transfer substance, the DNA fragment to be detected and the capture probe;

(6) coupling the obtained complex to a magnetic bead modified by an affinity substance M5 through an affinity substance M6 marked on a terminal signal substance or a signal converter to form a signal magnetic bead;

(7) detecting the signal quantity of the signal magnetic beads, and calculating the content of the DNA fragments to be detected in the DNA sample; wherein the content of the first and second substances,

the capture probe is a DNA fragment, and the capture probe contains a partial nucleotide sequence B of the DNA fragment to be detected in the DNA sample₁Complementary paired DNA fragments;

the partial nucleotide sequence B₁Containing a site to be detected;

the intermediate and the terminal signal substance are DNA fragments, and the nucleotide sequence L at one end of the intermediate₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pairing;

nucleotide sequence L of the other end of the said relay₂Partial nucleotide sequence C of terminal signaler₁Complementary pairing;

the signal converter is a DNA fragment, and part or all of the fragment of the signal converter and part of the nucleotide sequence C of the terminal signal converter₃And (4) complementary pairing.

In a specific embodiment of the above scheme, the following step (5a) is further included between the step (5) and the step (6): reacting the complex of the terminal signaler and the signal converter with a chip having a substrate to react the substrate with a partial nucleotide sequence C of the terminal signaler₂Hybridizing to obtain a complex of the chip, the substrate, the terminal signal substance and the signal converter; wherein the content of the first and second substances,

the substrate is a DNA fragment, and part or all of the fragment of the substrate and part of the nucleotide sequence C of the terminal signal substance₂Complementary pairing;

the chip also contains a magnetic sensor.

In a specific embodiment of the above scheme, in the step (2), the step (3) and the step (4), after the solid phase carriers are separated, the solid phase carriers are respectively washed by using a washing solution.

(1) coupling a capture probe to an unmodified solid phase support;

(2) the solid phase carrier coupled with the capture probe and the DNA sample areAdding the DNA fragment to be detected into the hybridization solution, and enabling the capture probe and a part of the nucleotide sequence B of the DNA fragment to be detected in the DNA sample₁Hybridizing; after the reaction is finished, separating the solid phase carrier coupled with the DNA fragment to be detected and the capture probe;

(4) adding the solid phase carrier separated in the step (3) and a terminal signal substance into a hybridization solution to enable the nucleotide sequence L at the other end of the transit substance₂Partial nucleotide sequence C of terminal signaler₁Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with a terminal signal substance, a transfer substance, a DNA fragment to be detected and a capture probe; after washing the unbound terminal signal substance, the solid phase carrier is subjected to appropriate temperature treatment, so that the terminal signal substance is separated from the solid phase carrier and enters a solution, and the solid phase carrier coupled with the transfer product, the DNA fragment to be detected and the capture probe is removed;

(5) reacting the solution containing the terminal signal substance obtained in the step (4) with a chip with a substrate to allow the substrate to react with a partial nucleotide sequence C of the terminal signal substance₂Hybridizing to form a complex of the chip, the substrate and the terminal signal substance;

(6) coupling the obtained complex to magnetic beads modified by an affinity substance M5 through the affinity substance M6 marked on the terminal signal substance to form signal magnetic beads;

the partial nucleotide sequence B₁Containing a site to be detected;

the intermediate and the terminal signal substance are both DNA fragments, and one of the intermediateTerminal nucleotide sequence L₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pairing;

the signal converter is a DNA fragment, and part or all of the fragment of the signal converter and part of the nucleotide sequence C of the terminal signal converter₃Complementary pairing;

the chip also contains a magnetic sensor.

In a specific embodiment of the above scheme, in the step (2) and the step (3), after the solid phase carriers are separated, the solid phase carriers are washed by a washing solution respectively.

In a specific embodiment of the above scheme, in the step (2), the reaction temperature is 45 ℃ and the reaction time is 20 minutes; in the step (3) and the step (4), the reaction temperature is 25 ℃, and the reaction time is 20 minutes.

In a specific embodiment of the above scheme, in the step (7), a magnetic signal detection device is used to detect the signal quantity of the signal magnetic beads; wherein the device is at least one selected from the group consisting of a hall element, a magnetoresistance effect element (magnetoresistance sensor) which may be selected from a GMR sensor (giant magnetoresistance sensor) and a TMR sensor (tunnel magnetoresistance sensor).

In a specific embodiment of the above scheme, the method for obtaining the DNA fragment to be detected in the DNA sample comprises: collecting blood of human body, extracting DNA in blood, and fragmenting the extracted DNA.

In a specific embodiment of the above protocol, the extracted DNA is fragmented by enzymatic or ultrasonic methods.

In another aspect, the present disclosure relates to a DNA sample detection kit comprising a capture probe, a primary signal amplifier, a secondary signal amplifier, an unmodified solid support, a magnetic bead modified with an affinity substance M5, and a signal converter; wherein the content of the first and second substances,

the primary signal amplifier includes a first relay having a nucleotide sequence L at one end₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pairing; nucleotide sequence L of the other end of the first transfer product₂Containing m₁Nucleotide fragment L₃；

The secondary signal amplifier comprises a first terminal signal containing a nucleotide sequence C₁And nucleotide sequence C₂Nucleotide sequence C₂Containing n₁A nucleotide sequence C which is complementary to a part or all of the DNA fragment of the signal transducer₃；

the terminal signal substance or the signal converter is marked with an affinity substance M6, and the affinity substance M6 and the affinity substance M5 have affinity;

the nucleotide fragment L₃And the nucleotide sequence C₁Complementary pairing;

m₁and n₁Are all positive integers.

In a particular embodiment of the above scheme, optionally, m₁And n₁Not simultaneously 1.

In a specific embodiment of the above protocol, the nucleotide sequence L of the first relay is₂The end of (a) is modified with an affinity substance M2; the primary signal amplifier further comprises an affinity substance M1 and a second intermediate; in the second placeThe product contains m₂Nucleotide fragment L₃，m₂Is a positive integer; the 5 'end and/or the 3' end of the second transfer product is modified with an affinity substance M2; the affinity substance M2 and the affinity substance M1 have affinity; the first and second relays are linked to affinity substance M1 via affinity substance M2.

In a specific embodiment of the above scheme, the terminal of the first terminal signaler is modified with an affinity substance M4; the secondary signal amplifier further comprises an affinity substance M3 and a second terminal signal substance, the second terminal signal substance comprising n₂Nucleotide sequence C₃，n₂Is a positive integer; the 5 'end and/or the 3' end of the second terminal signaler is modified with an affinity substance M4; the affinity substance M3 and the affinity substance M4 have affinity; the first and second terminal signalants are linked to the affinity substance M3 via affinity substance M4.

In a specific embodiment of the above aspect, m is₁(ii) 5, and/or said n₁＝2。

In another aspect, the present disclosure relates to a DNA sample detection kit, which is characterized in that the DNA sample detection kit comprises a capture probe, a primary signal amplifier, a secondary signal amplifier, an unmodified solid phase carrier, a magnetic bead modified with an affinity substance M5, and a signal converter; wherein the content of the first and second substances,

The secondary signal amplifier comprises a first terminalA signal substance, said first terminal signal substance comprising a nucleotide sequence C₁And nucleotide sequence C₂Nucleotide sequence C₂Containing n₁A nucleotide sequence C which is complementary to a part or all of the DNA fragment of the signal transducer₃；

The signal converter is a DNA fragment, and part or all of the fragment of the signal converter and part of the nucleotide sequence C of the terminal signal converter₃Complementary pairing; and

nucleotide sequence L of the first transfer product₂The end of (a) is modified with an affinity substance M2; the primary signal amplifier further comprises an affinity substance M1 and a second intermediate; the second transfer product contains m₂Nucleotide fragment L₃(ii) a The 5 'end and/or the 3' end of the second transfer product is modified with an affinity substance M2; the affinity substance M2 and the affinity substance M1 have affinity, and the first transfer substance and the second transfer substance are connected to the affinity substance M1 through the affinity substance M2; or, the end of the first terminal signal object is modified with an affinity substance M4; the secondary signal amplifier further comprises an affinity substance M3 and a second terminal signal substance, the second terminal signal substance comprising n₂Nucleotide sequence C₃(ii) a The 5 'end and/or the 3' end of the second terminal signaler is modified with an affinity substance M4; the affinity substance M3 and the affinity substance M4 have affinity, and the first terminal signaler and the second terminal signaler are connected to the affinity substance M3 through the affinity substance M4;

m1 and n1 are both positive integers; m is₂And n₂Are all positive integers;

optionally, m is₁And n₁And is also 1.

In a specific embodiment of the foregoing technical solution, Tm1 is a nucleotide sequence B of a capture probe and a DNA fragment to be detected₁The temperature at which bound base pairs separate; tm2 is the nucleotide sequence L of the primary signal amplifier₁And nucleotide sequence B of DNA fragment to be detected₂The temperature at which bound base pairs separate; tm3 is the nucleotide sequence L of the primary signal amplifier₃Nucleotide sequence C with secondary signal amplifier₁The temperature at which bound base pairs separate; tm4 is nucleotide sequence C of secondary signal amplifier₃The temperature at which base pair separation occurs with the signal transducer; the numerical value of Tm3 is the lowest among Tm1, Tm2, Tm3 and Tm 4.

In a specific embodiment of the aforementioned technical solution, the kit further comprises a chip with a substrate, the substrate is a DNA fragment, and the first terminal signal substance further comprises a nucleotide sequence C₄Partial or complete fragments of said substrate and nucleotide sequence C₄And (4) complementary pairing.

In a specific embodiment of the foregoing embodiment, the nucleotide sequence of the second relay and the nucleotide sequence L at the other end of the first relay₂The same is true.

In a specific embodiment of the foregoing embodiment, the nucleotide sequence of the second terminal signal substance and the nucleotide sequence C of the first terminal signal substance₂The same is true.

In a specific embodiment of the aforementioned technical solution, the affinity substance M1 or M3 or M5 is amino, polylysine, thiol, bovine serum albumin, avidin, agarose gel or polyacrylamide gel.

In a preferred embodiment, the affinity substance M1 is streptavidin and the affinity substance M2 is biotin; or the affinity substance M3 is streptavidin, and the affinity substance M4 is biotin; or the affinity substance M5 is streptavidin, and the affinity substance M6 is biotin.

In a specific embodiment of the aforementioned technical solution, the nucleotide sequence C₃Is longer than the nucleotide sequence C₁Length of (d).

In a specific embodiment of the foregoing technical solutionWherein said nucleotide sequence C₃Has a length of 16-60 bp, and the nucleotide sequence C₁The length of (a) is 10-15 bp.

In a preferred embodiment, the nucleotide sequence C₃The length of the DNA fragment is 25-60 bp.

In a specific embodiment of the above scheme, the nucleotide sequence C₄Is longer than the nucleotide sequence C₁Length of (d).

In a specific embodiment of the above scheme, the nucleotide sequence C₄Has a length of 16-60 bp, and the nucleotide sequence C₁The length of (a) is 10-15 bp.

In a preferred embodiment, the nucleotide sequence C₄The length of the DNA fragment is 25-60 bp.

In a specific embodiment of the above scheme, the substrate has a nucleotide sequence C₄The length of the complementary pairing nucleotide sequence is longer than the length of the remaining nucleotide sequence in the substrate.

In a specific embodiment of the foregoing technical solution, the solid phase carrier in the chip comprises Al₂O₃Glass, polymer and nylon.

In a specific embodiment of the foregoing technical solution, the DNA sample is derived from human blood.

