CN113046425A

CN113046425A - Universal gene detection method

Info

Publication number: CN113046425A
Application number: CN201911385442.1A
Authority: CN
Inventors: 魏宏泉; 孙相鑫
Original assignee: Tuoyuan Onebio Technology Co ltd
Current assignee: Tuoyuan Onebio Technology Co ltd
Priority date: 2019-12-28
Filing date: 2019-12-28
Publication date: 2021-06-29

Abstract

The invention provides a universal gene detection method, wherein amplification products generated in the amplification process are all fixed on a fixed phase, and the detection environment is not polluted.

Description

Universal gene detection method

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a universal gene detection method.

Background

The gene is a basic unit of heredity, is a DNA or RNA sequence carrying genetic information, transfers the genetic information to the next generation through replication, and guides the synthesis of protein to express the genetic information carried by the gene, thereby controlling the character expression of an individual organism. The gene detection is a technology for detecting DNA through blood, other body fluids or cells, and is a method for detecting DNA molecular information in cells of a detected person by taking peripheral venous blood or other tissue cells of the detected person through specific equipment and analyzing whether the gene type, the gene defect and the expression function contained in the cells are normal or not, so that people can know the gene information of themselves, and the etiology is clear or the risk of a certain disease of the body is predicted. Common techniques for gene detection include qPCR techniques and gene chip techniques. Wherein the qPCR technique comprises: SYBRGREEN dye method and Taqman probe method. The general strategy of gene chip technology is to fix the Capture Probe (Capture Probe) on the surface of solid phase carrier (chip), then add the nucleic acid to be detected on the chip, and through denaturation and annealing, make the target gene to be detected and the Capture Probe specifically hybridize, so that the Capture Probe specifically captures the target gene. Then, a Detection probe (Detection probe) is added to the system, and the Detection probe hybridizes to the target gene. The detection probe is generally an oligonucleotide sequence with one end labeled in various ways, such as enzyme labeling, gold labeling followed by silver nitrate staining, or fluorescein labeling. Since the sensitivity of the gene chip technology itself is relatively low, in most cases, the target gene in the sample needs to be amplified first, and the means for amplification can be selected from nucleic acid amplification technologies such as PCR, NASBA, LAMP, etc. After the amplification is finished, the hybridization detection of the gene chip is carried out.

During the PCR, a large amount of amplification products of the target gene are generated. These products are not immobilized on a solid support and readily form aerosols. And the whole detected operation environment is polluted. Therefore, PCR assays for clinical samples must be performed in standard PCR assay laboratories. The standard PCR detection laboratory has mandatory requirements on construction, has high investment cost and is not suitable for primary medical units. The specific implementation process of the two methods is complex, more experimental steps and professional technicians are needed for operation, the whole detection time is long, and the method is not beneficial to design of a POCT detection mode.

In addition, the signal display strategies of the qPCR technology and the gene chip technology are indirect, and for the PCR technology and the gene chip technology, two signal display modes are provided, one mode is SYBRGREEN dye which is fluorescent dye combined with minor groove of a DNA double chain, and the double chain formed in the PCR amplification process can be combined with the SYBRGREEN dye to emit fluorescence for detection. For the gene chip detection technique, the signal is generally collected by labeling the detection probe, as mentioned above, silver staining the gold particle labeled on the detection probe, or adding a substrate of an enzyme labeled on the detection probe to perform a color development reaction, or detecting fluorescein attached to the detection probe. All these detection means lead to the complexity of the detection steps in the actual operation process, and may lead to the reduction of sensitivity, or the occurrence of false positive and false negative phenomena, which may not accurately reflect the true condition of the target gene or amplification product.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a universal gene detection method, a gene detection kit and application of the gene detection kit in detection of a 1555 locus of a deafness gene.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

in a first aspect of the present invention, there is provided a universal gene detection method, comprising the steps of: preparing a PCR reaction system, executing a PCR reaction program, processing a reaction product after the PCR reaction is finished, detecting a fluorescent signal, and judging a result; the PCR reaction system comprises an immobilized primer, dNTP, a PCR buffer solution, an Extaq enzyme, a DNA sample and deionized water; the fixed primer comprises a fixed phase and a primer fixed on the fixed phase, and the dNTP is marked by fluorescein.

The primer fixed on the solid phase is an ARMS primer with sequence specificity, only a specific target gene can be amplified under the condition of proper PCR reaction, the sequences of other genotypes cannot be amplified, because the primer is fixed on the solid phase, dNTP in a reaction system is marked by fluorescein, and an amplification product is fixed on the solid phase and contains the fluorescein. After the PCR reaction is carried out for multiple rounds, the reaction system is sucked, and the negative and positive can be judged by directly detecting whether fluorescence exists on the stationary phase after rinsing.

Preferably, the primer is hybridized by a general nucleotide sequence and a specific nucleotide sequence.

