Functionalized magnetic beads and a fungal mRNA extraction kit and method comprising the same

Abstract

The application discloses a kind of functionalized magnetic beads, which are magnetic microspheres formed by wrapping magnetic beads with bifunctional nucleic acid, and can specifically bind mRNA.A fungal mRNA extraction kit composed of functionalized magnetic beads includes grinding material, lysing solution I, lysing solution II, magnetic bead binding solution, washing solution I, washing solution II and eluent.A mRNA extraction method includes sequentially adding grinding material, lysing solution I, lysing solution II, magnetic bead binding solution, washing solution I, washing solution II and eluent after sample pretreatment to obtain mRNA product.The application breaks through the traditional mRNA extraction method, obtains high-quality mRNA, and has the advantages of simple operation, safety, high sensitivity, good reproducibility and low cost.The mRNA obtained by the application can provide high-quality templates for RT-PCR detection, improve the specificity and sensitivity of detection, and has wide application prospects in the fields of clinical diagnosis of diseases, judicial identification, food safety detection, microbial detection and molecular biology.

Description

Functionalized magnetic beads and their constituent fungal mRNA extraction kits and extraction methods Technical Field

[0001] This invention belongs to the field of molecular biology technology, and specifically relates to a functionalized magnetic bead and a fungal mRNA extraction kit and extraction method thereof. Background Technology

[0002] Fungi are a class of microorganisms with eukaryotic cell structures, widely distributed in nature. Some fungi can cause human diseases, making fungal detection crucial for disease diagnosis, treatment, and public health. mRNA (messenger RNA) is a single-stranded RNA transcribed from one strand of DNA using a base-pairing principle. As a key medium for transmitting genetic information from DNA to protein synthesis, changes in mRNA expression levels can precisely reflect cellular physiological states and the progression of diseases. Clinicians, by using mRNA detection, possess a precise "diagnostic key," overcoming the limitations of traditional methods and quickly and accurately identifying the type of infection. PCR detection at the mRNA expression level is a valuable and effective technique for improving PCR sensitivity, significantly increasing the reliability of test results and injecting strong momentum into the development of life science research and clinical diagnosis.

[0003] The commonly used RT-PCR method first extracts total RNA from tissues or cells, then uses the mRNA within as a template to reverse transcribe it into cDNA for PCR amplification. However, mRNA content is low, accounting for only 1% to 5% of total cellular RNA. It is also characterized by high metabolic activity, a short half-life, and easy degradation, making mRNA extraction and preservation difficult and unsuitable for analyzing trace amounts of nucleic acid samples. Therefore, obtaining high-quality mRNA is a crucial step for the successful execution of downstream experiments.

[0004] In recent years, magnetic bead extraction has been favored in related fields due to its high efficiency in nucleic acid adsorption, convenient operation, rapid process, and ease of automation and high-throughput nucleic acid extraction. However, in practical applications, it has also exposed a series of problems, such as the need for high extraction efficiency in terms of magnetic bead performance, particle size, and surface modification, the susceptibility to cross-contamination, and limitations in application scope due to specific samples and nucleic acid types. For example, Chinese patent document CN 118291449 A discloses a reagent kit and method for mRNA extraction based on composite magnetic beads. This technology prepares composite magnetic beads encapsulated by nano-metal-organic frameworks (NMOFs), adsorbs mRNA reverse transcription primers by the composite magnetic beads to form a composite magnetic material containing specific reverse transcription primers, and then mixes the sample lysis buffer with the composite magnetic material to enrich mRNA for subsequent experiments. This technology has the following shortcomings: ① It does not explicitly mention whether the mRNA reverse transcription primers are oligomeric (dT) primers, random primers, or gene-specific primers. However, regardless of the primer used, each has its own disadvantages, resulting in low mRNA extraction efficiency. ② Biological sample lysis products are complex, containing a large amount of cell debris, proteins, carbohydrates, lipids, and other impurities. Without prior impurity removal, directly adding composite magnetic materials to the lysis products to enrich mRNA is akin to searching for a needle in a haystack. Furthermore, the viscous liquid nature of the post-lysis solution also affects the magnetic adsorption process, hindering effective mRNA enrichment. ③ If genomic DNA is not removed in the initial steps after sample lysis, the composite magnetic material will bind to both mRNA and genomic DNA, resulting in poor mRNA purity. Therefore, there is an urgent need to establish a practically applicable mRNA extraction method to provide high-quality templates for RT-PCR and improve detection specificity and sensitivity. Summary of the Invention

[0005] One of the objectives of this invention is to provide a functionalized magnetic bead that can achieve efficient and specific adsorption of mRNA.