In a specific embodiment of the foregoing technical solution, the DNA fragment to be detected in the DNA sample is a DNA fragment with a mutation site.

In a specific embodiment of the above scheme, the nucleotide sequence of the DNA fragment to be detected in the DNA sample is shown as SEQ ID NO. 13; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 14; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 15; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 16; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 17; the nucleotide sequence of the substrate is shown as SEQ ID NO. 18.

In a specific embodiment of the above scheme, the nucleotide sequence of the DNA fragment to be detected in the DNA sample is shown as SEQ ID NO. 19; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 20; the nucleotide sequence of the transfer product is shown as SEQ ID NO: 21; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 22; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 23; the nucleotide sequence of the substrate is shown as SEQ ID NO. 24.

In a specific embodiment of the above scheme, the kit further comprises a standard solution of the DNA fragment to be detected; preferably, the detection kit further comprises a hybridization solution and a cleaning solution.

In another aspect, the present disclosure relates to a method for detecting DNA using a DNA sample detection kit, comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

(3) adding the solid phase carrier separated in the step (2) and the primary signal amplifier into a hybridization solution to enable the nucleotide sequence L at one end of the first transfer product₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Hybridizing; after the reaction is finished, separating the solid phase carrier coupled with the primary signal amplifier, the DNA fragment to be detected and the capture probeA body;

(4) adding the solid phase carrier separated in the step (3) and a secondary signal amplifier into a hybridization solution to enable the nucleotide sequence L in the primary signal amplifier to₃With nucleotide sequence C in a secondary signal amplifier₁Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with a secondary signal amplifier, a primary signal amplifier, a DNA fragment to be detected and a capture probe;

(5) adding the solid phase carrier and the signal converter separated in the step (4) into the hybridization solution to enable the signal converter and the nucleotide sequence C of the secondary signal amplifier₃Hybridizing to form a complex of the secondary signal amplifier and the signal converter, and separating a solid phase carrier coupled with the primary signal amplifier, the DNA fragment to be detected and the capture probe;

(6) coupling the obtained complex to magnetic beads modified by an affinity substance M5 through the affinity substance M6 modified on the signal converter to form signal magnetic beads;

(7) and detecting the signal quantity of the signal magnetic beads, and calculating the content of the DNA fragment to be detected in the DNA sample.

In a specific embodiment of the above scheme, the following step (5a) is further included between the step (5) and the step (6): reacting the complex of the secondary signal amplifier and the signal converter with a chip having a substrate, and reacting the substrate with the nucleotide sequence C in the first terminal signal₄And hybridizing to obtain a complex of the chip, the substrate, the secondary signal amplifier and the signal converter.

In another embodiment, the present disclosure also provides a use of the aforementioned kit for preparing a kit for detection of a disease marker, prediction of a therapeutic effect of a drug, and/or prediction of a magnitude of a side effect of a therapy of a drug.

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment, the present disclosure unifies the analyte by a biotransformation process outside the test system when determining the DNA fragments to be tested, such that the substrate of the chip corresponds to only the unified standard. Therefore, the unified chip and the substrate can detect a plurality of target objects, the preparation of the chip is simplified, and the cross contamination of different substrates is avoided.

In another embodiment, the disclosure provides a method of amplifying a signal by designing at least one nucleotide sequence C in a primary signal amplifier and a secondary signal amplifier₁Complementary paired sequences to hybridize a plurality of secondary signal amplifiers to the primary signal amplifier; the plurality of signal transducers is further hybridized to the secondary signal amplifier by designing at least one nucleotide sequence on the secondary signal amplifier that is complementary to the signal transducer. Therefore, under the condition of the same amount of DNA fragments to be detected, the obtained compound of the secondary signal amplifier and the signal converter is multiplied, and the final test signal is also multiplied, so that the sensitivity of the determination is improved by multiple times.

In another embodiment, the present disclosure modifies affinity substances at the ends of the transitions (first and second transitions) or the terminal signals (first and second terminal signals), and a molecule of DNA fragment to be tested can indirectly bind to a plurality of secondary signal amplifiers by using a high affinity between the affinity substances, so that the complex of the secondary signal amplifier and the signal transition is amplified multiple times and the final test signal is also amplified multiple times, thereby further improving the sensitivity.

Drawings

FIG. 1 is a schematic diagram of a method for detecting a DNA sample in example 1 of the present disclosure.

FIG. 2 is a nucleotide sequence result chart of the detection of G380R mutation site of FGFR3 gene in example 1 of the present disclosure.

FIG. 3 is a schematic diagram of a method for detecting a DNA sample in example 3 of the present disclosure.

FIG. 4 is a schematic structural diagram of a sequence in example 3 of the present disclosure;

FIG. 5 is a schematic diagram of the detection of DNA using the signal amplification system of example 3 according to the present disclosure;

FIG. 6 is a schematic diagram of the structure of the sequence in example 4 of the present disclosure;

FIG. 7 is a schematic diagram of the structure of the sequence in example 5 of the present disclosure;

fig. 8 is a schematic structural diagram of a primary signal amplifier in embodiment 5 of the present disclosure.

Detailed Description

Definition of

In the claims and/or the description of the present disclosure, the words "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one", and "one or more than one".

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Also, the terms "comprising," "having," "including," or "containing" are intended to be inclusive and mean that there may be additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

In the prior art, when detecting various disease targets, a corresponding specific combined detection chip is mainly utilized, and the chip and a substrate must correspond to the target detection object. Thus, the preparation process of the chip is complicated and difficult to control, and the mutual contamination of substrates of adjacent chips may occur, which may eventually lead to inaccurate or unstable detection results.

To overcome these disadvantages, in one aspect, the present disclosure provides a DNA sample detection kit comprising a capture probe, a mediator and a terminal signal substance, an unmodified solid support, and a magnetic bead modified with an affinity substance M5; the capture probe is a DNA fragment, the capture probe contains a DNA fragment which is complementary and matched with a partial nucleotide sequence B1 of the DNA fragment to be detected in the DNA sample, and the partial nucleotide sequence B1 contains a site to be detected;the intermediate and the terminal signal substance are DNA fragments, and the nucleotide sequence L at one end of the intermediate₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pair, nucleotide sequence L at the other end of the said intermediate₂Partial nucleotide sequence C of terminal signaler₁Complementary pairing;

the kit also comprises a chip with a substrate, wherein the substrate is a DNA fragment, and part or all of the fragment E of the substrate₁Partial nucleotide sequence C of terminal signaler₂Complementary pairing; and/or the kit also comprises a signal converter, the signal converter is a DNA fragment, and part or all of the fragment of the signal converter and part of the nucleotide sequence C of the terminal signal converter₃Complementary pairing;

the terminal signal substance or the signal converter is marked with an affinity substance M6, and the affinity substance M6 and the affinity substance M5 have affinity.

In the technical scheme, the detection principle of the detection kit for the DNA sample of the present disclosure is shown in fig. 1. In FIG. 1, Tm represents the temperature at which the bound base pairs are separated, and the higher Tm, the greater the binding force of the bound 2 sequences, and the more stable it is. Tm1 is the nucleotide sequence B of the capture probe and the DNA fragment to be detected₁The temperature at which bound base pairs separate; tm2 is nucleotide sequence L of transfer product₁And nucleotide sequence B of DNA fragment to be detected₂The temperature at which bound base pairs separate; tm3 is nucleotide sequence L of transfer product₂The temperature at which base pair separation occurs in association with nucleotide sequence C1 of the terminal signal; tm4 nucleotide sequence C of terminal signal substance₃Temperature at which base pair separation occurs in association with the signal transducer. Among Tm1, Tm2, Tm3 and Tm4, Tm3 is the smallest (e.g., Tm 4. apprxeq.Tm 1. apprxeq.Tm 2)>Tm3) so that only the nucleotide sequence L is achieved at a certain temperature (e.g.65 ℃)₂Is cleaved from the binding base of the terminal signal substance nucleotide sequence C1.

In a specific implementation manner of the foregoing technical solution, the detection principle of the present disclosure is specifically: hybridizing a capture probe designed specifically with a DNA fragment to be detected in a DNA sample so as to capture the DNA fragment to be detected; then, the hybrids of the capture probes and the DNA fragments to be detected are converted into detection signals with unified design through the transfer objects and the terminal signal objects; the detection signal is separated from the complex body through a set condition and enters a test system; finally, the probe signal is bound to the magnetic beads by the high affinity between the affinity substance M5 and the affinity substance M6, and the signal of the magnetic beads is detected.

In a specific embodiment of the above technical scheme, the detection kit of the present disclosure solves the problems of tedious preparation of chips and contamination of substrates of adjacent chips; meanwhile, the controllability of the process is facilitated.

In one embodiment of the above technical solution, the kit of the present disclosure can be used for detection of DNA samples.

In one aspect, the present disclosure provides a signal amplification system for DNA detection, the amplification system comprising: a capture probe, a primary signal amplifier, and a secondary signal amplifier; the capture probe, primary signal amplifier and secondary signal amplifier are all DNA fragments.

In another embodiment, the present disclosure also provides a DNA sample detection kit, which includes a capture probe, a primary signal amplifier, a secondary signal amplifier, an unmodified solid support, a magnetic bead modified with an affinity substance M5, and a signal converter; wherein the content of the first and second substances,

the capture probe is a DNA fragment, and the capture probe contains a partial nucleotide sequence B of the DNA fragment to be detected in the DNA sample₁Complementary paired DNA fragments, said partial nucleotide sequence B₁Containing a site to be detected; the primary signal amplifier includes a first relay having a nucleotide sequence L at one end₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pairing; nucleotide sequence L of the other end of the first transfer product₂Containing m₁Nucleotide fragment L₃(ii) a The secondary signal amplifier comprises a first terminal signal containing a nucleotide sequence C₁And nucleotide sequence C₂Nucleotide sequence C₂Containing n₁A nucleotide sequence C which is complementary to a part or all of the DNA fragment of the signal transducer₃(ii) a The signal converter is a DNA fragment, and part or all of the fragment of the signal converter and part of the nucleotide sequence C of the terminal signal converter₃Complementary pairing; the terminal signal substance or the signal converter is marked with an affinity substance M6, and the affinity substance M6 and the affinity substance M5 have affinity; the nucleotide fragment L₃And the nucleotide sequence C₁Complementary pairing; m1 and n1 are both positive integers.

In a preferred embodiment of the above solution, m1 and n1 are not both 1.

In another embodiment, the present disclosure also provides a DNA sample detection kit, which includes a capture probe, a primary signal amplifier, a secondary signal amplifier, an unmodified solid support, a magnetic bead modified with an affinity substance M5, and a signal converter; wherein the capture probe is a DNA fragment, and the capture probe contains a partial nucleotide sequence B of the DNA fragment to be detected in the DNA sample₁Complementary paired DNA fragments, said partial nucleotide sequence B₁Containing a site to be detected; the primary signal amplifier includes a first relay having a nucleotide sequence L at one end₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pairing; nucleotide sequence L of the other end of the first transfer product₂Containing m₁Nucleotide fragment L₃(ii) a The secondary signal amplifier comprises a first terminal signal containing a nucleotide sequence C₁And nucleotide sequence C₂Nucleotide sequence C₂Containing n₁A nucleotide sequence C which is complementary to a part or all of the DNA fragment of the signal transducer₃(ii) a The signal converter is a DNA fragment, and part or all of the fragment of the signal converter and part of the nucleotide sequence C of the terminal signal converter₃And (4) complementary pairing.