Preferably, a tag sequence is added to the 5' end of the primer.

Preferably, the 5' end of the primer is further modified by an amine group.

Preferably, the stationary phase is selected from magnetic particles or gene chips.

Preferably, the surface of the stationary phase is labeled with carboxyl or aldehyde groups. The primer is fixed on the stationary phase through an amide condensation reaction.

Preferably, the dntps include dATP, dCTP, dGTP, dUTP, and dTTP; wherein one or more of dATP, dCTP, dGTP, dUTP and dTTP is labeled with fluorescein.

Preferably, the PCR reaction system comprises the following components in parts by volume: 5 parts of fixed primer, 2 parts of PCR buffer solution, 0.5 part of dATP, 0.5 part of dCTP, 0.5 part of dGTP, 0.5 part of dTTP, 0.5 part of Extaq enzyme, 1 part of DNA sample with the concentration of 50ng/ul and 9.5 parts of deionized water.

Compared with the prior art, the invention has the beneficial effects that:

the primers adopted by the invention are fixed on the stationary phase, so that amplification products generated in the amplification process are also all fixed on the stationary phase, the detection environment cannot be polluted, aerosol cannot occur, and the requirement on the detection environment is greatly reduced.

The dNTP is labeled by fluorescein, and the extended sequence is doped with the dNTP with the fluorescein label in the PCR single-strand amplification process of the fixed primer, so that the signal of a single-strand amplification product is greatly enhanced by the process. Thus, the number of cycles required for the entire reaction is small, and typically 1-5 cycles are performed to generate a very strong signal, which greatly reduces the time required to detect the reaction. The universal gene detection method or the gene detection kit provided by the invention does not need to add a capture probe additionally during operation, does not need to carry out silver staining on gold particles marked on the capture probe, or carry out chromogenic reaction on a substrate of enzyme marked on the capture probe, or detect fluorescein connected on the capture probe, and the omitted detection means can greatly simplify the detection steps in the actual operation process, simultaneously can improve the sensitivity, avoid the phenomena of false positive and false negative, and can accurately react the real situation of a target gene or an amplification product.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Reagent consumables:

PCR buffer, purchased from health as a century Biotechnology Limited, product number: CW 0680M.

The Extaq enzyme, purchased from health as a century Biotechnology Limited, product number: CW 0680M.

Carboxyl magnetic particles, purchased from pureimiger biotechnology limited, with product number: PB 0001.

dATP, available from Beijing Solaibao science and technology Inc., has the product number: a PC 2301.

dCTP, purchased from Beijing Solaibao science and technology Co., Ltd, with the product number: a PC 2303.

dGTP, purchased from Beijing Solaibao science and technology Co., Ltd, with the product number: a PC 2304.

dTTP, purchased from Beijing Solaibao science and technology Co., Ltd, with product number: a PC 2302.

Coupling buffer, purchased from pureimiger biotechnology ltd, product number: PB 0001.

Quenching buffer, purchased from pureimiger biotechnology ltd, product number: PB 0001.

Storage buffer, purchased from pureimiger biotechnology limited, with product number: PB 0001.

This example provides a universal gene detection method, comprising the steps of: preparing a PCR reaction system, executing a PCR reaction program, processing a reaction product after the PCR reaction is finished, detecting a fluorescent signal, and judging a result; the PCR reaction system comprises an immobilized primer, dNTP, a PCR buffer solution, an Extaq enzyme, a DNA sample and deionized water; the fixed primer comprises a fixed phase and a primer fixed on the fixed phase, and the dNTP is marked by fluorescein.

In the embodiment, the primer fixed on the solid phase is an ARMS primer with sequence specificity, under the appropriate PCR reaction condition, only a specific target gene can be amplified, and the sequences of other genotypes can not be amplified. After the PCR reaction is carried out for multiple rounds, the reaction system is sucked, and the negative and positive can be judged by directly detecting whether fluorescence exists on the stationary phase after rinsing.

In some embodiments, the primer has a tag sequence added to its 5 'end, the primer has its 5' end further modified with an amine group, and the stationary phase is selected from magnetic particles.

In some embodiments, the stationary phase is selected from a gene chip.

In some embodiments, the magnetic particle surface is labeled with carboxyl or aldehyde groups, and the primer is immobilized on the surface of the magnetic particle through an amide condensation reaction.

