Functional magnetic beads, fungal mRNA extraction kit composed of functional magnetic beads and extraction method

Abstract

The invention relates to a functionalized magnetic bead which is a magnetic bead formed by wrapping a magnetic bead with bifunctional nucleic acid and can be specifically combined with mRNA (messenger ribonucleic acid). The fungal mRNA extraction kit composed of the functionalized magnetic beads comprises a grinding material, a lysis solution I, a lysis solution II, a magnetic bead combination solution, a cleaning solution I, a cleaning solution II and an eluent. The mRNA extraction method comprises the following steps: sequentially adding the grinding material, the lysis solution I, the lysis solution II, the magnetic bead binding solution, the cleaning solution I, the cleaning solution II and the eluent after sample pretreatment to obtain an mRNA product. The traditional mRNA extraction method is broken through, high-quality mRNA is obtained, and the method has the advantages of simplicity in operation, safety, no toxicity, high sensitivity, good repeatability and low cost. The obtained mRNA can provide a high-quality template for RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection, the detection specificity and sensitivity are improved, and the mRNA has a wide application prospect in the fields of clinical diagnosis of diseases, judicial expertise, food safety detection, microbiological detection, molecular biology and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a fungal mRNA extraction kit and an extraction method comprising functionalized magnetic beads and the like. Background Art

[0002] Fungi are a class of microorganisms with eukaryotic cell structures that are widespread in nature. Some fungi can cause human disease, making fungal detection crucial for disease diagnosis, treatment, and public health. mRNA (messenger RNA) is a single strand transcribed from a DNA strand using base pairing as a template. It serves as a crucial mediator in the transfer of genetic information from DNA to protein synthesis. Changes in its expression can accurately reflect the physiological state of cells and the progression of disease. Clinicians possess a precise "diagnostic key" by detecting mRNA, enabling them to overcome the limitations of traditional testing methods and quickly and accurately identify the type of infection. PCR testing based on mRNA expression levels is a valuable and effective means of improving PCR sensitivity, significantly enhancing the confidence of test results and providing a powerful impetus for advancements in life science research, clinical diagnostics, and other fields.

[0003] The commonly used RT-PCR method first extracts total RNA from tissues or cells, then uses the mRNA as a template for reverse transcription into cDNA for PCR amplification. However, mRNA is present in low concentrations, accounting for only 1% to 5% of total cellular RNA. Its metabolic activity, short half-life, and susceptibility to degradation make its extraction and preservation challenging, making it difficult to analyze trace amounts of nucleic acid samples. Therefore, obtaining high-quality mRNA is crucial for the success of downstream experiments.

[0004] In recent years, magnetic bead extraction has gained popularity in related fields due to its efficient nucleic acid adsorption, convenient operation, rapid process, and ease of automation and high-throughput nucleic acid extraction. However, practical applications have also exposed a series of problems, including limitations in magnetic bead performance, particle size, and surface modification, which require efficient extraction, cross-contamination, and limited application by specific samples and nucleic acid types. For example, Chinese patent document CN 118291449 A discloses an mRNA extraction kit and method based on composite magnetic beads. This technology prepares composite magnetic beads coated with nanometal-organic frameworks (NMOFs). The composite magnetic beads adsorb mRNA reverse transcription primers to form a composite magnetic material containing specific reverse transcription primers. Sample lysate is then mixed with the composite magnetic material to enrich mRNA for subsequent experiments. This technology has the following shortcomings: ① It does not clearly specify whether the mRNA reverse transcription primer should be an oligo(dT) primer, a random primer, or a gene-specific primer. Regardless of the primer type, each has its own shortcomings, resulting in low mRNA extraction efficiency. ② The composition of biological sample lysis products is complex, including a large amount of impurities such as cell debris, proteins, sugars, and lipids. If the composite magnetic material is directly added to the lysis product to enrich mRNA without first performing a decontamination step, it is like looking for a needle in a haystack. In addition, the solution after sample lysis is a viscous liquid, which also affects the magnetic adsorption process and cannot achieve effective enrichment of mRNA. ③ Failure to remove genomic DNA in the early steps after sample lysis will cause the composite magnetic material to bind to genomic DNA while also binding to mRNA, resulting in poor purity of the extracted mRNA. Therefore, there is an urgent need to establish an mRNA extraction method with practical application value to provide high-quality templates for RT-PCR and improve detection specificity and sensitivity. Summary of the Invention

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

[0006] The second purpose of the present invention is to provide a fungal mRNA extraction kit and extraction method composed of functionalized magnetic beads, which does not require the use of organic substances such as trizol, phenol, and chloroform for extraction. It is safe and non-toxic, simple and fast to operate, and has a stable and good extraction effect. It is suitable for extracting mRNA from various fungi.