In one embodiment of the above embodiment, the nucleotide sequence of said first transfer agentColumn L₂The end of (a) is modified with an affinity substance M2; the primary signal amplifier further comprises an affinity substance M1 and a second intermediate; the second transfer product contains m₂Nucleotide fragment L₃(ii) a The 5 'end and/or the 3' end of the second transfer product is modified with an affinity substance M2; the affinity substance M2 and the affinity substance M1 have affinity, and the first transfer substance and the second transfer substance are connected to the affinity substance M1 through the affinity substance M2.

In another embodiment of the above technical solution, the terminal of the first terminal signaler is modified with an affinity substance M4; the secondary signal amplifier further comprises an affinity substance M3 and a second terminal signal substance, the second terminal signal substance comprising n₂Nucleotide sequence C₃(ii) a The 5 'end and/or the 3' end of the second terminal signaler is modified with an affinity substance M4; the affinity substance M3 and the affinity substance M4 have affinity, and the first terminal signaler and the second terminal signaler are connected to the affinity substance M3 through the affinity substance M4; the terminal signal substance or the signal converter is marked with an affinity substance M6, and the affinity substance M6 and the affinity substance M5 have affinity; the nucleotide fragment L₃And the nucleotide sequence C₁Complementary pairing; m1 and n1 are both positive integers; m is₂And n₂Are all positive integers.

In a preferred embodiment of the above solution, m1 and n1 are both 1.

In one embodiment of the above protocol, a first intermediate and at least one second intermediate can be attached to a primary signal amplifier, the nucleotide fragment L of which is attached to the primary signal amplifier, by modifying the affinity substance M2 at the end of the sequence and attaching both affinity substances M2 to M1₃Will be increased to connect more first termination beacons. For example, when the affinity substance M1 is streptavidin and the affinity substance M2 is biotin, a streptavidin molecule can be linked to 4 biotin molecules, so that when one end of the second intermediate modifies the affinity substance M2, the primary signal amplifier contains 1 first intermediate and 3 second intermediates, which contain (M) thereon₁+3m₂) Nucleotide fragment L₃。

In one embodiment of the above technical solution, the signal converter is a nucleotide fragment connected with a detection signal substance (e.g. magnetic beads). Capture probe and nucleotide sequence B in DNA fragment to be detected₁Complementary pairing, so as to capture the DNA fragment to be detected; the first transit product is formed by the nucleotide fragment L₁And the nucleotide sequence B in the DNA fragment to be detected₂Complementary pairing by nucleotide fragment L₃Nucleotide sequence C related to first terminal signal substance₁Complementary pairing; nucleotide sequence C of the first terminal signaler₃Complementary pairing with a signal transducer. When the first transit product contains two or more nucleotide fragments L₃In this case, a molecule of the DNA fragment to be tested will connect two or more first terminal signalizers, so that the connected signal convertants are also multiplied.

In another embodiment of the above embodiment, when the first terminal signaler comprises two or more nucleotide sequences C₃The signal converter connected is also multiplied. It can be seen that when the first intermediate contains m₁Nucleotide fragment L₃The first terminal signaler contains only 1 nucleotide sequence C₃When the signal is amplified by m₁Doubling; when the first transfer product contains only 1 nucleotide fragment L₃When the first termination signal object contains only n₁Nucleotide sequence C₃While the signal will amplify n₁Doubling; when the first transfer product contains m₁Nucleotide fragment L₃The first termination signal object contains n only₁Nucleotide sequence C₃When the signal is amplified by m₁×n₁And (4) doubling.

In one embodiment of the above technical solution, the nucleotide sequence L of the other end of the first transit substance₂In, if m₁Greater than 1, any two adjacent nucleotide fragments L₃May contain no nucleotides in between, or may contain several nucleotides, such as 1, 2, 3 … … nucleotides; nucleotide sequence C of the first terminal signal substance₂In, if n₁Greater than 1, renTwo adjacent nucleotide fragments C₃There may be no nucleotides in between, or several nucleotides, such as 1, 2, 3 … … nucleotides.

In a preferred embodiment of the above technical solution, the nucleotide sequence L of the first transfer product₂The end of (a) is modified with an affinity substance M2; the primary signal amplifier further comprises an affinity substance M1 and a second intermediate; the second transfer product contains m₂Nucleotide fragment L₃，m₂Is a positive integer; the 5 'end and/or the 3' end of the second transfer product is modified with an affinity substance M2; the affinity substance M2 and the affinity substance M1 have affinity; the first and second relays are linked to affinity substance M1 via affinity substance M2.

In another embodiment of the above protocol, a first intermediate and at least one second intermediate can be attached to a primary signal amplifier, the nucleotide fragment L attached to the primary signal amplifier being obtained by modifying the affinity substance M2 at the end of the sequence and attaching both affinity substances M2 to M1₃Will be increased to connect more first termination beacons. For example, when the affinity substance M1 is streptavidin and the affinity substance M2 is biotin, a streptavidin molecule can be linked to 4 biotin molecules, so that when one end of the second intermediate modifies the affinity substance M2, the primary signal amplifier contains 1 first intermediate and 3 second intermediates, which contain (M) thereon₁+3m₂) Nucleotide fragment L₃。

In one embodiment of the above technical solution, the nucleotide sequence of the second relay and the nucleotide sequence L at the other end of the first relay₂The same is true. At this time, when one end of the second transfer product is modified with the affinity substance M2, the nucleotide fragment L linked to the primary signal amplifier₃Is amplified by 4 times compared with the non-modified affinity substance, so that the total signal is amplified by 4m₁n₁Doubling; when both ends of the second transfer were modified with affinity M2, the total signal was amplified (3)^X-1)m₁n₁2 times, X denotes the connected stageAnd (4) counting.

In one embodiment of the above technical solution, the terminal of the first terminal signaler is modified with an affinity substance M4; the secondary signal amplifier further comprises an affinity substance M3 and a second terminal signal substance, the second terminal signal substance comprising n₂Nucleotide sequence C₃，n₂Is a positive integer; the 5 'end and/or the 3' end of the second terminal signaler is modified with an affinity substance M4; the affinity substance M3 and the affinity substance M4 have affinity; the first and second terminal signalants are linked to the affinity substance M3 via affinity substance M4.

In another embodiment of the above technical solution, the affinity substance M4 is modified at the ends of the first terminal signal and the second terminal signal, and the affinity substance M4 is linked to M3, so that the nucleotide sequence C of the first terminal signal is₃And thus more signal transitions. For example, when the affinity substance M3 is streptavidin and the affinity substance M4 is biotin, a molecular chain of streptavidin can be linked to 4 molecules of biotin, so that when one end of the second terminal signaler is modified with the affinity substance M4, the secondary signal amplifier contains 1 first terminal signaler and 3 second terminal signalers, each of which contains (n is a member of the group consisting of₁+3n₂) Nucleotide fragment L₃. At this time, the nucleotide fragment C ligated to the secondary signal amplifier₃Is amplified by 4 times compared with the non-modified affinity substance, so that the total signal is amplified by 4m₁n₁And (4) doubling.

In one embodiment of the above technical solution, the nucleotide sequence of the second terminal signal substance and the nucleotide sequence C of the first terminal signal substance₂The same is true. At this time, when one end of the second terminal signal was modified with the affinity substance M4, the signal was amplified 4-fold compared with that without the affinity modification.

In one embodiment of the above technical solution, m is₁(ii) 5, and/or said n₁2. When m is₁When the signal is 5 times, amplifying the signal by 5 times; when n is₁When the signal is 2 times, amplifying the signal by 2 times; when m is₁＝5，n₁At 2, the signal is amplified by a factor of 10.

In one embodiment, the present disclosure modifies affinity substances at the ends of the transitions (the first transition and the second transition) or the terminal signals (the first terminal signal and the second terminal signal), and a molecule of DNA fragment to be tested can indirectly bind to a plurality of secondary signal amplifiers by using high affinity between the affinity substances, so that the complex between the secondary signal amplifier and the signal transition is amplified by multiple times, and the final test signal is also amplified by multiple times, thereby further improving the sensitivity.

In one embodiment of the above technical solution, the nucleotide sequence of the second relay and the nucleotide sequence L at the other end of the first relay₂The same is true.

In one embodiment of the above technical solution, the nucleotide sequence of the second terminal signal substance and the nucleotide sequence C of the first terminal signal substance₂The same is true.

In one embodiment of the above embodiment, the capture probe has a partial nucleotide sequence B₁If the length of the complementary paired DNA fragments is too short, the stability of the combination of the two is poor; although the longer the length of the DNA fragment, the more part of the nucleotide sequence B₁The higher the stability of the binding, but if it is too long, it is difficult to distinguish between the wild-type gene and the mutant-type gene.

In one embodiment of the above technical solution, the capture probe has a partial nucleotide sequence B₁The length of the complementary paired DNA fragments is 6-20 bp. For example, the neutralizing partial nucleotide sequence B in the capture probe₁The length of complementary paired DNA fragments is 6bp, 8bp, 10bp, 12bp, 16bp, 18bp, 20bp and the like.

In a preferred embodiment of the above technical solution, the capture probe has a partial nucleotide sequence B₁The length of the complementary paired DNA fragments is 10-15 bp.

In a specific embodiment of the above technical solution, in order to improve the stability of the hybrid of the capture probe and the DNA fragment to be detected, the capture probe is provided with a modifier capable of enhancing the binding force of base pairing. The modifier is more favorable for distinguishing the wild type gene from the mutant type gene.

In a preferred embodiment of the above technical solution, the modifier is at least one of PNA, LNA, MNA, ANA, TNA, CeNA, GNA, XNA, HNA, INA, and BNA. Of course, the modification is not limited to the substance, and those skilled in the art can routinely select other modifications that enhance the binding force of base pairing.

In addition, the present disclosure also provides a kit for DNA detection, which includes the above signal amplification system, further includes an unmodified solid phase carrier, a magnetic bead modified by an affinity substance M5, and a signal converter; the signal converter is a DNA fragment, and part or all of the DNA fragment and the nucleotide sequence C of the signal converter₃Complementary pair, the signal converter is modified with an affinity substance M6, and the affinity substance M5 and the affinity substance M6 have affinity.

In one embodiment, the detection principle of the kit of the present disclosure is shown in fig. 3. In FIG. 3, Tm represents the temperature at which the bound base pairs are separated, and the higher Tm, the greater the binding force of the bound 2 sequences, and the more stable it is. Tm1 is the nucleotide sequence B of the capture probe and the DNA fragment to be detected₁The temperature at which bound base pairs separate; tm2 is the nucleotide sequence L of the primary signal amplifier₁And nucleotide sequence B of DNA fragment to be detected₂The temperature at which bound base pairs separate; tm3 is the nucleotide sequence L of the primary signal amplifier₃Nucleotide sequence C with secondary signal amplifier₁The temperature at which bound base pairs separate; tm4 is nucleotide sequence C of secondary signal amplifier₃Temperature at which base pair separation occurs in association with the signal transducer. Among Tm1, Tm2, Tm3 and Tm4, Tm3 is the smallest (e.g., Tm 4. apprxeq.Tm 1. apprxeq.Tm 2)>Tm3) so that only the nucleotide sequence L is achieved at a certain temperature (e.g.65 ℃)₃Is cleaved from the binding base of the nucleotide sequence C1 of the secondary signal amplifier.