Preferably, the preparation method of the immobilized primer comprises the following steps:

(1) taking 0.5mg of carboxyl magnetic particles, washing the carboxyl magnetic particles for 2 times by using PBS buffer solution, adding 1ml of PBS buffer solution, oscillating and resuspending, adding 10mg of EDC and 10mg of NHS activated carboxyl, after 30min, carrying out magnetic adsorption, removing supernatant, and washing the carboxyl magnetic particles for 3 times by using the PBS buffer solution to obtain activated carboxyl magnetic particles;

(2) adding 80ul of coupling buffer solution, 20ul of ARMS primer PNA buffer solution with the concentration of 50nmol/ml and 100ul of primer into the activated carboxyl magnetic particles, and performing shake incubation for 2 hours at room temperature to obtain a product;

(3) adding 500ul of quenching buffer solution into the product, carrying out vortex for 20 seconds, carrying out magnetic separation, and removing the supernatant;

(4) repeating the step (3) for three times;

(5) 100ul of storage buffer was added to obtain immobilized primers, which were stored at 4 ℃ for further use.

In some embodiments, the dntps include dATP, dCTP, dGTP, dUTP, and dTTP; wherein one or more of dATP, dCTP, dGTP, dUTP and dTTP is labeled with fluorescein.

In some embodiments, the fluorescein is selected from one or more of fluorescein isothiocyanate, phycoerythrin, polymetaxanthin chlorophyll protein, propidium iodide, or allophycocyanin.

In some embodiments, the fluorescein is selected from fluorescein isothiocyanate.

In some embodiments, the PCR reaction system comprises, in parts by volume: 5 parts of fixed primer, 2 parts of PCR buffer solution, 0.5 part of dATP, 0.5 part of dCTP, 0.5 part of dGTP, 0.5 part of dTTP, 0.5 part of Extaq enzyme, 1 part of DNA sample with the concentration of 50ng/ul and 9.5 parts of deionized water.

In some embodiments, the PCR reaction program comprises the steps of: taking two PCR reaction tubes, respectively adding the PCR reaction systems into the PCR reaction tubes, preheating for 3 minutes at 95 ℃ on a PCR instrument to fully denature the DNA sample, then entering amplification cycles, keeping the temperature at 95 ℃ for 15 seconds in each cycle to denature the DNA sample, then reducing the temperature to 56 ℃ and keeping the temperature for 30 seconds to fully anneal the primers and the DNA sample; after 5 cycles at 72 ℃ for 30s, the reaction product was obtained at 72 ℃ for 60 s.

In some embodiments, the process of treating the reaction product comprises:

1) placing the reaction product on a magnetic frame for adsorption for 3min, collecting magnetic particles, and removing supernatant;

2) adding 500ul of deionized water, shaking gently, rinsing the magnetic particles, then placing on a magnetic frame for adsorption for 3min, collecting the magnetic particles, discarding the supernatant, repeating the step for three times, and suspending the magnetic particles in 20ul of deionized water.

For the specific structure of the magnetic frame, see: lixingwang et al "magnetic rack with imbibition function", CN 206232701U; magnetic racks such as Wei-Hongquan, CN 206244805U; wuyanfeng, etc., CN 206345848U; "biomolecule extraction device", CN206477001U, Sun Conn et al. The entire contents of each of these publications are incorporated herein by reference.

In some embodiments, the fluorescein is selected from FITC fluorescein.

The embodiment also provides a gene detection kit, which comprises an immobilized primer, a PCR buffer solution, dNTP, an Extaq enzyme and deionized water.

In some embodiments, the immobilized primer comprises a stationary phase and a primer immobilized on the stationary phase, and the dntps are labeled with fluorescein.

In some embodiments, the nucleotide sequence of the primer is:

5’-gccgaccgccgattctgc GCATTTATATAGAGGAGA-3’

or 5'-gccgaccgccgattctgcGCATTTATATAGAGGAGG-3'.

In some embodiments, a tag sequence is added to the 5' end of the primer.

In some embodiments, the 5' end of the primer is further modified with an amine group.

In this embodiment, a tag sequence is added to the 5 'end of the primer, the 5' end of the primer is modified by an amine group, and the stationary phase is selected from magnetic particles.

In some embodiments, the stationary phase is selected from a gene chip.

In this embodiment, the stationary phase is labeled with a carboxyl group or an aldehyde group on the surface.

In this example, the primer was immobilized on the magnetic particle by an amide condensation reaction.

(2) adding 80ul of coupling buffer solution, 20ul of PNA buffer solution with the concentration of 50nmol/ml and 100ul of primers into the activated carboxyl magnetic particles, and performing shake incubation for 2 hours at room temperature to obtain a product;

(4) repeating the step (3) for three times;

The primer fixed on the solid phase is an ARMS primer with sequence specificity, only a specific target gene can be amplified under the condition of adjusting to a proper PCR reaction condition, the sequences of other genotypes can not be amplified, because the primer is fixed on the solid phase, dNTP in a reaction system is marked by fluorescein, a single-stranded amplification product can be fixed on the solid phase and contains the fluorescein. After the PCR reaction is carried out for multiple rounds, the reaction system is sucked, and the negative and positive can be judged by directly detecting whether fluorescence exists on the stationary phase after rinsing.