[0006] The second objective of this invention is to provide a fungal mRNA extraction kit and method composed of functionalized magnetic beads, which eliminates the need for extraction with organic substances such as trizol, phenol, and chloroform. It is safe and non-toxic, simple and quick to operate, and provides stable and good extraction results, making it suitable for the extraction of mRNA from various fungi.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] A functionalized magnetic bead refers to a magnetic microsphere formed by encapsulating a magnetic bead with a bifunctional nucleic acid, which can specifically bind to mRNA; the magnetic bead is a toluenesulfonyl magnetic bead with a particle size of 0.5 μm to 1.0 μm; the bifunctional nucleic acid is a double-stranded DNA sequence, one end of which is covalently coupled to the toluenesulfonyl magnetic bead via a 5' NH2- group, and the other end specifically binds to mRNA via a poly-T group, and the double-stranded sequence contains an EcoRI restriction site; the double-stranded DNA includes oligonucleotide I and oligonucleotide II, where oligonucleotide I consists of 25 deoxyribonucleotides.

[0009] Its sequence is: 5'NH2- CACCAGAGAGCTGGAATTCGAGTGC-3';

[0010] Oligonucleotide II consists of 32 deoxynucleotides.

[0011] Its sequence is: 5'-TTTTTTTTTTTTTTGCACTCGAATTCCAGGT-3',

[0012] Oligonucleotide I and oligonucleotide II partially complement each other to form a double strand.

[0013] A fungal mRNA extraction kit composed of functionalized magnetic beads includes grinding material, lysis buffer I, lysis buffer II, magnetic bead binding solution, washing buffer I, washing buffer II, and elution buffer; the specific preparation of each component is as follows:

[0014] Abrasive material: Glass beads with a particle size of 200~400μm, which are soaked in 0.1% DEPC water and then sterilized by autoclaving;

[0015] Lysis buffer I consists of: 5-8 mol / L guanidine isothiocyanate, 0.1%-1.0% Triton X-100 (v / v), 1-2% sucrose, 50-100 mmol / L EDTA, 25-50 mmol / L Tris-HCl, and sterile DEPC water as solvent;

[0016] Lysis buffer II comprises: 0.1%–0.15% SDS, 0.5–1.0 mol / L NaCl, 10–200 mmol / L sodium acetate, 200–500 mmol / L EDTA, 0.1–0.6 mg / mL heparin, and pH 5.0–6.0;

[0017] The magnetic bead binding solution includes: 0.5-1.0 mol / L NaCl, 10-15 mg / mL functionalized magnetic beads, 1.2-1.5 mol / L guanidine hydrochloride, 20-30% anhydrous ethanol (v / v), 30-40% isopropanol (v / v), and pH 5.5-6.5.

[0018] Cleaning solution I comprises: 40-55% ethanol solution (volume fraction), 0.5-1.2 mol / L NaCl, 1-2 mol / L guanidine hydrochloride, and 0.2-0.3 mol / L NaHCO3;

[0019] Cleaning solution II consists of: 80% ethanol and 1-2 mol / L NaCl;

[0020] Eluent: 30-50 mmol / L Tris-HCl, 10-20 mmol / L MgCl2, 100-150 mmol / L NaCl, 0.01-0.02% Triton X-100, 0.1-0.3 mg / mL BSA, 2000-3500 U / mL EcoRI, 0.1-0.6 mg / mL heparin, pH 7.5-8.2.