In one technical solution, the detection principle of the present disclosure is specifically: hybridizing a capture probe designed specifically with a DNA fragment to be detected in a DNA sample so as to capture the DNA fragment to be detected; then, the hybrid of the capture probe and the DNA fragment to be detected is converted into a uniformly designed detection signal through the primary signal amplifier and the secondary signal amplifier; the detection signal is separated from the complex body through a set condition and enters a test system; finally, the probe signal is bound to the magnetic beads by the high affinity between the affinity substances, and the signal of the magnetic beads is detected. More importantly, the detection kit disclosed by the invention utilizes the signal amplification system disclosed by the invention, so that the sensitivity is greatly improved.

In one embodiment of the above technical solution, the affinity substance M1 and the affinity substance M2 are a pair of substances having specific binding action (i.e. affinity action), and when the affinity substance M1 is determined, the corresponding affinity substance M2 can be selected according to the affinity action; when affinity substance M2 is determined, the corresponding affinity substance M1 may be selected according to affinity interaction. For example, the affinity substance M1 is preferably, but not limited to, amino, polylysine, thiol, bovine serum albumin, avidin, agarose gel or polyacrylamide gel. More preferably, the affinity substance M1 is streptavidin and the affinity substance M2 is biotin.

In one embodiment of the above technical solution, the affinity substance M3 and the affinity substance M4 are a pair of substances having specific binding action (i.e. affinity action), and when the affinity substance M3 is determined, the corresponding affinity substance M4 can be selected according to the affinity action; when affinity substance M4 is determined, the corresponding affinity substance M3 can also be selected on the basis of the affinity interaction. For example, the affinity substance M3 is preferably, but not limited to, amino, polylysine, thiol, bovine serum albumin, avidin, agarose gel or polyacrylamide gel. More preferably, the affinity substance M3 is streptavidin and the affinity substance M4 is biotin.

In one embodiment of the above technical solution, when the kit comprises a signal converter, the affinity substance M6 can be labeled on the terminal signal substance and can also be labeled on the signal converter; when the kit does not include a signal converter, the affinity substance M6 is labeled on the terminal signal. Wherein the affinity substance M5 and the affinity substance M6 are a pair of substances having specific binding action (i.e., affinity action), and when the affinity substance M5 is determined, the corresponding affinity substance M6 can be selected according to the affinity action; when affinity substance M6 is determined, the corresponding affinity substance M5 can also be selected on the basis of the affinity interaction. For example, the affinity substance M5 may preferably be, but not limited to, amino, polylysine, thiol, bovine serum albumin, avidin, agarose gel, or polyacrylamide gel. When the affinity substance M5 is avidin, the affinity substance M6 is biotin. More preferably, the affinity substance M5 is streptavidin and the affinity substance M6 is biotin.

In a preferred embodiment of the detection kit of the present disclosure, the unmodified solid support is a magnetic bead or a microparticle made of glass or nylon. More preferably, the unmodified solid support is a magnetic bead.

As a preferred embodiment of the detection kit of the present disclosure, the carrier in the chip is selected from inorganic materials or polymer materials (e.g., glass, nylon). It should be noted that the solid phase carrier in the chip can not be selected from magnetic beads. This is because when the kit of the present disclosure is used, the chip is connected to the terminal signal substance, and the terminal signal substance is also directly or indirectly connected to the signal magnetic bead (magnetic bead modified by the affinity substance M5), and if the solid phase carrier in the chip is selected from the magnetic bead, it will interfere with the signal of the signal magnetic bead, thereby resulting in inaccurate measurement result of the DNA sample.

In a particular embodiment, the solid support, e.g., an inorganic material or a polymeric material, in the chip has one or more layers, e.g., two, three or four layers.

In a specific embodiment, the solid support in the chip has three layers. Specifically, the upper layer attached to the substrate comprises at least one of the following components: phosphoric acid, silane coupling agents or other ingredients that may be attached to the substrate; the middle layer of the solid phase carrier is covered with Al₂O₃(ii) a The lower layer of the solid support comprises a GMR sensor.

In one embodiment, the binding between the terminal signaler and the substrate or signal converter is made more stable than the binding between the terminal signaler and the mediatorAnd (3) the separation of the terminal signal substance from the transfer substance is facilitated, and when the kit comprises a chip with a substrate, the partial nucleotide sequence C of the terminal signal substance₂A partial nucleotide sequence C longer than the terminal signaler₁Length of (d); when the kit comprises a signal converter, a partial nucleotide sequence C of the terminal signal substance₃A partial nucleotide sequence C longer than the terminal signaler₁Length of (d). In the above manner, the segment L can be made₂And fragment C₁And separating the target fragment from other fragments for detection without separation among other fragments.

As a preferred embodiment of the DNA detection kit of the present disclosure, the partial nucleotide sequence C of the terminal signal substance₁The length of (a) is 10-15 bp; partial nucleotide sequence C of the terminal signaler₂The length of the probe is 16-60 bp; partial nucleotide sequence C of the terminal signaler₃The length of the probe is 16-60 bp.

As a more preferred embodiment of the detection kit of the present disclosure, the partial nucleotide sequence C of the terminal signal substance₂The length of the probe is 25-60 bp; partial nucleotide sequence C of the terminal signaler₃The length of the DNA fragment is 25-60 bp.

In view of stability of direct base binding, the kit comprises a chip having a substrate in which a nucleotide sequence C is bonded₂Complementary paired nucleotide sequences (nucleotide sequence E in FIG. 1)₁) Is longer than the remaining nucleotide sequence in the substrate (nucleotide sequence E in FIG. 1)₂) Length of (d).

As a preferred embodiment of the detection kit of the present disclosure, the DNA sample is derived from human blood.

In a preferred embodiment of the above technical solution, the present disclosure is applicable to the detection of disease markers, such as gene mutations.

As a preferred embodiment of the detection kit of the present disclosure, the DNA fragment to be detected in the DNA sample is a DNA fragment with a mutation site.

In a specific embodiment of the above technical solution, it is found that mutation of the transmembrane region of fibroblast growth factor receptor 3 (FGFR 3) results in Achondroplasia (ACH). At present, more than 97% of mutations reported in foreign countries and taiwan regions are more than 1138 th nucleotide of 10 th exon of FGFR3 gene. Of these, 95% are G to A base transitions, and the remainder are G to C transversions. Both mutations resulted in the substitution of glycine 380 of FGFR3 with arginine (G380R).

As a specific embodiment of the detection kit disclosed by the disclosure, the DNA fragment to be detected in the DNA sample is an FGFR3 gene fragment containing a G380R mutation site.

In the foregoing specific embodiment, the nucleotide sequence of the DNA fragment to be tested in the DNA sample is as follows:

the nucleotide sequence of the DNA fragment to be detected in the DNA sample is shown as SEQ ID NO 1; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 2; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 3; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 4; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 5; the nucleotide sequence of the substrate is shown as SEQ ID NO. 6.

In another preferred embodiment, the present disclosure is applicable to the detection of SNP sites, such as the detection of CYP2C19 × 2 (G681A).

In a specific embodiment of the above technical scheme, it was found that CYP2C19 participates in the formation of clopidogrel active metabolites and intermediate metabolites. The CYP2C19 gene has genetic diversity, so that the human population has three phenotypes of fast metabolism (EM), Intermediate Metabolism (IM) and slow metabolism (PM). The pharmacokinetics and antiplatelet effect of clopidogrel active metabolites are different along with the difference of CYP2C19 genotypes, and CYP2C19 x 2(G681A) is the most common genetic variation type in Chinese population, which can cause shear mutation of transcription protein, further leads to enzyme activity loss, and leads to slow metabolism (PM) phenotype in population. After a CYP2C19 slow metabolism type (PM) patient applies conventional dose of clopidogrel, the production of active metabolites in vivo is reduced, and the inhibition effect on platelets is reduced. That is, patients with a slow metabolism (PM) phenotype have an increased rate of cardiovascular events over Intermediate Metabolism (IM) patients under conventional dose therapy, and therefore, the use of other antiplatelet drugs or the adjustment of clopidogrel dosage may be considered.

Meanwhile, common drugs metabolized via CYP2C19 enzyme include at least: valproic acid, diazepam, phenytoin sodium, phenobarbital, fluoxetine, amitriptyline, trimipramine, phosphoramide, progesterone, proguanil, rifampin, lansoprazole, nelfinavir. Therefore, it is necessary to perform the CYP2C19 gene assay for improving the therapeutic effect and reducing the toxic side effect whenever the drug is metabolized by the CYP2C19 enzyme.

As a specific embodiment of the detection kit disclosed by the disclosure, the DNA fragment to be detected in the DNA sample is a CYP2C19 gene fragment containing G681A site.

Thus, in one aspect, the methods and products of the present disclosure may be used to predict the therapeutic effect and/or magnitude of side effects of a drug.

In another aspect, the methods and products of the present disclosure may provide a reference for selection of a treatment regimen.

In a specific embodiment, if the presence of CYP2C19 x 2(G681A) is detected in the patient by the above-described kit, the physician is prompted to consider the use of other antiplatelet drugs or to adjust the dose of clopidogrel.

the nucleotide sequence of the DNA fragment to be detected in the DNA sample is shown as SEQ ID NO. 7; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 8; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 9; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 10; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 11; the nucleotide sequence of the substrate is shown as SEQ ID NO. 12.

In a specific embodiment of the above protocol, the kit further comprises a standard solution of the DNA fragment to be tested. Through the standard solution of the DNA fragment to be detected, a standard curve about the signal quantity of the signal magnetic beads and the DNA fragment to be detected can be obtained, so that the DNA fragment to be detected in the DNA sample is quantitatively analyzed.

In a specific embodiment of the above embodiment, the kit further comprises a hybridization solution and a washing solution.

In one embodiment, the present disclosure also provides a method for detecting a DNA sample using the above kit, the method comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

(2) adding the solid phase carrier coupled with the capture probe and the DNA fragment to be detected in the DNA sample into a hybridization solution, so that the capture probe is hybridized with a part of the nucleotide sequence B1 of the DNA fragment to be detected in the DNA sample; after the reaction is finished, separating the solid phase carrier coupled with the DNA fragment to be detected and the capture probe;

(5) adding the solid phase carrier and the signal converter separated in the step (4) into the hybridization solution to ensure that the signal converter and the partial nucleotide sequence C of the terminal signal converter₃Hybridizing to form a terminal signal substance and a signal conversion substanceSeparating the solid phase carrier coupled with the transfer product, the DNA fragment to be detected and the capture probe;

When a signal transducer is present in the kit of the present disclosure, the DNA sample can be assayed using the methods described above. In the detection method of the present disclosure, a magnetic sensor, a GMR sensor, or a TMR sensor may be used for detection.

As a specific embodiment of the method for detecting a DNA sample of the present disclosure, the following step (5a) is further included between the step (5) and the step (6): reacting the complex of the terminal signaler and the signal converter with a chip having a substrate to react the substrate with a partial nucleotide sequence C of the terminal signaler₂Hybridizing to obtain a complex of the chip, the substrate, the terminal signal substance and the signal converter.

In one embodiment of the method for detecting a DNA sample according to the present disclosure, in the step (2), the step (3), and the step (4), after the solid phase carriers are separated, the solid phase carriers are washed with a washing solution respectively.

In another embodiment, the present disclosure also provides a method for detecting a DNA sample using the above kit, the method comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

When no signal converter is present in the kit of the present disclosure, the DNA sample can be assayed using the methods described above. In the absence of a signal transducer, the terminal signal is separated by appropriate temperature treatment and then reacted with a substrate. Wherein, the appropriate temperature is to ensure that the terminal signal substance is separated from the binding base pair of the transfer substance, but the DNA fragment to be detected is not separated from the binding base pair between the transfer substance and the capture probe, for example, 65 ℃ is selected as the appropriate temperature.