[0007] In order to achieve the above object, the present invention adopts the following technical means: Functionalized magnetic beads are magnetic microspheres formed by coating magnetic beads with bifunctional nucleic acids, which can specifically bind to mRNA. The magnetic beads are tosyl magnetic beads with a particle size of 0.5μm to 1.0μm. The bifunctional nucleic acid is a double-stranded DNA sequence, one end of which is covalently coupled to the tosyl magnetic beads via a 5' NH2- group and the other end specifically binds to mRNA via a poly-T. The double-stranded sequence contains an EcoRI restriction site. The double-stranded DNA comprises oligonucleotide I and oligonucleotide II, wherein oligonucleotide I is composed of 25 deoxynucleotides. Its sequence is: 5'NH2- CACCAGAGAGCTGGAATTCGAGTGC-3'; Oligonucleotide II consists of 32 deoxynucleotides. Its sequence is: 5'- TTTTTTTTTTTTTTTGCACTCGAATTCCAGGT-3', Oligonucleotide I and oligonucleotide II are partially complementary to form a double strand.

[0008] A fungal mRNA extraction kit composed of functionalized magnetic beads includes grinding material, lysis solution I, lysis solution II, magnetic bead binding solution, cleaning solution I, cleaning solution II, and eluent; the specific preparation of each component is as follows: Grinding material: Glass beads with a particle size of 200-400 μm, soaked in 0.1% DEPC water and sterilized by high pressure; Lysis buffer I includes: 5-8 mol / L guanidine thiocyanate, 0.1%-1.0% Triton X-100, 1-2% sucrose, 50-100 mmol / L EDTA, 25-50 mmol / L Tris-HCl, and sterile DEPC water. Lysis buffer II includes: 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, pH 5.0-6.0; 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% by volume anhydrous ethanol, 30-40% by volume isopropanol, and pH 5.5-6.5; Cleaning solution I includes: 40-55% ethanol solution by volume, 0.5-1.2 mol / L NaCl, 1-2 mol / L guanidine hydrochloride, and 0.2-0.3 mol / L NaHCO3; Cleaning solution II includes: 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% 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.

[0009] A fungal mRNA extraction kit comprising functionalized magnetic beads comprises the following steps: (1) Sample pretreatment: Place the cultured cells in a centrifuge tube and centrifuge at 12,000 rpm for 30 seconds. Discard the supernatant and collect the precipitate. (2) Add the ground material and 300 μL of lysis buffer I to the above precipitate, place on an oscillator at 1000 rpm for 2 min, place 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; (3) Add 200 μL of lysis buffer II, mix thoroughly by inverting, incubate in a 55-60°C water bath for 10 min, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube. (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix well by pipetting, and place at room temperature for 2-3 minutes. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack. (5) Add 1 mL of cleaning solution I to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic stand for 1 min, allow the magnetic beads to completely absorb to the tube wall, and then discard the supernatant; (6) Add 1 mL of cleaning solution II to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic rack for 1 min, allow the magnetic beads to be completely absorbed by the tube wall, then discard the supernatant, remove the centrifuge tube from the magnetic rack, and dry it at room temperature for 3-5 min until there is no liquid left in the centrifuge tube; (7) Add 50-100 μL of elution buffer to the centrifuge tube, mix gently, elute at 37°C for 15-20 minutes, then place on a magnetic stand for 1-2 minutes. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the mRNA product.

[0010] Compared with the prior art, the present invention has the following advantages: 1. The present invention provides a novel functional composite magnetic bead, which is a magnetic microsphere formed by wrapping tosyl magnetic beads with bifunctional nucleic acid, and can efficiently and specifically adsorb mRNA. Specifically, it has the following advantages: The tosyl magnetic beads used in the present 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 the present invention have a surface covalently modified bifunctional nucleic acid. The bifunctional nucleic acid is a double-stranded DNA sequence containing the EcoRI (restriction endonuclease) cleavage sequence GAATTC. One end of the bifunctional nucleic acid is covalently coupled to the tosyl magnetic beads via a 5' NH2- group, and the other end specifically binds to mRNA via poly-T. This allows the composite magnetic beads to directionally and specifically identify mRNA in a solution, thereby obtaining high-concentration, high-purity mRNA.

