Primer probe combination for detecting fungi, kit, detection method and application

Abstract

The invention provides a primer probe combination, a kit, a detection method and application for detecting fungi. The primer probe combination comprises primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and a single-chain fluorescent probe with a nucleotide sequence as shown in SEQ ID NO: 3. The primer probe combination for detecting fungi provided by the invention is good in specificity, and the primer group has the advantage of wide detection range, and can detect all strains containing target sequences amplified by the primer group, including common fungi in the fermentation process.

Description

Technical Field

[0001] The present invention belongs to the technical field of fungal detection, and in particular relates to a primer-probe combination, a kit, a detection method and an application for detecting fungi. Background Art

[0002] In the process of Baijiu brewing, fungi play a crucial role, especially in the koji-making and fermentation stages. One of the core steps in Baijiu brewing is koji-making, which relies on a variety of fungi, especially Aspergillus ( Aspergillus spp.), Rhizopus ( Rhizopus spp.) and yeast ( Saccharomyces spp.). These fungi secrete a rich enzyme system, such as amylase, protease and cellulase, to degrade macromolecular substances such as starch, protein and cellulose in raw materials into fermentable small-molecule sugars and amino acids, providing the necessary substrates for subsequent alcohol fermentation. In the fermentation stage, yeast is the main alcohol producer, which converts glucose into ethanol and carbon dioxide through the glycolysis pathway. In addition, some fungi can also produce a variety of flavor substances, such as esters, alcohols and acid compounds, which play an important role in the aroma and taste of Baijiu. For example, esters are one of the main aroma components in Baijiu, and their formation is closely related to the metabolic activities of yeast and mold. Fungi, with their unique metabolic and conversion abilities, drive the fermentation process, and the change of their content directly affects the quality, efficacy and market competitiveness of fermentation products. Therefore, the rapid quantitative detection of fungi is of great significance for the precise control of fermentation and digital intelligent fermentation.

[0003] At present, there are many defects in the existing fungal detection methods. The traditional microbial culture method, although intuitive, is time-consuming. It takes several weeks or even months for some fungi to form visible colonies, seriously delaying the detection, and is easily interfered by environmental factors, with cumbersome operations. The microscopic examination method can quickly provide fungal morphological images, but has low sensitivity and is prone to missed detection of fungi with low content and atypical morphology. The detection reagents developed based on the principle of specific binding of antigen-antibody can achieve a certain degree of quantitative detection, but the antibody specificity is significantly limited. One antibody only targets a few fungi, and the broad-spectrum detection ability is insufficient. With the development of molecular biology, nucleic acid amplification technologies based on PCR, such as real-time fluorescence quantitative PCR and multiplex PCR, have been widely used in fungal detection. They rely on nucleic acid recognition and amplification to improve the detection accuracy and depth. However, the PCR technology requires cycles of heating and cooling, relies on sophisticated and expensive instrument equipment, and has a long detection time and depends on professional technical personnel, which is not suitable for rapid point-of-care testing.

[0004] Recombinase polymerase-mediated isothermal amplification technology (RPA technology) is a novel nucleic acid amplification technology that gets rid of the dependence on traditional thermal cycling and efficiently amplifies nucleic acids under the constant temperature condition of 37°C - 42°C. It is easy to operate. After sample treatment, reagents are added, and the reaction can be initiated using a small constant temperature device or even the body temperature environment. Amplification is completed within 5 - 20 minutes, significantly shortening the detection time and meeting the requirements of real-time detection. Its reaction system is simple, has low requirements for sample purity, is less inhibited by impurities, and has high accuracy and reliability, which is conducive to accurately controlling the dynamics of fermenting fungi. After retrieval, no report on the use of recombinase polymerase isothermal amplification technology for the detection of general brewing fungi has been found. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and proposes a primer-probe combination, kit, detection method, and application for detecting fungi.

[0006] To achieve the above object, the technical solution of the present invention is realized as follows: In the first aspect, the present invention provides a primer-probe combination for detecting fungi, including primers with nucleotide sequences shown in SEQ ID NO: 1 - 2 and a single-stranded fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 3.

[0007] The above primer-probe combination is designed for the conserved region of the 18S rDNA gene of fungi related to brewing. Among them, the nucleotide sequence of the conserved region of the fungal 18S rDNA gene is shown in SEQ ID NO: 8.

[0008] In the present invention, tetrahydrofuran (THF) is inserted into the sequence of the single-stranded fluorescent probe as a cleavage site. Fluorescent groups and quenching groups are labeled on both sides of tetrahydrofuran, with a spacer of 1 - 10 nt between the fluorescent group and the quenching group, and a modification group is labeled at the 3' end of the single-stranded fluorescent probe; The fluorescent group is selected from FAM, TET, NED, ROX, CY3, CY5, VIC, JOE, HEX, Texas RED, LCRED460; the quenching group is selected from MGB, TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1, BHQ2; the modification group is selected from amino group, phosphate group, C3-Spacer.

[0009] In some specific embodiments: the fluorescent group is FAM, the quenching group is BHQ, the spacer between the fluorescent group and the quenching group is 3 nt, and the modification group is C3-Spacer.

[0010] In some specific embodiments: the length of the single-stranded fluorescent probe is 45 - 60 nt.