In a preferred embodiment of the method for detecting a DNA sample according to the present disclosure, in the step (2) and the step (3), after the solid phase carriers are separated, the solid phase carriers are washed with a washing solution, respectively.

As a preferred embodiment of the method for detecting a DNA sample according to the present disclosure, in the step (2), the reaction temperature is 45 ℃ and the reaction time is 20 minutes; in the step (3) and the step (4), the reaction temperature is 25 ℃, and the reaction time is 20 minutes.

In another embodiment, the present disclosure also provides a method for detecting DNA using the above kit, comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

(3) adding the solid phase carrier separated in the step (2) and the primary signal amplifier into a hybridization solution to enable the nucleotide sequence L at one end of the first transfer product₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with the primary signal amplifier, the DNA fragment to be detected and the capture probe;

It should be noted that the detection method of the present disclosure includes using an apparatus for detecting a magnetic signal. The device is at least one selected from the group consisting of a hall element, a magnetoresistance effect element (magnetoresistance sensor) which may be selected from a GMR sensor (giant magnetoresistance sensor) and a TMR sensor (tunnel magnetoresistance sensor).

As a preferred embodiment of the method for detecting DNA according to the present disclosure, the following step (5a) is further included between the steps (5) and (6): reacting the complex of the secondary signal amplifier and the signal converter with a chip having a substrate, and reacting the substrate with the nucleotide sequence C in the first terminal signal₄And hybridizing to obtain a complex of the chip, the substrate, the secondary signal amplifier and the signal converter.

In a preferred embodiment of the method for detecting DNA of the present disclosure, in the step (2), the step (3) and the step (4), after the solid phase carrier is separated, the solid phase carrier is washed with a washing solution.

In a preferred embodiment of the method for detecting DNA according to the present disclosure, in the step (7), the signal amount of the signal magnetic beads is detected by using a magnetic signal detection device.

As a preferred embodiment of the method for detecting DNA of the present disclosure, the method for obtaining a DNA fragment to be detected in a DNA sample comprises: collecting blood of human body, extracting DNA in blood, and fragmenting the extracted DNA.

As a more preferred embodiment of the method for detecting DNA according to the present disclosure, the extracted DNA is fragmented by an enzymatic cleavage method or an ultrasonic method.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

All reagents used in the examples were commercially available unless otherwise noted.

Example 1

This example is used for detecting the mutation from nucleotide 1138 of exon 10 of FGFR3 from nucleotide G to C (G380R), and the detection kit of this example includes: the kit comprises a capture probe, a transfer product, a terminal signal substance, a signal converter, unmodified magnetic beads, magnetic beads modified by an affinity substance M5, a glass chip with a substrate, a standard solution of a DNA fragment to be detected, a hybridization solution and a cleaning solution.

In this example, the nucleotide sequence of the DNA fragment to be detected is: 5'-CTTTGCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACCGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCT-3' (shown in SEQ ID NO: 1); the nucleotide sequence of the capture probe is as follows: 5'-AGCCCACCCGGTAGCTGAGG-3' (shown in SEQ ID NO: 2); the nucleotide sequence of the transfer product is as follows: 5'-ACACACTGCCCGCCTCGTCAGCCTC GTC TCT GTG TCG TGC-3' (shown in SEQ ID NO: 3); the nucleotide sequence of the terminal signaler is as follows: 5'-CACACACGAGACGCAGACGCCGAGCACAGACGAGCAGCACGACACAGAGACGCAGCACCGAC-3' (shown in SEQ ID NO: 4); the nucleotide sequence of the signal converter is as follows: 5'-GTCTGTGCTCGGCGTCTGCGTCTCGTGTGTGCTGCGTGGCGT GTG TGT GC-3' (shown in SEQ ID NO: 5); the nucleotide sequence of the substrate is: 5'-GT CGGTGCTGCGTCTCTGTGTCGTGCTGCT-3' (shown in SEQ ID NO: 6).

The nucleotide sequence is shown in FIG. 2, the 11 th nucleotide in the partial nucleotide sequence B1 of the DNA fragment to be detected is a mutation site, and the capture probe is complementary and paired with the partial nucleotide sequence B1 of the DNA fragment to be detected; nucleotide sequence L at one end of the Transferon₁Partial nucleotide sequence B of DNA fragment to be detected₂Complementary pairing, nucleotide sequence L at the other end of the transfer₂Partial nucleotide sequence C of terminal signaler₁Complementary pairing; partial fragment D of a Signal transducer₁Partial nucleotide sequence C of terminal signaler₃Complementary pairing; partial nucleotide sequence C of substrate and terminal signal₂And (4) complementary pairing.

Wherein, the capture probe is modified by LNA, and the terminal signal substance is marked with biotin; the affinity substance M5 is streptavidin; the hybridization solution contains the following components: 50% deionized formamide, 2 XSSC, 5 XDenhard's, 2% SDS and deionized water; the cleaning solution contains the following components: 50mM Tris-Citric acid (pH6.0),0.5M NaCl and 0.01% Tween 20.

The method for detecting the G380R mutation of the FGFR3 gene by adopting the kit in the embodiment comprises the following steps:

(1) collecting blood of a human body, extracting DNA in the blood, and fragmenting the extracted DNA through enzyme digestion to obtain a DNA fragment to be detected;

(2) coupling the capture probe to an unmodified magnetic bead (for transfer), adsorbing the magnetic bead by using a magnet, discarding waste liquid, and cleaning the unadsorbed capture probe to obtain the magnetic bead coupled with the capture probe;

(3) adding 10pmol of magnetic beads coupled with the capture probe and the DNA fragment to be detected in the DNA sample into 50 μ l of hybridization solution, and reacting at 45 ℃ for 20 minutes to allow the capture probe to hybridize with a partial nucleotide sequence B1 of the DNA fragment to be detected in the DNA sample; after the reaction is finished, sucking out the magnetic beads by a magnet to obtain the magnetic beads coupled with the DNA fragments to be detected and the capture probes, and cleaning the magnetic beads in 100 mu l of cleaning solution;

(4) adding the magnetic beads obtained in the step (3) and 10pmol of the transfer to 50. mu.l of a hybridization solution, and reacting at 25 ℃ for 20 minutes to obtain a nucleotide sequence L at one end of the transfer₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Hybridizing; after the reaction is finished, sucking out the magnetic beads by a magnet to obtain the magnetic beads coupled with the transfer product, the DNA fragment to be detected and the capture probe, and cleaning the magnetic beads in 100 mu l of cleaning solution;

(5) adding the magnetic beads obtained in the step (4) and 10pmol of a terminal signal substance to 50. mu.l of a hybridization solution, and reacting at 25 ℃ for 20 minutes to obtain a nucleotide sequence L at the other end of the intermediate₂Partial nucleotide sequence C of terminal signaler₁Hybridizing; after the reaction is finished, sucking out the magnetic beads by a magnet to obtain the magnetic beads coupled with the terminal signal substance, the transfer substance, the DNA fragment to be detected and the capture probe, and cleaning the magnetic beads in 100 mu l of cleaning solution;

(6) adding the magnetic beads separated in the step (5) and the signal converter into a hybridization solution to allow the signal converter and a partial nucleotide sequence C of the terminal signal₃Hybridizing to form a complex of a terminal signal substance and a signal converter, allowing the complex to enter a solution, adsorbing magnetic beads by a magnet, and separating the magnetic beads coupled with the transfer substance, the DNA fragment to be detected and the capture probe; carefully pipetting the solution containing the complex for use;

(7) reacting a solution containing a complex of the terminal signaler and the signal converter with a glass chip having a substrate, to thereby obtain a partial nucleotide sequence C of the substrate and the terminal signaler₂Hybridizing to obtain a solution containing a chip, a substrate, a terminal signal substance and a signal converter complex;

(8) coupling the obtained complex on magnetic beads modified by streptavidin through biotin marked on a terminal signal substance to form signal magnetic beads;

(9) and detecting the signal quantity of the signal magnetic beads, and calculating the content of the DNA fragment to be detected in the DNA sample.

In the method for detecting the G380R mutation in the FGFR3 gene, biotin may be labeled not on the terminal signaling substance but on the signal transducer. In addition, if the step (7) is omitted, the G380R mutation of the FGFR3 gene can be detected; alternatively, the G380R mutation in the FGFR3 gene can be detected by labeling biotin onto the terminal signaling substance according to this example, omitting step (6), separating the terminal signaling substance from the magnetic beads after step (5), and then performing the steps (7) to (9) on the solution containing the terminal signaling substance and the glass chip with the substrate. Of course, when the corresponding operation steps are omitted, the kit may omit the corresponding substances.

Example 2

This example was used to determine whether the CYP2C19 gene fragment in the DNA sample contained the G681A mutation. The detection kit of the embodiment comprises: the kit comprises a capture probe, a transfer product, a terminal signal substance, a signal converter, unmodified magnetic beads, magnetic beads modified by an affinity substance M5, a glass chip with a substrate, a standard solution of a DNA fragment to be detected, a hybridization solution and a cleaning solution.

In this example, the nucleotide sequence of the DNA fragment to be detected is: 5'-AATTACAACCAGAGCTTGGCATATTGTATCTATACCTTTATTAAATGCTTTTAATTTAATAAATTATTGTTTTCTCTTAGATATGCAATAATTTTCCCACTATCATTGATTATTTCCCGGGAACCCATAACAAATTACTTAAAAACCTTGCTTTTATGGAAAGTGATATTTTGGAGAAAG-3' (shown in SEQ ID NO: 7); the nucleotide sequence of the capture probe is as follows: 5'-TATGGGTTCCCGGGAAATAAT-3' (shown in SEQ ID NO: 8); the nucleotide sequence of the transfer product is as follows: 5'-GGTATAGATACAATATGCCAAGCTCTGGTTGGTCTCTGTGTCGTGC-3' (shown in SEQ ID NO: 9); the nucleotide sequence of the terminal signaler is as follows: 5'-CACACACGAGACGCAGACGCCGAGCACAGACGAGCAGCACGACACAGAGACGCAGCACCGAC-3' (shown in SEQ ID NO: 10); the nucleotide sequence of the signal converter is as follows: 5'-GTCTGTGCTCGGCGTCTGCGTCTCGTGTGTGCTGCGTGGCGTGTGTGTGC-3' (shown in SEQ ID NO: 11); the nucleotide sequence of the substrate is: 5'-GTCGGTGCTGCGTCTCTGTGTCGTGCTGCT-3' (shown in SEQ ID NO: 12).

The experimental procedure of example 2 was compared with that of example 1, except that the sequence of the specific DNA used was partially different, and the other experimental protocol and experimental conditions were the same as those of example 1.

Example 3

This example is used for detecting the mutation of 1138 th nucleotide G to C of exon 10 of FGFR3 gene (G380R), and the detection kit of this example comprises a signal amplification system, which comprises a capture probe, a primary signal amplifier and a secondary signal amplifier.