[0021] An extraction method for fungal mRNA using a kit composed of functionalized magnetic beads includes the following steps:

[0022] (1) Sample pretreatment: The cultured cells were placed in a centrifuge tube, centrifuged at 12000 r / min for 30 seconds, the supernatant was discarded and the precipitate was collected;

[0023] (2) Add grinding material and 300 μL of lysis buffer I to the above precipitate, place it in a shaker at 1000 r / min for 2 min, place it on ice for 2 min, and repeat this step twice; centrifuge at 5000 rpm for 2 min, and aspirate the supernatant into a clean centrifuge tube;

[0024] (3) Add 200 μL of lysis buffer II, invert and mix well, bathe in a water bath at 55~60℃ for 10 min, centrifuge at 12000 r / min for 5 min, and transfer the supernatant to a new centrifuge tube;

[0025] (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix by blowing and stirring, let stand at room temperature for 2-3 minutes, then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely attracted to the tube wall, use a pipette to remove the supernatant and remove the centrifuge tube from the magnetic rack.

[0026] (5) Add 1 mL of cleaning solution I to the centrifuge tube, mix by blowing and blowing, place the centrifuge tube on the magnetic rack for 1 min, so that the magnetic beads are completely attracted to the tube wall, and then discard the supernatant.

[0027] (6) Add 1 mL of cleaning solution II to the centrifuge tube, mix by blowing and blowing, place the centrifuge tube on the magnetic rack for 1 min, so that the magnetic beads are completely attracted to the tube wall, then remove the supernatant, remove the centrifuge tube from the magnetic rack, and dry at room temperature for 3-5 min until there is no liquid residue in the centrifuge tube.

[0028] (7) Add 50-100 μL of elution buffer to the centrifuge tube, mix gently, elute at 37°C for 15-20 min, then place on a magnetic rack for 1-2 min. After the magnetic beads are completely drawn onto the centrifuge tube wall, carefully aspirate the supernatant into a new centrifuge tube to obtain the mRNA product.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1. This invention provides a novel functionalized composite magnetic bead, which is a magnetic microsphere formed by encapsulating toluenesulfonyl magnetic beads with bifunctional nucleic acids. It can efficiently and specifically adsorb mRNA, and specifically has the following advantages: The toluenesulfonyl magnetic beads used in this invention are superparamagnetic pre-activated functional magnetic microspheres with fast magnetic response, good hydrophilicity and biocompatibility, and can achieve high loading and covalent coupling of functional nucleic acids on their surface. The functionalized magnetic beads provided by this invention have a surface covalently modified bifunctional nucleic acid. The bifunctional nucleic acid is a double-stranded DNA sequence containing the EcoRI (restriction endonuclease) digestion sequence GAATTC. One end of the bifunctional nucleic acid is covalently coupled to a toluenesulfonyl magnetic bead via 5' NH2-, and the other end specifically binds to mRNA via polyT. This allows the composite magnetic beads to directionally and specifically recognize mRNA in solution, resulting in high concentrations and high purity of mRNA.

[0031] 2. Fungal cell walls are composed of a multi-layered, interwoven network of chitin and β-glucan, resulting in high mechanical strength and toughness, which increases the difficulty of lysis. Based on the structural characteristics of fungal cells, this invention provides two highly efficient cell lysis buffers, I and II. Through the synergistic effect of the grinding material and the two lysis buffer components, cells can be effectively lysed after two lysis processes. While ensuring a large release of mRNA, the guanidine isothiocyanate and heparin in the lysis buffers effectively inhibit RNase activity, preventing mRNA degradation and protecting the integrity and stability of the mRNA. This lays the foundation for extracting mRNA with higher concentrations and purity. It also avoids the use of the toxic reagent trizol.

[0032] 3. The elution buffer provided by this invention is a rationally formulated system of multiple components. While ensuring that EcoRI (restriction endonuclease) specifically recognizes the GAATTC sequence in double-stranded DNA, it simultaneously completes double-strand cleavage, thereby releasing mRNA. High-quality mRNA is then obtained through a magnetic adsorption process. The heparin in this elution buffer system effectively inhibits mRNA enzyme activity, ensuring mRNA integrity. Furthermore, the Triton X-100, MgCl2, BSA, and other components remaining in the buffer system can reduce primer dimer formation in subsequent RT-PCR, improving reaction efficiency and specificity, reducing template loss and primer dimer formation, thus improving PCR results and achieving a dual benefit.