[0011] 2. The fungal cell wall is a multi-layered mesh structure formed by chitin and β-glucan, which makes the fungal cell wall highly mechanically strong and very tough, increasing the difficulty of lysing it. Based on the structural characteristics of fungal cells, the present invention provides a highly efficient cell lysis solution I and lysis solution II. Under the synergistic effect of the grinding material and the two lysis solution components, the cells can be effectively lysed after two lysis steps. While ensuring the release of large amounts of mRNA, the guanidine isothiocyanate and heparin in the lysis solution can effectively inhibit the activity of RNases, prevent mRNA degradation, and protect the integrity and stability of mRNA, laying the foundation for the extraction of mRNA with higher concentration and purity. It also avoids the use of the toxic reagent trizol.

[0012] 3. The eluent provided by the present invention is a rationally formulated system of multiple components. While ensuring that EcoRI (a restriction endonuclease) specifically recognizes the GAATTC sequence in double-stranded DNA, it also completes double-strand cleavage, thereby releasing mRNA. High-quality mRNA is obtained through magnetic adsorption. The heparin in this eluent buffer system effectively inhibits mRNA enzyme activity, ensuring mRNA integrity. Furthermore, components such as Triton X-100, MgCl2, and BSA retained 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, and thus improving PCR results, achieving a two-pronged effect.

[0013] 4. Fungal mRNA has a polyadenylic acid (polyA) tail consisting of 30 to 200 nucleotide residues at its 3′-end. This polyA tail is crucial for maintaining the structural stability of the mRNA, and its length determines its half-life. Over time, this polyA tail gradually shortens. The mRNA extracted in this invention is bound to the composite magnetic beads and then cleaved by EcoRI. The resulting double-stranded 3′-end maintains the mRNA's structural stability, preventing degradation and preserving its biological activity.

[0014] 5. The present invention offers the advantages of simple operation, safety, nontoxicity, high sensitivity, good reproducibility, and low cost. This breakthrough in traditional mRNA extraction methods enables the extraction of mRNA from trace samples. The mRNA obtained by this method can provide a high-quality template for RT-PCR detection, improving the specificity and sensitivity of detection. It has broad application prospects in clinical disease diagnosis, forensic identification, food safety testing, microbiological testing, molecular biology, and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the UV absorption spectrum of Trichoderma mRNA extracted using the method in Example 4; Figure 2 This is the UV absorption spectrum of Saccharomyces cerevisiae mRNA extracted using the method of Example 5; Figure 3 This is the RT-qPCR amplification curve of the cox3 gene and CHS2 gene of Saccharomyces cerevisiae in Example 5; Figure 4 RT-qPCR melting curves of CHS2 gene for mRNA extracted using different methods; Figure 5 RT-qPCR amplification curves of CHS2 gene after mRNA extraction using different methods. DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] The basic component of the present invention, the specific composition of the functionalized magnetic beads is as follows: the functionalized magnetic beads refer to magnetic microspheres formed by wrapping the magnetic beads with bifunctional nucleic acids, which can specifically bind to mRNA; the magnetic beads are tosyl magnetic beads with a particle size of 0.5μm to 1.0μm. The bifunctional nucleic acid is a double-stranded DNA sequence, one end of which is covalently coupled to the tosyl magnetic beads through 5' NH2-, and the other end specifically binds to mRNA through poly-T, and the double-stranded sequence contains an EcoRI restriction site. The double-stranded DNA includes oligonucleotide I and oligonucleotide II, and oligonucleotide I is composed of 25 deoxynucleotides. Its sequence is: 5'NH2- CACCAGAGAGCTGGAATTCGAGTGC-3'; Oligonucleotide II consists of 32 deoxynucleotides. Its sequence is: 5'- TTTTTTTTTTTTTTTGCACTCGAATTCCAGGT-3', Oligonucleotide I and oligonucleotide II are partially complementary to form a double strand.

[0018] The present invention will be further described below with reference to specific examples.

[0019] Example 1, a fungal mRNA extraction kit composed of functionalized magnetic beads, comprising a grinding material, lysis solution I, lysis solution II, magnetic bead binding solution, cleaning solution I, cleaning solution II, and eluent; the specific preparation of each component is as follows: Grinding material: 200 μm glass beads, soaked in 0.1% DEPC water and sterilized by high pressure; Lysis buffer I includes: 5 M guanidine thiocyanate, 0.1% Triton X-100, 1% sucrose, 50 mmol / L EDTA, 25 mmol / L Tris-HCl, and sterile DEPC water; Lysis buffer II includes: 0.1% SDS, 0.5 mol / L NaCl, 100 mmol / L sodium acetate, 200 mmol / L EDTA, 0.1 mg / mL heparin, pH 5.0.