Example 1

In this embodiment, the detection of the 1555 locus of the deafness gene is taken as an example, and the universal gene detection method of the present invention is further described in detail.

The embodiment provides a method for detecting a 1555 locus of a deafness gene, which comprises the following steps:

1) taking 5ul of fixed primer, 2 ul of PCR buffer solution, 0.5ul of dATP, 0.5ul of dCTP, 0.5ul of dGTP, 0.5ul of dTTP, 0.5ul of Extaq enzyme and 9.5 ul of deionized water, and adding the materials into a PCR reaction tube to obtain a PCR reaction system; the fixed primer is formed by combining carboxyl magnetic particles and a primer;

2) taking 1 ul of DNA sample with the concentration of 50ng/ul, adding the DNA sample into the PCR reaction system, preheating the PCR reaction system on a PCR instrument at 95 ℃ for 1-3 minutes, executing a single-strand PCR process, specifically combining a fixed primer with the DNA sample, annealing, extending the primer along the DNA template, rinsing, removing the DNA template, and obtaining a reaction product;

3) and detecting the fluorescence value of the reaction product, and judging the detection result.

The sequence of the primer in this example is:

1555-W：5’-gccgaccgccgattctgc GCATTTATATAGAGGAGA-3’

or 1555-M: 5'-gccgaccgccgattctgc GCATTTATATAGAGGAGG-3' are provided.

Sequence gccgaccgccgattctgc is a universal nucleotide sequence; the sequence GCATTTATATAGAGGAGA or GCATTTATATAGAGGAGG is a specific nucleotide sequence, the general nucleotide sequence does not specifically bind with the DNA sample, the specific nucleotide sequence specifically binds with the DNA sample, and the primer is added with the general nucleotide sequence, so that the specific nucleotide sequence has sufficient space to bind with the DNA sample. In this example, the 5' end of the primer is modified with an amino group, and the modified primer can be bound to a solid support through the amino group.

The preparation method of the immobilized primer comprises the following steps:

(4) repeating the step (3) for three times;

The step of treating the reaction product of this example included:

While embodiments of the present invention have been described above, the present invention is not limited to the specific embodiments and applications described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art who have the benefit of this description will be able to devise various modifications without departing from the scope of the appended claims.

Claims

1. A universal gene detection method, comprising the steps of: preparing a PCR reaction system, executing a PCR reaction program, processing a reaction product after the PCR reaction is finished, detecting a fluorescent signal, and judging a result; the PCR reaction system comprises an immobilized primer, dNTP, a PCR buffer solution, an Extaq enzyme, a DNA sample and deionized water; the fixed primer is formed by combining a fixed phase and the primer, and the dNTP is marked by fluorescein.

2. The method for detecting a universal gene according to claim 1, wherein the primer is composed of a universal nucleotide sequence and a specific nucleotide sequence.

3. The method for detecting a universal gene according to claim 1, wherein a tag sequence is added to the 5' end of the primer.

4. The method for detecting a universal gene according to claim 1, wherein the 5' -end of the primer is modified with an amino group.

5. The universal gene detection method according to claim 1, wherein the stationary phase is selected from magnetic particles, microspheres or gene chip matrix materials.

6. The universal gene detection method according to claim 5, wherein the stationary phase is labeled with a carboxyl group or an aldehyde group on the surface.

7. The universal gene detection method according to claim 1, wherein the dntps include dATP, dCTP, dGTP, dUTP, and dTTP; wherein one or more of dATP, dCTP, dGTP, dUTP and dTTP is labeled with fluorescein.

8. The universal gene detection method according to claim 1, wherein the PCR reaction system comprises, in parts by volume: 5 parts of fixed primer, 2 parts of PCR buffer solution, 0.5 part of dATP, 0.5 part of dCTP, 0.5 part of dGTP, 0.5 part of dTTP, 0.5 part of Extaq enzyme, 1 part of DNA sample with the concentration of 50ng/ul and 9.5 parts of deionized water.

9. The universal gene detection method according to claim 1, wherein the PCR reaction procedure comprises the steps of: taking two PCR reaction tubes, respectively adding the PCR reaction systems into the PCR reaction tubes, preheating for 3 minutes at 95 ℃ on a PCR instrument to fully denature the DNA sample, then entering amplification cycles, keeping the temperature at 95 ℃ for 15 seconds in each cycle to denature the DNA sample, then reducing the temperature to 56 ℃ and keeping the temperature for 30 seconds to fully anneal the primers and the DNA sample; after 5 cycles at 72 ℃ for 30s, the reaction product was obtained at 72 ℃ for 60 s.

10. The universal gene detection method according to claim 1, wherein the fluorescein is selected from one or more of fluorescein isothiocyanate, phycoerythrin, polymethacrylflavin chlorophyll protein, propidium iodide and allophycocyanin.