[0033] 4. Fungal mRNA has a polyadenylated (polyA) tail of 30-200 nucleotide residues at its 3′ end. This polyA tail is related to maintaining the structural stability of the mRNA, and its length determines the half-life of the mRNA. As the mRNA persists, this polyA tail gradually shortens. The mRNA extracted in this invention has its 3′ end bound to a composite magnetic bead and then cleaved by EcoRI. The extracted mRNA has a double-stranded 3′ end, which can maintain the structural stability of the mRNA, prevent degradation, and thus preserve RNA biological activity.

[0034] 5. This invention boasts advantages such as simple operation, safety and non-toxicity, high sensitivity, good repeatability, and low cost. It breaks through traditional mRNA extraction methods, pioneering the extraction of mRNA from trace samples. The mRNA obtained by this invention can provide a high-quality template for RT-PCR detection, improving the specificity and sensitivity of the detection, and has broad application prospects in fields such as clinical disease diagnosis, forensic identification, food safety testing, microbial detection, and molecular biology. Attached Figure Description

[0035] Figure 1 shows the ultraviolet absorption spectrum of Trichoderma mRNA extracted by the method in Example 4;

[0036] Figure 2 shows the ultraviolet absorption spectrum of Saccharomyces cerevisiae mRNA extracted by the method in Example 5;

[0037] Figure 3 shows the RT-qPCR amplification curves of the cox3 and CHS2 genes of Saccharomyces cerevisiae in Example 5.

[0038] Figure 4 shows the RT-qPCR melting curves of the CHS2 gene after mRNA extraction using different methods.

[0039] Figure 5 shows the RT-qPCR amplification curves of the CHS2 gene using mRNA extracted by different methods. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The basic component of this invention, the functionalized magnetic beads, is specifically composed of magnetic microspheres formed by encapsulating a bifunctional nucleic acid, which can specifically bind to mRNA. The magnetic beads are toluenesulfonyl magnetic beads with a particle size of 0.5 μm to 1.0 μm. The bifunctional nucleic acid is a double-stranded DNA sequence, one end of which is covalently coupled to the toluenesulfonyl magnetic bead via a 5' NH2- group, and the other end specifically binds to mRNA via a poly-T group. This double-stranded sequence contains an EcoRI restriction site. The double-stranded DNA includes oligonucleotide I and oligonucleotide II, where oligonucleotide I consists of 25 deoxyribonucleotides.

[0042] Its sequence is: 5'NH2- CACCAGAGAGCTGGAATTCGAGTGC-3';

[0043] Oligonucleotide II consists of 32 deoxynucleotides.

[0044] Its sequence is: 5'-TTTTTTTTTTTTTTGCACTCGAATTCCAGGT-3',

[0045] Oligonucleotide I and oligonucleotide II partially complement each other to form a double strand.

[0046] The present invention will be further illustrated by specific embodiments below.

[0047] Example 1: A fungal mRNA extraction kit composed of functionalized magnetic beads, comprising grinding material, lysis buffer I, lysis buffer II, magnetic bead binding solution, washing buffer I, washing buffer II, and elution buffer; the specific preparation of each component is as follows:

[0048] Abrasive material: Glass beads with a particle size of 200μm, which are soaked in 0.1% DEPC water and then autoclaved.

[0049] Lysis buffer I consisted of: 5M guanidine isothiocyanate, 0.1% Triton X-100 (v / v), 1% sucrose, 50 mmol / L EDTA, 25 mmol / L Tris-HCl, and sterile DEPC water as the solvent.

[0050] Lysis buffer II consists of: 0.1% SDS, 0.5 mol / L NaCl, 100 mmol / L sodium acetate, 200 mmol / L EDTA, 0.1 mg / mL heparin, and pH 5.0.

[0051] The magnetic bead binding solution consists of: 0.5 mol / L NaCl, 10 mg / mL functionalized magnetic beads, 1.2 mol / L guanidine hydrochloride, 20% anhydrous ethanol (v / v), 30% isopropanol (v / v), and pH 5.5.