[0020] The magnetic bead binding solution includes: 0.5 mol / L NaCl, 10 mg / mL functionalized magnetic beads, 1.2 mol / L guanidine hydrochloride, 20% anhydrous ethanol by volume, 30% isopropanol by volume, and pH 5.5.

[0021] Cleaning solution I includes: 40% ethanol solution, 0.5 mol / L NaCl, 1 mol / L guanidine hydrochloride, and 0.2 mol / L NaHCO3; Cleaning solution II includes: 80% ethanol, 1 mol / L NaCl; Eluent: 30 mmol / L Tris-HCl, 10 mmol / L MgCl2, 100 mmol / L NaCl, 0.01% TritonX-100, 0.1 mg / mL BSA, 2000 U / mL EcoRI, 0.1 mg / mL heparin, pH 7.5.

[0022] Example 2, a fungal mRNA extraction kit composed of functionalized magnetic beads, including grinding material, lysis solution I, lysis solution II, magnetic bead binding solution, cleaning solution I, cleaning solution II, and eluent, the specific preparation of each component is as follows: Grinding material: Glass beads with a particle size of approximately 400 μm, soaked in 0.1% DEPC water and sterilized under high pressure; Lysis buffer I includes: 8 M guanidine thiocyanate, 1.0% Triton X100, 2% sucrose, 100 mmol / L EDTA, 50 mmol / L Tris-HCl, and sterile DEPC water. Lysis buffer II includes: 0.15% SDS, 1.0 mol / L NaCl, 200 mmol / L sodium acetate, 500 mmol / L EDTA, 0.6 mg / mL heparin, pH 6.0.

[0023] The magnetic bead binding solution includes: 1.0 mol / L NaCl, 15 mg / mL functionalized magnetic beads, 1.5 mol / L guanidine hydrochloride, 30% by volume anhydrous ethanol, 40% by volume isopropanol, and pH 6.5.

[0024] Cleaning solution I includes: 55% by volume ethanol solution, 1.2 mol / L NaCl, 2 mol / L guanidine hydrochloride, and 0.3 mol / L NaHCO3; Cleaning solution II includes: 80% ethanol, 2 mol / L NaCl; Eluent: 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 E-CoRI, 0.6 mg / mL heparin, pH 8.2.

[0025] Example 3, a fungal mRNA extraction kit composed of functionalized magnetic beads, including grinding material, lysis solution I, lysis solution II, magnetic bead binding solution, cleaning solution I, cleaning solution II, and eluent, the specific preparation of each component is as follows: Grinding material: glass beads with a particle size of about 300 μm, soaked in 0.1% DEPC water and sterilized by high pressure; Lysis buffer I includes: 6 M guanidine thiocyanate, 0.5% Triton X100, 1.5% sucrose, 75 mmol / L EDTA, 40 mmol / L Tris-HCl, and sterile DEPC water. Lysis buffer II includes: 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.

[0026] The magnetic bead binding solution includes: 0.8 mol / L NaCl, 12 mg / mL functionalized magnetic beads, 1.3 mol / L guanidine hydrochloride, 25% anhydrous ethanol by volume, 35% isopropanol by volume, and pH 6.0.

[0027] Cleaning solution I includes: 50% ethanol solution, 1.0 mol / L NaCl, 1.5 mol / L guanidine hydrochloride, and 0.25 mol / L NaHCO3; Cleaning solution II includes: 80% ethanol, 1.5 mol / L NaCl; Eluent: 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.

[0028] Example 4, a method for extracting mRNA using the kit prepared in Example 1, comprising the following steps: (1) Sample pretreatment: Take 2 mL of Trichoderma cells cultured at 37°C for 60 h, place them in a centrifuge tube, centrifuge at 12,000 rpm for 30 seconds, discard the supernatant, and collect the precipitate.

[0029] (2) Add the ground material and 300 μL of Lysis Buffer I to the above precipitate, place on an oscillator at 1000 rpm for 2 minutes, place on ice for 2 minutes, and repeat this step twice. Centrifuge at 5000 rpm for 2 minutes, and aspirate the supernatant into a clean centrifuge tube.

[0030] (3) Add 200 μL of lysis buffer II, mix thoroughly by inverting, incubate in a 55-60°C water bath for 10 min, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube.