Wherein, the nucleotide sequence of the DNA fragment to be detected is as follows: 5'-CTTTGCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACCGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCT-3' (shown in SEQ ID NO: 13); the nucleotide sequence of the capture probe is as follows: 5'-AGCCCACCCGGTAGCTGAGG-3' (shown as SEQ ID NO: 14), the 10 th basic group in the capture probe is combined with the mutation site to be detected; the primary signal amplifier is a first transfer product, and the nucleotide sequence of the first transfer product is as follows: 5'-ACACACTGCCCGCCTCGTCAGCCTC GTC TCT GTG TCG TGCATCG GTC TCT GTG TCG TGCATCGGTC TCT GTG TCG TGCATCGGTC TCT GTG TCG TGCATCG GTC TCT GTG TCG TGC-3' (shown in SEQ ID NO: 15); the secondary signal amplifier is a first terminal signal, and the nucleotide sequence of the first terminal signal is as follows: 5'-CACACACGAGACGCAGACGCCGAGCACAGACATCGCACACACGAGACGCAGACGCCGAGCACAGACGAGCAGCACGACACAGAGACGCAGCACCGAC-3' (shown in SEQ ID NO: 16).

As shown in FIG. 4, the capture probe and the nucleotide sequence B in the DNA fragment to be detected₁Complementary pairing, nucleotide B₁The 11 th basic group in the sequence is a site to be detected; nucleotide sequence L at one end of the first transfer₁And nucleotide sequence B of DNA fragment to be detected₂Complementary pairing; nucleotide sequence at the other end of the first transfer (excluding nucleotide sequence L in the first transfer)₁Other nucleotide sequence) contains a 5-nucleotide fragment L₃(ii) a The first terminal signaler comprises the nucleotide sequence C₁And nucleotide sequence C₂Nucleotide sequence C₂Containing 2 nucleotide sequences C which are complementary pairs to partial DNA fragments of the signal transducer₃(ii) a Nucleotide fragment L₃And the nucleotide sequence C₁And (4) complementary pairing.

With the signal amplification system, the signal amplification is 5 × 2 to 10 times.

The kit for detecting the FGFR3 gene G380R comprises the signal amplification system, unmodified magnetic beads, streptavidin-modified magnetic beads, a signal converter and a glass chip with a substrate; the signal converter and the substrate are DNA fragments, and the nucleotide sequence of the signal converter is as follows: 5'-GTCTGTGCTCGGCGTCTGCGTCTCGTGTGTGCTGCGTGGCGT GTG TGT GC-3' (shown in SEQ ID NO: 17), biotin is labeled on the signal transducer; the nucleotide sequence of the substrate is: 5'-GTCGGTGCTGCGTCTCTGTGTCGTGCTGCT-3' (shown in SEQ ID NO: 18); as shown in FIG. 4, a partial DNA fragment D of the signal transducer₁Nucleotide sequence C related to first terminal signal substance₃Complementary pair, nucleotide sequence C of substrate and first terminal signal₄And (4) complementary pairing.

The method for detecting the FGFR3 gene G380R comprises the following steps:

(3) adding the magnetic beads coupled with the capture probes and the DNA fragments to be detected in the DNA sample into 50 mul of hybridization solution, and reacting for 20 minutes at 45 ℃ to ensure that the capture probes and the partial nucleotide sequences B of the DNA fragments to be detected in the DNA sample₁Hybridizing; after the reaction is finished, sucking out the magnetic beads by a magnet to obtain the magnetic beads coupled with the DNA fragments to be detected and the capture probes, and cleaning the magnetic beads in 100 mu l of cleaning solution;

(4) adding the magnetic beads obtained in the step (3) and the primary signal amplifier into 50 mu L of hybridization solution, and reacting at 25 ℃ for 20 minutes to ensure that the nucleotide sequence L of the primary signal amplifier₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Hybridizing; after the reaction is finished, sucking out the magnetic beads by a magnet to obtain the magnetic beads coupled with the primary signal amplifier, the DNA fragment to be detected and the capture probe, and cleaning the magnetic beads in 100 mu l of cleaning solution;

(5) adding the magnetic beads obtained in the step (4) and the secondary signal amplifier into 50 mu L of hybridization solution, and reacting at 25 ℃ for 20 minutes to ensure that the nucleotide sequence L of the primary signal amplifier₃Nucleotide sequence C with secondary signal amplifier₁Hybridizing; after the reaction is finished, sucking out the magnetic beads by a magnet to obtain the magnetic beads coupled with the secondary signal amplifier, the primary signal amplifier, the DNA fragment to be detected and the capture probe, and cleaning the magnetic beads in 100 mu l of cleaning solution;

(6) adding the magnetic beads separated in the step (5) and the signal converter into a hybridization solution to enable the signal converter and the nucleotide sequence C of the secondary signal amplifier₃Hybridizing to form a complex of a secondary signal amplifier and a signal converter, allowing the complex to enter a solution, adsorbing magnetic beads by a magnet, and separating the magnetic beads coupled with the primary signal amplifier, the DNA fragment to be detected and the capture probe; carefully pipetting the solution containing the complex for use;

(7) will contain secondary signal amplification and signalReacting the solution of the complex of the transducer with a glass chip having a substrate, the substrate being reacted with the nucleotide sequence C of the secondary signal amplifier₄Hybridizing to obtain a solution containing a glass chip, a substrate, a secondary signal amplifier and a signal converter complex;

(8) coupling the obtained complex on magnetic beads modified by streptavidin through biotin marked on a signal converter to form signal magnetic beads;

A schematic diagram of DNA detection using the signal amplification system of this example is shown in FIG. 5. In FIG. 5, A denotes a capture probe, B denotes a DNA fragment to be detected, L denotes a primary signal amplifier, C denotes a secondary signal amplifier, D denotes a signal converter, and S denotes a signal (for example, streptavidin-modified magnetic beads).

Example 4 (comparative example)

Wherein, the DNA fragment to be detected and the capture probe are the same as those in example 3; the primary signal amplifier consists of a first transfer substance, a second transfer substance and streptavidin, and the nucleotide sequence of the first transfer substance is as follows: 5'-ACACACTGCCCGCCTCGTCAGCCTC GTC TCT GTG TCG TGC-biotin (shown in SEQ ID NO: 19), wherein the 3' end of the first transfer is modified with biotin; the nucleotide sequence of the second transfer is: 5'-GTC TCT GTG TCG TGC-biotin (shown in SEQ ID NO: 20), and the 3' end of the second transfer is modified with biotin; in the primary signal amplifier, 1 molecule of the first transfer substance and 3 molecules of the second transfer substance are connected to streptavidin through biotin; the secondary signal amplifier is a first terminal signal, and the nucleotide sequence of the first terminal signal is as follows: 5'-CACACACGAGACGCAGACGCCGAGCACAGACGAGCAGCACGACAC AGAGACGCAGCACCGAC-3' (shown in SEQ ID NO: 21).

As shown in fig. 6; first of allNucleotide sequence L at one end of the Transferon₁And nucleotide sequence B of DNA fragment to be detected₂Complementary pairing; the first terminal signaler comprises the nucleotide sequence C₁And nucleotide sequence C₃Nucleotide sequence C₃Complementary pairing with a portion of the DNA fragment of the signal transducer; nucleotide fragment L in first and second transitions₃And the nucleotide sequence C₁And (4) complementary pairing.

By adopting the signal amplification system, the signal is amplified by 4 times.

The kit for detecting the FGFR3 gene G380R comprises the signal amplification system, unmodified magnetic beads, streptavidin-modified magnetic beads, a signal converter and a glass chip with a substrate; the signal transducer and substrate were the same as in example 3.

The method for detecting the FGFR3 gene G380R in this example is the same as that in example 3.

Example 5

The present embodiment differs from embodiment 4 only in that: the nucleotide sequence of the first transfer is: 5' -ACACACTGCCCGCCTCGTCAGCCTCGTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGC-biotin (shown in SEQ ID NO: 15); the nucleotide sequence of the second transfer is: 5' -GTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGCATCGGTCTCTGTGTCGTGC-biotin (shown in SEQ ID NO: 22). As shown in FIG. 7, the nucleotide sequence L at one end of the first relay product₁And nucleotide sequence B of DNA fragment to be detected₂Complementary pairing; nucleotide sequence L at the other end of the first transfer₂(first transfer product except nucleotide sequence L₁Other nucleotide sequence) contains a 5-nucleotide fragment L₃(ii) a The second transit contains a 5 nucleotide fragment L₃. The true bookThe schematic structure of the primary signal amplifier of the embodiment is shown in fig. 8.

With the signal amplification system, the signal amplification is 4 × 5 to 20 times.

The kit for detecting the FGFR3 gene G380R of this example was the same as in example 4 except for the first intermediate and the second intermediate, and the method for detecting the FGFR3 gene G380R of this example was the same as in example 3.

Example 6

The present embodiment is different from embodiment 5 only in the following points: biotin is modified at both ends of the second transfer product. X represents the connection stage number, and if only the first intermediate product exists, X is 1; when the first relay is connected with 3 second relays through 1 streptavidin, X is 2; when each second intermediate is linked to 3 second intermediates via 1 streptavidin moiety, X is 3, and so on … …, 1 molecule of the first intermediate and (3) in the primary signal amplifier^XThe-3)/2 molecules of the second intermediate are connected to streptavidin through biotin, wherein the amount of the streptavidin is 1/3 of the second intermediate. With the signal amplification system of the present embodiment, the signal can be amplified (3)^X-1) 5 × 1/2 times.

The kit for detecting the FGFR3 gene G380R of this example was the same as in example 5 except for the second intermediate transfer, and the method for detecting the FGFR3 gene G380R of this example was the same as in example 3.

Example 7

This example was conducted to examine whether the CYP2C19 gene segment of the DNA sample contained the G681A mutation. The detection kit of this embodiment comprises a signal amplification system comprising a capture probe, a primary signal amplifier and a secondary signal amplifier.

This example is compared with example 3 only in that the DNA fragment to be tested is different from the specific sequences of the specifically used DNA capture probe, primary signal amplifier and secondary signal amplifier, and other experimental conditions are identical.

In the specific reagent used in this example, the nucleotide sequence of the DNA fragment to be detected is: 5'-AATTACAACCAGAGCTTGGCATATTGTATCTATACCTTTATTAAATGCTTTTAATTTAATAAATTATTGT TTTCTCTTAG ATATGCAATA ATTTTCCCACTATCATTGATTATTTCCCGGGAACCCATAACAAATTACTTAAAAACCTTGCTTTTATGGA AAGTGATATT TTGGAGAAAG-3' (shown in SEQ ID NO: 23); the nucleotide sequence of the capture probe is as follows: 5'-TATGGGTTC CCGGGAAATA AT-3' (shown as SEQ ID NO: 24), the 10 th basic group in the capture probe is combined with the mutation site to be detected; the primary signal amplifier is a first transfer product, and the nucleotide sequence of the first transfer product is as follows: 5'-GGTATA GATACAATAT GCCAAGCTCT GGTTG GTC TCT GTG TCG TGCATCG GTC TCT GTG TCG TGCATCG GTC TCT GTG TCG TGCATCG GTC TCT GTG TCG TGCATCG GTC TCT GTG TCG TGC-3' (shown in SEQ ID NO: 25); the secondary signal amplifier is a first terminal signal, and the nucleotide sequence of the first terminal signal is as follows: 5'-CACACACGAGACGCAGACGCCGAGCACAGAC ATCGCACACACGAGACGCAGACGCCGAGCACAGACGAGCAGCACGACACAGAGACGCAGCACCG AC-3' (shown in SEQ ID NO: 26).

The kit for detecting whether the CYP2C19 gene segment of the DNA sample contains the G681A mutation comprises the signal amplification system, and can also comprise unmodified magnetic beads, streptavidin-modified magnetic beads, a signal converter and a glass chip with a substrate. Wherein, the signal converter and the substrate are DNA fragments, and the nucleotide sequence of the signal converter is as follows: 5'-GTC TGT GCT CGG CGT CTG CGT CTC GTG TGT GCT GCG TGG CGT GTG TGT GC-3' (shown in SEQ ID NO: 27), biotin being labeled on the signal transducer; the nucleotide sequence of the substrate is: 5'-GTCGGTGCTGCGTCTCTGTGTCGTGCTGCT-3' (shown in SEQ ID NO: 28).