[0052] Cleaning solution I consists of: a 40% (v / v) ethanol solution, 0.5 mol / L NaCl, 1 mol / L guanidine hydrochloride, and 0.2 mol / L NaHCO3;

[0053] Cleaning solution II consists of: 80% ethanol and 1 mol / L NaCl;

[0054] Elution buffer: 30 mmol / L Tris-HCl, 10 mmol / L MgCl2, 100 mmol / L NaCl, 0.01% Triton X-100, 0.1 mg / mL BSA, 2000 U / mL EcoRI, 0.1 mg / mL heparin, pH 7.5.

[0055] Example 2: A fungal mRNA extraction kit composed of functionalized magnetic beads, comprising grinding material, lysis buffer I, lysis buffer II, magnetic bead binding solution, washing buffer I, washing buffer II, and elution buffer. The specific preparation of each component is as follows:

[0056] Abrasive material: Glass beads with a particle size of approximately 400 μm, which are soaked in 0.1% DEPC water and then autoclaved.

[0057] Lysis buffer I consisted of: 8M guanidine isothiocyanate, 1.0% Triton X100 (v / v), 2% sucrose, 100 mmol / L EDTA, 50 mmol / L Tris-HCl, and sterile DEPC water as the solvent.

[0058] Lysis buffer II consists of: 0.15% SDS, 1.0 mol / L NaCl, 200 mmol / L sodium acetate, 500 mmol / L EDTA, 0.6 mg / mL heparin, and pH 6.0.

[0059] The magnetic bead binding solution consists of: 1.0 mol / L NaCl, 15 mg / mL functionalized magnetic beads, 1.5 mol / L guanidine hydrochloride, 30% anhydrous ethanol (v / v), 40% isopropanol (v / v), and pH 6.5.

[0060] Cleaning solution I consists of: 55% ethanol solution, 1.2 mol / L NaCl, 2 mol / L guanidine hydrochloride, and 0.3 mol / L NaHCO3;

[0061] Cleaning solution II consists of: 80% ethanol and 2 mol / L NaCl;

[0062] Elution buffer: 50 mmol / L Tris-HCl, 20 mmol / L MgCl2, 150 mmol / L NaCl, 0.02% Triton X-100, 0.3 mg / mL BSA, 3500 U / mL LEcoRI, 0.6 mg / mL heparin, pH 8.2.

[0063] Example 3: A fungal mRNA extraction kit composed of functionalized magnetic beads, comprising grinding material, lysis buffer I, lysis buffer II, magnetic bead binding solution, washing buffer I, washing buffer II, and elution buffer. The specific preparation of each component is as follows:

[0064] Abrasive material: Glass beads with a particle size of approximately 300 μm, which are soaked in 0.1% DEPC water and then autoclaved.

[0065] Lysis buffer I consisted of: 6M guanidine isothiocyanate, 0.5% Triton X100 (v / v), 1.5% sucrose, 75 mmol / L EDTA, 40 mmol / L Tris-HCl, and sterile DEPC water as solvent.

[0066] Lysis buffer II consists of: 0.12% SDS, 0.8 mol / L NaCl, 100 mmol / L sodium acetate, 300 mmol / L EDTA, 0.3 mg / mL heparin, and pH 5.5.

[0067] The magnetic bead binding solution consists of: 0.8 mol / L NaCl, 12 mg / mL functionalized magnetic beads, 1.3 mol / L guanidine hydrochloride, 25% anhydrous ethanol (v / v), 35% isopropanol (v / v), and pH 6.0.

[0068] Cleaning solution I consists of: a 50% (v / v) ethanol solution, 1.0 mol / L NaCl, 1.5 mol / L guanidine hydrochloride, and 0.25 mol / L NaHCO3;

[0069] Cleaning solution II consists of: 80% ethanol and 1.5 mol / L NaCl;

[0070] Elution buffer: 40 mmol / L Tris-HCl, 15 mmol / L MgCl2, 120 mmol / L NaCl, 0.015% Triton X-100, 0.2 mg / mL BSA, 3000 U / mL EcoRI, 0.4 mg / mL heparin, pH 8.0.

[0071] Example 4 describes a method for extracting mRNA using the kit prepared in Example 1, comprising the following steps:

[0072] (1) Sample pretreatment: Take 2 mL of Trichoderma cells cultured at 37℃ for 60 h with shaking, place them in a centrifuge tube, centrifuge at 12000 r / min for 30 seconds, discard the supernatant and collect the precipitate.