[0031] (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix well by pipetting, and place at room temperature for 2 to 3 minutes. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack.

[0032] (5) Add 1 mL of cleaning solution I to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic stand for 1 min, allow the magnetic beads to be completely absorbed to the tube wall, and then discard the supernatant.

[0033] (6) Add 1 mL of cleaning solution II to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic rack for 1 minute, allow the magnetic beads to be completely absorbed by the tube wall, then discard the supernatant, remove the centrifuge tube from the magnetic rack, and dry it at room temperature for 3 to 5 minutes until there is no liquid left in the centrifuge tube.

[0034] (7) Add 100 μL of elution buffer to the centrifuge tube, mix gently, elute at 37°C for 15-20 minutes, then place on a magnetic stand for 1-2 minutes. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the mRNA product.

[0035] (8) Determine the mRNA concentration and purity using a UV spectrophotometer. Figure 1 The ultraviolet absorption spectrum of the mRNA extracted from Trichoderma sp. is shown in the figure. The extracted mRNA has high concentration (150.36 μg / mL) and good purity (A 260 / 280 =1.89).

[0036] Example 5, a method for extracting mRNA using the kit prepared in Example 3, comprising the following steps: (1) Sample pretreatment: Take 2 mL of Saccharomyces cerevisiae cells cultured at 37°C for 48 h and place them in a centrifuge tube. Centrifuge at 12,000 rpm for 30 seconds, discard the supernatant, and collect the precipitate.

[0037] (2) Add the ground material and 300 μL of Lysis Buffer I to the above precipitate, place on an oscillator at 1000 rpm for 2 minutes, place on ice for 2 minutes, and repeat this step twice. Centrifuge at 5000 rpm for 2 minutes, and aspirate the supernatant into a clean centrifuge tube.

[0038] (3) Add 200 μL of lysis buffer II, mix thoroughly by inverting, incubate in a 55-60°C water bath for 10 min, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube.

[0039] (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix well by pipetting, and place at room temperature for 2 to 3 minutes. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack.

[0040] (5) Add 1 mL of cleaning solution I to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic stand for 1 min, allow the magnetic beads to be completely absorbed to the tube wall, and then discard the supernatant.

[0041] (6) Add 1 mL of cleaning solution II to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic rack for 1 minute, allow the magnetic beads to be completely absorbed by the tube wall, then discard the supernatant, remove the centrifuge tube from the magnetic rack, and dry it at room temperature for 3 to 5 minutes until there is no liquid left in the centrifuge tube.

[0042] (7) Add 100 μL of elution buffer to the centrifuge tube, mix gently, elute at 37°C for 15-20 minutes, then place on a magnetic stand for 1-2 minutes. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the mRNA product.

[0043] (8) Determine the mRNA concentration and purity using a UV spectrophotometer. Figure 2 This is the ultraviolet absorption spectrum of the mRNA extracted from Saccharomyces cerevisiae in the present invention. The extracted mRNA has a high concentration (173.36 μg / mL) and good purity (A 260 / 280 =1.85).

[0044] (9) RT-qPCR assay. The extracted mRNA was immediately reverse transcribed to obtain cDNA. Using the cDNA as a template, the Hifair® III qPCR SYBR Green Kit was used to amplify the Saccharomyces cerevisiae cytochrome oxidase subunit 3 gene (COX3) and chitin synthase gene (CHS2) using the SYBR Green I dye method. The mRNA extracted by the kit of the present invention was evaluated. The primer sequences are shown in Table 1, and the reaction system is shown in Table 2.

[0045] Table 1 RT-qPCR primer sequences for Saccharomyces cerevisiae Table 2 qPCR reaction system qPCR amplification used a two-step method with the following reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, and annealing at 55°C for 20 s, for a total of 35 cycles. After the reaction was completed, the Ct value was obtained based on the fluorescence signal data.

[0046] (10) Figure 3 This is a graph showing the RT-qPCR amplification curves of the COX3 and CHS2 genes of Saccharomyces cerevisiae in Example 5. As can be seen from the figure, the Ct values of the amplified genes of the present invention are approximately 20-22.

[0047] The beneficial effects of the present invention are further illustrated below in conjunction with comparative examples.

[0048] In Comparative Example 1, 2 mL of a Saccharomyces cerevisiae cell sample cultured at 37°C with shaking for 60 hours was taken and treated using the classic Trizol method and the Omega EZNA® Total RNA Kit I (R6831) kit, respectively, to obtain a total RNA preparation solution. Subsequently, the total RNA preparation solution was further purified using an oligo(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 the present invention, and the results were analyzed by ultraviolet spectrophotometry. The results are shown in Table 3. The results show that the mRNA extracted using the kit of the present invention has higher concentration and purity than the mRNA extracted using the Trizol method and the commercially available Omega kit.