Example 8 (Effect example)

This effect demonstrates the experimental results of example 1 and compares the sensitivity of detection with the experimental and comparative kits.

The experimental kit is different from the kit in example 3 only in that: the nucleotide sequence of the first terminal signal is: 5'-CACACACGAGACGCAGACGCCGAGCACAGACGAGCAGCACGACACAGAGACGCAGCACCGAC-3' (SEQ ID NO: 21), and in this effect example, biotin is labeled on the first terminal signal. That is, the expansion factor of the comparative kit should theoretically be 5 × 1 to 5 times as compared with the kit used in example 3 of the present disclosure.

The comparative and experimental kits differ only in that: the nucleotide sequence of the first transfer is: 5'-ACACACTGCCCGCCTCGTCAGCCTC GTC TCT GTG TCG TGC-3' (shown in SEQ ID NO: 19). That is, the comparative kit is the same as that used in example 1 of the present disclosure.

In the effect example, a standard curve is firstly manufactured by adopting a contrast kit, and the specific steps are as follows:

1.5 tube experimental group: respectively reacting 10pmol of capture probe (Bead-AM1) combined with magnetic beads with 1pmol, 5pmol, 10pmol, 50pmol and 100pmol of DNA fragments to be detected (BM1) in a hybridization solution to respectively obtain unequal amounts of Bead-AM1-BM1 (a compound of the capture probe combined with the magnetic beads and the DNA fragments to be detected);

respectively adding 50pmol of a first transfer substance (normal P0(L)) in a contrast detection kit into the Bead-AM1-BM1, and reacting in a hybridization solution to obtain the Bead-AM1-BM1-L (a compound of a capture probe combined with magnetic beads, a DNA fragment to be detected and the first transfer substance);

respectively adding 50pmol of biotin-bound first terminal signal substance (C/biotin) into the Bead-AM1-BM1-L, reacting in a hybridization solution to obtain the Bead-AM1-BM1-L-C/biotin (a complex of the capture probe bound with the magnetic Bead, the DNA fragment to be detected, the first transfer substance and the first terminal signal substance)

Testing the quantity of C/biotin on the bead to output signal gradients of different BM1 concentration gradients, and making a standard curve; the test results are shown in table 2.

Then, an experimental kit is adopted for comparison, and the specific steps are as follows:

1.1 tube experimental group, 10pmol of Bead-AM1 and 1pmol of BM1 are reacted in a hybridization solution to obtain Bead-AM1-BM 1;

adding 50pmol of a first transfer substance (new P0(L1)) in the detection kit disclosed by the disclosure into the Bead-AM1-BM1, and reacting in a hybridization solution to obtain the Bead-AM1-BM 1-L1;

50pmol of C/biotin is added into the Bead-AM1-BM1-L1, the mixture is reacted in a hybridization solution to obtain the Bead-AM1-BM1-L1-C/biotin, the amount of the C/biotin on the Bead is tested to output a BM1 signal amplified by L1, and the test results are shown in Table 2.

In the effect example, the dosage of the related substances in the standard curve preparation and the experimental kit comparison process is shown in table 1. The test results of the effect example are shown in table 2.

TABLE 1

TABLE 2

	1	2	3	4	5	6
							BM1	1P	5	10	50	100	1
OD	16.87	19.23	21.09	21.36	22.59	19.25

Wherein, the tubes 1-5 are used as a control group for making a standard curve. Tube 6 is the experimental group for experimental effect comparison.

As can be seen from Table 2, tubes 1 to 5 exhibited a good linear relationship, demonstrating that the kit of example 1 can be used for the detection of gene mutations.

Meanwhile, as can be seen from table 2, since the OD value of the tube 6 and the OD value of the tube 2 are almost the same, they can be directly used for comparison. That is, the result of the test with the test kit, in which the concentration of the DNA fragment to be tested (BM1) was 1pmol, was consistent with the result of the test with the control kit, in which the concentration of the DNA fragment to be tested (BM1) was 5 pmol. Therefore, when the experimental kit is adopted, the sensitivity is improved by 5 times.

The above examples of the present disclosure are merely examples provided for clearly illustrating the present disclosure and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Sequence listing

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<213> Intelligent people

<400> 23

aattacaacc agagcttggc atattgtatc tataccttta ttaaatgctt ttaatttaat 60

aaattattgt tttctcttag atatgcaata attttcccac tatcattgat tatttcccgg 120

gaacccataa caaattactt aaaaaccttg cttttatgga aagtgatatt ttggagaaag 180

<210> 24

<211> 21

<212> DNA

<213> Artificial sequence

<400> 24

tatgggttcc cgggaaataa t 21

<210> 25

<211> 122

<212> DNA

<213> Artificial sequence

<400> 25

ggtatagata caatatgcca agctctggtt ggtctctgtg tcgtgcatcg gtctctgtgt 60

cgtgcatcgg tctctgtgtc gtgcatcggt ctctgtgtcg tgcatcggtc tctgtgtcgt 120

gc 122

<210> 26

<211> 97

<212> DNA

<213> Artificial sequence

<400> 26

cacacacgag acgcagacgc cgagcacaga catcgcacac acgagacgca gacgccgagc 60

acagacgagc agcacgacac agagacgcag caccgac 97

<210> 27

<211> 50

<212> DNA

<213> Artificial sequence

<400> 27

gtctgtgctc ggcgtctgcg tctcgtgtgt gctgcgtggc gtgtgtgtgc 50

<210> 28

<211> 30

<212> DNA

<213> Artificial sequence

<400> 28

gtcggtgctg cgtctctgtg tcgtgctgct 30

Claims

1. The detection kit for the DNA sample is characterized by comprising a capture probe, a transfer product, a terminal signal product, an unmodified solid phase carrier and a magnetic bead modified by an affinity substance M5; wherein the content of the first and second substances,

the intermediate and the terminal signal substance are DNA fragments, and the nucleotide sequence L at one end of the intermediate₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Complementary pair, nucleotide sequence L at the other end of the said intermediate₂Partial nucleotide sequence C of terminal signaler₁Complementary pairing; and

2. The kit for detecting a DNA sample according to claim 1, wherein Tm1 is a partial nucleotide sequence B between the capture probe and the DNA fragment to be detected₁The temperature at which bound base pairs separate; tm2 is the nucleotide sequence L of the transfer product₁And partial nucleotide sequence B of the DNA fragment to be detected₂The temperature at which bound base pairs separate; tm3 is the nucleotide sequence L of the transfer product₂Partial nucleotide sequence C of terminal signaler₁The temperature at which bound base pairs separate; tm4 is partial nucleotide sequence C of the terminal signal substance₃The temperature at which base pair separation occurs with the signal transducer; the numerical value of Tm3 is the lowest among Tm1, Tm2, Tm3 and Tm 4.

3. The kit for detecting a DNA sample according to claim 1, wherein the kit comprises a chip having a substrate, and wherein the partial nucleotide sequence C of the terminal signaling substance is₂A partial nucleotide sequence C longer than the terminal signaler₁Length of (d); when the kit comprises a signal converter, a partial nucleotide sequence C of the terminal signal substance₃A partial nucleotide sequence C longer than the terminal signaler₁Length of (d).

4. The kit for detecting a DNA sample according to claim 1, wherein the kit comprisesThen, the capture probe is a DNA fragment, and the neutralizing partial nucleotide sequence B in the capture probe₁The length of the complementary paired DNA fragments is 6-20 bp.

5. The kit for detecting a DNA sample according to claim 4, wherein the capture probe has a partial nucleotide sequence B₁The length of the complementary paired DNA fragments is 10-15 bp.

6. The kit for detecting a DNA sample according to any one of claims 1 to 5, wherein the capture probe has a modifier capable of enhancing the binding force of base pairing.

7. The kit for detecting a DNA sample according to claim 6, wherein the modifier is at least one of PNA, LNA, MNA, ANA, TNA, CeNA, GNA, XNA, HNA, INA, BNA.

8. The DNA sample detection kit of claim 1, wherein the unmodified solid support is a magnetic bead or a microparticle made of glass or nylon.

9. The kit for detecting a DNA sample according to claim 1, wherein the affinity substance M5 is amino, polylysine, thiol, bovine serum albumin, avidin, agarose gel or polyacrylamide gel; preferably, the affinity substance M5 is streptavidin, and the affinity substance M6 is biotin.

10. The kit for detecting a DNA sample according to claim 1, wherein the solid carrier in the chip comprises a material selected from the group consisting of Al₂O₃Glass, polymer and nylon.

11. The kit for detecting a DNA sample according to claim 1, wherein the partial nucleotide sequence C of the terminal signaling substance₁The length of (a) is 10-15 bp; partial nucleotide sequence C of the terminal signaler₂The length of the probe is 16-60 bp; partial nucleotide sequence C of the terminal signaler₃The length of the probe is 16-60 bp.

12. The kit for detecting a DNA sample according to claim 1, wherein the partial nucleotide sequence C of the terminal signaling substance₂The length of the probe is 25-60 bp; partial nucleotide sequence C of the terminal signaler₃The length of the DNA fragment is 25-60 bp.

13. The kit for detecting a DNA sample according to claim 1, wherein the kit comprises a chip having a substrate in which the nucleotide sequence C is homologous₂The length of the complementary pairing nucleotide sequence is longer than the length of the remaining nucleotide sequence in the substrate.

14. The kit for detecting a DNA sample according to claim 1, wherein the DNA sample is derived from human blood.

15. The kit for detecting a DNA sample according to claim 1 or 14, wherein the DNA fragment to be detected in the DNA sample is a DNA fragment having a mutation site.

16. The DNA sample detection kit of claim 15, wherein the DNA fragment to be detected in the DNA sample is an FGFR3 gene fragment containing a G380R mutation site.

17. The kit for detecting a DNA sample according to claim 16, wherein the nucleotide sequence of the DNA fragment to be detected in the DNA sample is represented by SEQ ID NO 1; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 2; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 3; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 4; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 5; the nucleotide sequence of the substrate is shown as SEQ ID NO. 6.

18. The kit for detecting a DNA sample according to claim 15, wherein the DNA fragment to be detected in the DNA sample is a CYP2C19 gene fragment containing G681A site.

19. The kit for detecting a DNA sample according to claim 18, wherein the nucleotide sequence of a DNA fragment to be detected in the DNA sample is represented by SEQ ID NO. 7; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 8; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 9; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 10; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 11; the nucleotide sequence of the substrate is shown as SEQ ID NO. 12.

20. The kit for detecting a DNA sample according to claim 1, further comprising a standard solution of a DNA fragment to be detected; preferably, the detection kit further comprises a hybridization solution and a cleaning solution.

21. The DNA sample detection kit of claim 19, wherein the hybridization solution comprises deionized formamide, 2 xssc, 5 xdenhard's, SDS and deionized water; the washing solution contains Tris-Citric, NaCl and Tween 20.