[0073] (2) Add grinding material and 300 μL of lysis buffer I to the above precipitate, place it in a shaker at 1000 r / min for 2 min, place it on ice for 2 min, and repeat this step twice. Centrifuge at 5000 rpm for 2 min, and aspirate the supernatant into a clean centrifuge tube.

[0074] (3) Add 200 μL of lysis buffer II, invert and mix well, bathe in water at 55~60℃ for 10 min, centrifuge at 12000 r / min for 5 min, and transfer the supernatant to a new centrifuge tube.

[0075] (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix by blowing and stirring, let stand at room temperature for 2-3 minutes, then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely attracted to the tube wall, use a pipette to remove the supernatant and remove the centrifuge tube from the magnetic rack.

[0076] (5) Add 1 mL of cleaning solution I to the centrifuge tube, mix by blowing and blowing, place the centrifuge tube on the magnetic rack for 1 minute, so that the magnetic beads are completely attracted to the tube wall, and then discard the supernatant.

[0077] (6) Add 1 mL of cleaning solution II to the centrifuge tube, mix by blowing and blowing, place the centrifuge tube on the magnetic rack for 1 min, so that the magnetic beads are completely attracted to the tube wall, then remove the supernatant, remove the centrifuge tube from the magnetic rack, and dry at room temperature for 3-5 min until there is no liquid residue in the centrifuge tube.

[0078] (7) Add 100 μL of elution buffer to the centrifuge tube, mix gently, elute at 37°C for 15-20 min, then place on a magnetic rack for 1-2 min. After the magnetic beads are completely drawn onto the centrifuge tube wall, carefully aspirate the supernatant into a new centrifuge tube to obtain the mRNA product.

[0079] (8) The concentration and purity of mRNA were determined by ultraviolet spectrophotometer. Figure 1 shows the ultraviolet absorption spectrum of the Trichoderma mRNA extracted in this invention. The extracted mRNA had a high concentration (150.36 μg / mL) and good purity (A).260 / 280 =1.89).

[0080] Example 5 describes a method for extracting mRNA using the kit prepared in Example 3, comprising the following steps:

[0081] (1) Sample pretreatment: Take 2 mL of Saccharomyces cerevisiae cells cultured at 37℃ for 48 h and place them in a centrifuge tube. Centrifuge at 12000 r / min for 30 seconds, discard the supernatant, and collect the precipitate.

[0082] (2) Add grinding material and 300 μL of lysis buffer I to the above precipitate, place it in a shaker at 1000 r / min for 2 min, place it on ice for 2 min, and repeat this step twice. Centrifuge at 5000 rpm for 2 min, and aspirate the supernatant into a clean centrifuge tube.

[0083] (3) Add 200 μL of lysis buffer II, invert and mix well, bathe in water at 55~60℃ for 10 min, centrifuge at 12000 r / min for 5 min, and transfer the supernatant to a new centrifuge tube.

[0084] (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix by blowing and stirring, let stand at room temperature for 2-3 minutes, then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely attracted to the tube wall, use a pipette to remove the supernatant and remove the centrifuge tube from the magnetic rack.

[0085] (5) Add 1 mL of cleaning solution I to the centrifuge tube, mix by blowing and blowing, place the centrifuge tube on the magnetic rack for 1 minute, so that the magnetic beads are completely attracted to the tube wall, and then discard the supernatant.

[0086] (6) Add 1 mL of cleaning solution II to the centrifuge tube, mix by blowing and blowing, place the centrifuge tube on the magnetic rack for 1 min, so that the magnetic beads are completely attracted to the tube wall, then remove the supernatant, remove the centrifuge tube from the magnetic rack, and dry at room temperature for 3-5 min until there is no liquid residue in the centrifuge tube.

[0087] (7) Add 100 μL of elution buffer to the centrifuge tube, mix gently, elute at 37°C for 15-20 min, then place on a magnetic rack for 1-2 min. After the magnetic beads are completely drawn onto the centrifuge tube wall, carefully aspirate the supernatant into a new centrifuge tube to obtain the mRNA product.