[0049] In addition, the Saccharomyces cerevisiae CHS2 gene was amplified by RT-qPCR, and the melting curves were all single peaks ( Figure 4 ), the amplification curve is as follows Figure 5 As shown in the figure, it can be seen that each amplification curve is smooth and orderly, but when the mRNA extracted by the present 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 the present invention has better quality.

[0050] Table 3 Comparison of mRNA extraction effects by different methods Comparative Example 2 compares the effectiveness of the functionalized magnetic beads of the present invention with traditional Oligo(dT) magnetic beads in mRNA extraction. Using the kit buffer system of Example 2 of the present invention, the conventional Oligo(dT) magnetic beads were substituted for the conventional Oligo(dT) magnetic beads in the magnetic bead binding solution of the control group, and the extraction effect was verified using the method of Example 5 of the present 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 demonstrate that the mRNA concentration and purity extracted using the functionalized magnetic beads of this kit are higher than those using the Oligo(dT) magnetic bead method.

[0051] Table 4 Comparison of mRNA extraction effects by different methods.

Claims

1. A functionalized magnetic bead, characterized in that: It refers to the magnetic microspheres formed by the coating of magnetic beads with bifunctional nucleic acids, which can specifically bind to mRNA; The magnetic beads are tosyl 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 tosyl magnetic beads 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. A functionalized magnetic bead according to claim 1, characterized in that The double-stranded DNA comprises oligonucleotide I and oligonucleotide II, wherein oligonucleotide I consists of 25 deoxynucleotides. Its sequence is: 5′NH2- CACCAGAGAGCTG GAATTCGAGTGC-3′; Oligonucleotide II consists of 32 deoxynucleotides. Its sequence is: 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, characterized in that: The kit consists of grinding material, lysis solution I, lysis solution II, magnetic bead binding solution, cleaning solution I, cleaning solution II, and elution solution; the specific preparation of each component is as follows: Grinding material: Glass beads with a particle size of 200-400 μm, soaked in 0.1% DEPC water and sterilized by high pressure; Lysis buffer I includes: 5-8 mol / L guanidine thiocyanate, 0.1%-1.0% Triton X-100, 1-2% sucrose, 50-100 mmol / L EDTA, 25-50 mmol / L Tris-HCl, and sterile DEPC water. Lysis buffer II includes: 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, pH 5.0-6.0; 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% by volume anhydrous ethanol, 30-40% by volume isopropanol, and pH 5.5-6.5; Cleaning solution I includes: 40-55% ethanol solution by volume, 0.5-1.2 mol / L NaCl, 1-2 mol / L guanidine hydrochloride, and 0.2-0.3 mol / L NaHCO3; Cleaning solution II includes: 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% 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. In the preparation of all the above solutions, sterile DEPC water was used as the solvent.

4. A fungal mRNA extraction kit comprising functionalized magnetic beads, characterized in that: The following steps are involved: (1) Sample pretreatment: Place the cultured cells in a centrifuge tube and centrifuge at 12,000 rpm for 30 seconds. Discard the supernatant and collect the precipitate. (2) Add the ground material and 300 μL of lysis buffer I to the above precipitate, place on an oscillator at 1000 rpm for 2 min, place 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; (3) Add 200 μL of lysis buffer II, mix by inversion, incubate in a 55-60°C water bath for 10 min, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube. (4) Add 500 μL of magnetic bead binding solution to the centrifuge tube, mix well by pipetting, and place at room temperature for 2-3 minutes. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack. (5) Add 1 mL of cleaning solution I to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic stand for 1 min, allow the magnetic beads to completely absorb to the tube wall, and then discard the supernatant; (6) Add 1 mL of cleaning solution II to the centrifuge tube, pipette and mix thoroughly, place the centrifuge tube on a magnetic rack for 1 min, allow the magnetic beads to be completely absorbed by the tube wall, then discard the supernatant, remove the centrifuge tube from the magnetic rack, and dry it at room temperature for 3-5 min until there is no liquid left in the centrifuge tube; (7) Add 50-100 μL of elution buffer to the centrifuge tube, mix gently, elute at 37°C for 15-20 minutes, then place on a magnetic stand for 1-2 minutes. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the mRNA product.

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