22. A method for detecting a DNA sample, comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

(3) adding the solid phase carrier and the transfer substance separated in the step (2) into a hybridization solution to ensure that a core at one end of the transfer substanceNucleotide sequence L₁And partial nucleotide sequence B of DNA fragment to be detected in DNA sample₂Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with the transfer product, the DNA fragment to be detected and the capture probe;

the partial nucleotide sequence B₁Containing a site to be detected;

23. The method for detecting a DNA sample according to claim 22, further comprising a step (5a) between the step (5) and the step (6), wherein the step (5a) comprises reacting the complex of the terminal signaler and the signal converter with a chip having a substrate, and allowing the substrate to react with a part of the nucleotide sequence C of the terminal signaler₂Hybridizing to obtain a complex of the chip, the substrate, the terminal signal substance and the signal converter; wherein the content of the first and second substances,

the chip also contains a magnetic sensor.

24. The method for detecting a DNA sample according to claim 22, wherein in the step (2), the step (3) and the step (4), after the solid carriers are separated, the solid carriers are washed with a washing solution, respectively.

25. A method for detecting a DNA sample, comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

(4) adding the solid phase carrier separated in the step (3) and a terminal signal substance into a hybridization solution to enable the nucleotide sequence L at the other end of the transit substance₂Partial nucleus of terminal signalerNucleotide sequence C₁Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with a terminal signal substance, a transfer substance, a DNA fragment to be detected and a capture probe; after washing the unbound terminal signal substance, the solid phase carrier is subjected to appropriate temperature treatment, so that the terminal signal substance is separated from the solid phase carrier and enters a solution, and the solid phase carrier coupled with the transfer product, the DNA fragment to be detected and the capture probe is removed;

the partial nucleotide sequence B₁Containing a site to be detected;

the chip also contains a magnetic sensor.

26. The method for detecting a DNA sample according to claim 25, wherein in the step (2) and the step (3), after the solid carriers are separated, the solid carriers are washed with a washing solution, respectively.

27. The method for detecting a DNA sample according to claim 22 or 25, wherein in the step (2), the reaction temperature is 45 ℃ and the reaction time is 20 minutes; in the step (3) and the step (4), the reaction temperature is 25 ℃, and the reaction time is 20 minutes.

28. The method for detecting a DNA sample as claimed in claim 22 or 25, wherein in the step (7), the signal quantity of the signal magnetic beads is detected by using a magnetic signal detection device;

wherein the device is at least one selected from the group consisting of a hall element, a magnetoresistance effect element (magnetoresistance sensor) which may be selected from a GMR sensor (giant magnetoresistance sensor) and a TMR sensor (tunnel magnetoresistance sensor).

29. The method for detecting a DNA sample according to claim 22 or 25, wherein the DNA fragment to be detected in the DNA sample is obtained by: collecting blood of human body, extracting DNA in blood, and fragmenting the extracted DNA.

30. The method for detecting a DNA sample according to claim 29, wherein the extracted DNA is fragmented by an enzymatic cleavage method or an ultrasonic method.

31. The detection kit for the DNA sample is characterized by comprising a capture probe, a primary signal amplifier, a secondary signal amplifier, an unmodified solid phase carrier, a magnetic bead modified by an affinity substance M5 and a signal converter; wherein the content of the first and second substances,

m₁and n₁Are all positive integers;

optionally, m is₁And n₁Not simultaneously 1.

32. The kit for detecting a DNA sample according to claim 31, wherein the nucleotide sequence L of the first intermediate is₂The end of (a) is modified with an affinity substance M2; the primary signal amplifier further comprises an affinity substance M1 and a second intermediate; the second transfer product contains m₂Nucleotide fragment L₃，m₂Is a positive integer; the 5 'end and/or the 3' end of the second transfer product is modified with an affinity substance M2; the affinity substance M2 and the affinity substance M1 have affinity; the first and second relays are linked to affinity substance M1 via affinity substance M2.

33. The kit for detecting a DNA sample according to claim 31, wherein the first terminal signaler is modified at its end with an affinity substance M4; the secondary signal amplifier further comprises an affinity substance M3 and a second terminal signal substance, the second terminal signal substance comprising n₂Nucleotide sequence C₃，n₂Is a positive integer; the 5 'end and/or the 3' end of the second terminal signaler is modified with an affinity substance M4; the affinity substance M3 and the affinity substance M4 have affinity; the first and second terminal signalants are linked to the affinity substance M3 via affinity substance M4.

34. The kit for detecting a DNA sample according to claim 31, wherein m is₁(ii) 5, and/or said n₁＝2。

35. The detection kit for the DNA sample is characterized by comprising a capture probe, a primary signal amplifier, a secondary signal amplifier, an unmodified solid phase carrier, a magnetic bead modified by an affinity substance M5 and a signal converter; wherein the content of the first and second substances,

The secondary signal amplifier comprises a first terminal signal containing a nucleotide sequence C₁And nucleotide sequence C₂Nucleotide sequence C₂Containing n₁Part of and signal conversion objectNucleotide sequence C of complementary pairing of partial or whole DNA fragments₃；

m₁and n₁Are all positive integers; m is₂And n₂Are all positive integers;

optionally, m is₁And n₁And is also 1.

36. The kit for detecting a DNA sample according to claim 31 or 35, wherein Tm1 represents the nucleotide sequence B between the capture probe and the DNA fragment to be detected₁The temperature at which bound base pairs separate; tm2 is the nucleotide sequence L of the primary signal amplifier₁And D to be measuredNucleotide sequence B of NA fragment₂The temperature at which bound base pairs separate; tm3 is the nucleotide sequence L of the primary signal amplifier₃Nucleotide sequence C with secondary signal amplifier₁The temperature at which bound base pairs separate; tm4 is nucleotide sequence C of secondary signal amplifier₃The temperature at which base pair separation occurs with the signal transducer; the numerical value of Tm3 is the lowest among Tm1, Tm2, Tm3 and Tm 4.

37. The kit for detecting a DNA sample according to claim 31 or 35, wherein the kit further comprises a chip having a substrate which is a DNA fragment, and the first terminal signaling substance further comprises a nucleotide sequence C₄Partial or complete fragments of said substrate and nucleotide sequence C₄And (4) complementary pairing.

38. The kit for detecting a DNA sample according to claim 31 or 35, wherein the nucleotide sequence of the second intermediate and the nucleotide sequence L at the other end of the first intermediate are the same₂The same is true.

39. The kit for detecting a DNA sample according to claim 31 or 35, wherein the nucleotide sequence of the second terminal signal substance and the nucleotide sequence C of the first terminal signal substance are the same₂The same is true.

40. The kit for detecting a DNA sample according to any one of claims 32 to 33 or 35, wherein the affinity substance M1 or M3 or M5 is selected from amino group, polylysine, thiol group, bovine serum albumin, avidin, agarose gel or polyacrylamide gel; preferably, the affinity substance M1 is streptavidin, and the affinity substance M2 is biotin; or the affinity substance M3 is streptavidin, and the affinity substance M4 is biotin; or the affinity substance M5 is streptavidin, and the affinity substance M6 is biotin.

41. The kit for detecting a DNA sample according to claim 31 or 35, which comprisesThe nucleotide sequence C₃Is longer than the nucleotide sequence C₁Length of (d).

42. The kit for detecting a DNA sample according to claim 31 or 35, wherein the nucleotide sequence C is₃Has a length of 16-60 bp, and the nucleotide sequence C₁The length of (a) is 10-15 bp; preferably, the nucleotide sequence C₃The length of the DNA fragment is 25-60 bp.

43. The kit for detecting a DNA sample according to claim 37, wherein the nucleotide sequence C is₄Is longer than the nucleotide sequence C₁Length of (d).

44. The kit for detecting a DNA sample according to claim 37, wherein the nucleotide sequence C is₄Has a length of 16-60 bp, and the nucleotide sequence C₁The length of (a) is 10-15 bp; preferably, the nucleotide sequence C₄The length of the DNA fragment is 25-60 bp.

45. The kit for detecting a DNA sample according to claim 37, wherein the substrate has a nucleotide sequence C₄The length of the complementary pairing nucleotide sequence is longer than the length of the remaining nucleotide sequence in the substrate.

46. The kit for detecting a DNA sample according to claim 31 or 35, wherein the solid carrier in the chip comprises a material selected from the group consisting of Al₂O₃Glass, polymer and nylon.

47. The kit for detecting a DNA sample according to claim 31 or 35, wherein the DNA sample is derived from human blood.

48. The kit for detecting a DNA sample according to claim 31 or 35, wherein the DNA fragment to be detected in the DNA sample is a DNA fragment having a mutation site.

49. The DNA sample detection kit of claim 48, wherein the DNA fragment to be detected in the DNA sample is an FGFR3 gene fragment containing a G380R mutation site.

50. The kit for detecting a DNA sample according to claim 49, wherein the nucleotide sequence of the DNA fragment to be detected in the DNA sample is represented by SEQ ID NO. 13; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 14; the nucleotide sequence of the transfer product is shown as SEQ ID NO. 15; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 16; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 17; the nucleotide sequence of the substrate is shown as SEQ ID NO. 18.

51. The kit for detecting a DNA sample according to claim 48, wherein the DNA fragment to be detected in the DNA sample is a CYP2C19 gene fragment containing G681A site.

52. The kit for detecting a DNA sample according to claim 51, wherein the nucleotide sequence of the DNA fragment to be detected in the DNA sample is represented by SEQ ID NO. 19; the nucleotide sequence of the capture probe is shown as SEQ ID NO. 20; the nucleotide sequence of the transfer product is shown as SEQ ID NO: 21; the nucleotide sequence of the terminal signal substance is shown as SEQ ID NO. 22; the nucleotide sequence of the signal converter is shown as SEQ ID NO. 23; the nucleotide sequence of the substrate is shown as SEQ ID NO. 24.

53. The kit for detecting a DNA sample according to claim 48, further comprising a standard solution of a DNA fragment to be detected; preferably, the detection kit further comprises a hybridization solution and a cleaning solution.

54. The DNA sample detection kit of claim 53, wherein the hybridization solution comprises deionized formamide, 2 XSSC, 5 XDenhard's, SDS, and deionized water; the washing solution contains Tris-Citric, NaCl and Tween 20.

55. A method for detecting DNA using the DNA sample detection kit according to claim 31 or 35, comprising the steps of:

(1) coupling a capture probe to an unmodified solid phase support;

(2) adding the solid phase carrier coupled with the capture probe and the DNA fragment to be detected in the DNA sample into a hybridization solution, so that the capture probe and part of nucleotide B of the DNA fragment to be detected in the DNA sample₁Hybridizing; after the reaction is finished, separating the solid phase carrier coupled with the DNA fragment to be detected and the capture probe;

(3) adding the solid phase carrier separated in the step (2) and the primary signal amplifier into a hybridization solution to enable the nucleotide sequence L at one end of the first transfer product₁And partial nucleotide B of DNA fragment to be detected in DNA sample₂Hybridizing; after the reaction is finished, separating a solid phase carrier coupled with the primary signal amplifier, the DNA fragment to be detected and the capture probe;

(6) coupling the obtained complex to magnetic beads modified by an affinity substance M5 through an affinity substance M6 marked on the signal converter to form signal magnetic beads;

56. The method for detecting DNA according to claim 55, further comprising a step (5a) between the step (5) and the step (6), wherein the step (5a) comprises reacting the complex of the secondary signal amplifier and the signal converter with a chip having a substrate, and reacting the substrate with the nucleotide sequence C in the first terminal signal₄And hybridizing to obtain a complex of the chip, the substrate, the secondary signal amplifier and the signal converter.

57. Use of a kit according to any one of claims 1 to 21 or 31 to 54 in the manufacture of a kit for the detection of a disease marker, the prediction of the therapeutic effect of a drug and/or the side effects of a treatment with a drug.