[0088] (8) The concentration and purity of mRNA were determined by ultraviolet spectrophotometer. Figure 2 shows the ultraviolet absorption spectrum of the extracted Saccharomyces cerevisiae mRNA. The extracted mRNA had a high concentration (173.36 μg / mL) and good purity (A). 260 / 280 =1.85).

[0089] (9) RT-qPCR detection. The extracted mRNA was immediately reverse transcribed to obtain cDNA. Using the cDNA as a template, the gene for cytochrome oxidase subunit 3 (COX3) and the gene for chitin synthase 2 (CHS2) of *Saccharomyces cerevisiae* were amplified using the Hifair® Ⅲ qPCR SYBR Green Kit with SYBR Green I dye to evaluate the mRNA extracted by the kit of this invention. Primer sequences are shown in Table 1, and reaction system is shown in Table 2.

[0090] Table 1. Primer sequences for Saccharomyces cerevisiae RT-qPCR

[0091]

[0092] Table 2 qPCR reaction system

[0093]

[0094] qPCR amplification was performed using a two-step method. The reaction conditions were: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 15 s; 55 °C annealing for 20 s, for a total of 35 cycles. After the reaction, the Ct value was obtained based on the fluorescence signal data.

[0095] (10) Figure 3 is the RT-qPCR amplification curve of the COX3 gene and CHS2 gene of Saccharomyces cerevisiae in Example 5. As can be seen from the figure, the Ct value of the amplified gene in this invention is about 20~22.

[0096] The following comparative examples further illustrate the beneficial effects of the present invention.

[0097] Comparative Example 1: 2 mL of *Saccharomyces cerevisiae* cell samples cultured at 37°C with shaking for 60 h were taken and processed using the classic Trizol method and the Omega EZNA® Total RNA Kit I (R6831) to obtain total RNA preparation solutions. Subsequently, the total RNA preparation solutions were further purified using an oligomeric (dT) cellulose column to obtain mRNA. The mRNA extraction effects of these two methods were compared with the mRNA extraction effect of Example 5 of this invention, and the results were analyzed using ultraviolet spectrophotometry. The results are shown in Table 3. The results show that the mRNA extracted using the kit of this invention has higher concentration and purity than the mRNA extracted by the Trizol method and the commercially available Omega kit.

[0098] Furthermore, the *Saccharomyces cerevisiae* CHS2 gene was amplified by RT-qPCR, and the melting curves were all single peaks (Figure 4). The amplification curves are shown in Figure 5. As can be seen from the figures, the amplification curves are smooth and orderly. However, when the mRNA extracted by this invention is used for gene amplification, its Ct value is lower than that of the other two methods. This result fully verifies that the mRNA extracted by the method of this invention has superior quality.

[0099] Table 3 Comparison of the effects of different methods for mRNA extraction

[0100]

[0101] Comparative Example 2 compares the effectiveness of the functionalized magnetic beads of this invention with that of traditional Oligo(dT) magnetic beads in mRNA extraction. Using the reagent buffer system of Example 2 of this invention, the control group's magnetic bead binding solution was used instead of the commonly used Oligo(dT) magnetic beads, and the extraction effect was verified using the method of Example 5 of this invention. The mRNA concentration and purity were measured using a UV spectrophotometer, and the results of the extracted mRNA are shown in Table 4. The results show that the mRNA concentration and purity extracted by the functionalized magnetic beads in this kit are higher than those extracted by the Oligo(dT) magnetic bead method.

[0102] Table 4. Comparison of the effects of different methods for extracting mRNA.

[0103]

Claims

1. A functionalized magnetic bead, characterized in that, It refers to magnetic microspheres formed by encapsulating magnetic beads with bifunctional nucleic acids, which can specifically bind to mRNA; the magnetic beads are toluenesulfonyl magnetic beads with a particle size of 0.5μm~1.0μm; the bifunctional nucleic acid is a double-stranded DNA sequence, one end of which is covalently coupled to the toluenesulfonyl magnetic bead via 5' NH2-, and the other end specifically binds to mRNA via poly-T, and the double-stranded sequence contains an EcoRI restriction site.

2. The functionalized magnetic bead according to claim 1, characterized in that, The double-stranded DNA comprises oligonucleotide I and oligonucleotide II. Oligonucleotide I consists of 25 deoxynucleotides with the sequence: 5'NH2- CACCAGAGAGCTG GAATTCGAGTGC-3'; oligonucleotide II consists of 32 deoxynucleotides with the sequence: 5'- TTTTTTTTTTTTTTTGCACTCGAATTCCAGGT-3'. Oligonucleotide I and oligonucleotide II are partially complementary to form a double strand.

3. A fungal mRNA extraction kit composed of functionalized magnetic beads as described in claim 1, characterized in that, This kit consists of grinding media, lysis buffer I, lysis buffer II, magnetic bead binding buffer, washing buffer I, washing buffer II, and elution buffer. The specific preparation of each component is as follows: Grinding media: glass beads with a particle size of 200–400 μm, soaked in 0.1% DEPC water and autoclaved; Lysis buffer I includes: 5–8 mol / L guanidine isothiocyanate, 0.1%–1.0% Triton X-100 (v / v), 1–2% sucrose, 50–100 mmol / L EDTA, 25–50 mmol / L Tris-HCl, and sterile DEPC water as the solvent; Lysis buffer II includes: 0.1%–0.15% SDS, 0.5–1.0 mol / L NaCl, 10–200 mmol / L sodium acetate, and 200–500 mmol / L... The solution consists of: EDTA, 0.1–0.6 mg / mL heparin, pH 5.0–6.0; magnetic bead binding solution including: 0.5–1.0 mol / L NaCl, 10–15 mg / mL functionalized magnetic beads, 1.2–1.5 mol / L guanidine hydrochloride, 20–30% (v / v) anhydrous ethanol, 30–40% (v / v) isopropanol, pH 5.5–6.5; washing solution I including: 40–55% (v / v) ethanol solution, 0.5–1.2 mol / L NaCl, 1–2 mol / L guanidine hydrochloride, 0.2–0.3 mol / L NaHCO3; washing solution II including: 80% ethanol, 1–2 mol / L NaCl; eluent: 30–50 mmol / L Tris-HCl, 10–20 mmol / L MgCl2, 100–150 mmol / L NaCl, 0.01–0.02% HCl. Triton X-100, 0.1~0.3 mg / mL BSA, 2000~3500 U / mL EcoRI, 0.1~0.6 mg / mL heparin, pH 7.5~8.2; all solutions were prepared using sterile DEPC water as the solvent.

4. An extraction method for fungal mRNA extraction kit composed of functionalized magnetic beads as described in claim 3, characterized in that, Includes the following steps: (1) Sample pretreatment: Place the cultured cells in a centrifuge tube, centrifuge at 12000 r / min for 30 seconds, discard the supernatant and collect the precipitate; (2) Add grinding material and 300 μL of lysis buffer I to the above precipitate, place in a shaker at 1000 r / min for 2 min, place on ice for 2 min, repeat this step twice; centrifuge at 5000 rpm for 2 min, and aspirate the supernatant into a clean centrifuge tube; (3) Add 200 μL of lysis buffer II, invert and mix, incubate at 55~60℃ for 10 min, centrifuge at 12000 r / min for 5 min, and aspirate the supernatant to transfer to a new centrifuge tube; (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix by pipetting, place at room temperature for 2~3 min, then place the centrifuge tube on a magnetic rack, and after the magnetic beads are completely adsorbed onto the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack; (5) Add 1 mL of washing buffer I to the centrifuge tube, mix by pipetting. (6) Add 1 mL of washing solution II to the centrifuge tube, mix well by pipetting, place the centrifuge tube on the magnetic rack for 1 min, and then remove the supernatant after the magnetic beads are completely adsorbed onto the tube wall. Remove the centrifuge tube from the magnetic rack and dry it at room temperature for 3-5 min until there is no liquid residue in the centrifuge tube. (7) Add 50-100 μL of elution solution to the centrifuge tube, mix gently, and elute at 37°C for 15-20 min. Then place it on the magnetic rack for 1-2 min until the magnetic beads are completely adsorbed onto the centrifuge tube wall. Carefully aspirate the supernatant into a new centrifuge tube to obtain the mRNA product.

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