[0011] In some specific embodiments: A dSpacer (tetrahydrofuran, THF) is labeled at the middle position 30 - 35 nt away from the 5' end (any base, no special requirement), serving as the recognition site for exonuclease.

[0012] In some specific embodiments: A fluorescent group (FAM) is labeled on the T base upstream of the THF site, and a quenching group (BHQ1) is labeled on the T base downstream, with the distance between the two groups being 1 - 10 nt.

[0013] In some specific embodiments: THF is approximately 15 nt away from the 3' end, and a modification group (C3 - spacer) is labeled at the 3' end.

[0014] In the second aspect, the present invention also provides the application of the above primer - probe combination in the preparation of a kit for detecting fungi.

[0015] In some embodiments, the kit of the present invention further includes a primer pair and a single - strand fluorescent probe: the sequences of the primer pair are as shown in SEQ ID NO: 1 - 2; the sequence of the single - strand fluorescent probe is as shown in SEQ ID NO: 3.

[0016] In some specific embodiments, THF is inserted as a cleavage site in the sequence of the single - strand fluorescent probe, with a fluorescent group FAM and a quenching group BHQ1 labeled on both sides of THF respectively, and a modification group C3 - Spacer is labeled at the 3' end of the probe primer.

[0017] In some specific embodiments, the final concentrations of the primers are 0.001 μM - 30 μM respectively; the final concentration of the single - strand fluorescent probe is 0.001 μM - 20 μM.

[0018] In some specific embodiments, the kit further includes a rehydration buffer, magnesium acetate, RPA lyophilized enzyme powder, and ddH2O.

[0019] The RPA lyophilized enzyme powder is a mixture of a recombinase, a single - strand binding protein, a DNA polymerase, and a creatine kinase required for the RPA amplification reaction, and exists in the RPA reaction tube in the form of the RPA lyophilized enzyme powder.

[0020] In some specific embodiments, the kit includes: 32.9 μL of rehydration buffer, 0.5 μL of 20 μM upstream primer, 0.5 μL of 20 μM downstream primer, 0.3 μL of 10 μM single - strand fluorescent probe, 2 μL of the DNA sample to be tested, 9.8 μL of ddH2O, and 1 μL of magnesium acetate.

[0021] The present invention also provides a method for detecting fungi, which uses the primer - probe combination of the present invention to perform RPA (recombinase polymerase amplification) detection on a sample to be tested.

[0022] In some embodiments, the method for detecting fungi comprises the following steps: S1: Extract DNA from the test substance containing fungi to obtain a DNA template; S2: Use the extracted DNA as a template for RPA amplification, and use a primer-probe combination to interpret the results of the RPA amplification to identify the fungi in the test sample.

[0023] In some specific embodiments, the step S2 further includes configuring an RPA reaction system. The specific steps are as follows: sequentially add the following components to a reaction tube containing RPA freeze-dried enzyme powder: 32.9 μL of rehydration buffer, 0.5 μL of 20 μM forward primer, 0.5 μL of 20 μM reverse primer, 0.3 μL of 10 μM single-stranded fluorescent probe, 9.8 μL of ultrapure water. After vortex mixing, use a pipette to divide the solution prepared in the tube into two equal parts in an eight-strip row. Then, sequentially add 2 μL of DNA template to the eight-strip row. Finally, add 1 μL of magnesium acetate to the walls of the eight-strip row reaction tubes in sequence, and add it to the system by centrifugation to catalyze the occurrence of the reaction.

[0024] In some specific embodiments, the steps of RPA amplification are as follows: Place the reaction tube in a fluorescence quantitative PCR instrument, react at 37 °C for 5 - 20 min, collect fluorescence signals every 30 s, and construct an amplification curve by analyzing the fluorescence signal intensities collected at different time points through the instrument software. If an amplification curve is obtained, it is determined to be positive; otherwise, if there is no amplification curve, it is determined to be negative.

[0025] The above primer-probe combination, kit, and detection method can be used for, but are not limited to, the detection of fungi in wine products, beverages, fermented foods, tobacco leaves, etc.

[0026] Compared with the prior art, the present invention has the following advantages: (1) Through the NCBI database, the present invention retrieved more than 500 fungi related to brewing (such as the genus Saccharomyces, Aspergillus, Rhizopus, Penicillium, Mucor, Candida, etc.), and downloaded their 18S rDNA gene sequences. The software DNAman was used to perform sequence alignment on the downloaded large number of gene sequences, and homologous regions were accurately screened out from more than 500 sequences. Based on these highly conserved homologous regions, primers and probes for real-time fluorescence RPA detection of fungi were designed. The designed primers and probes have outstanding specificity and can widely and accurately cover various fungi common in the brewing process, such as the genus Saccharomyces (including Saccharomyces cerevisiae, Pichia pastoris, Zygosaccharomyces bailii, Candida utilis, etc.), the genus Aspergillus (including Aspergillus niger, Monascus purpureus, Aspergillus oryzae, etc.), the genus Rhizopus, the genus Mucor, the genus Penicillium, etc. In complex fermentation environment samples, by virtue of unique sequence design, the primers and probes can effectively avoid non-specific binding with nucleic acid sequences of other microorganisms and specifically amplify and detect only the target fungi.

[0027] (2) The primer set for detecting fungi provided by the present invention is designed with the partial conserved region sequence of fungal 18S rDNA as the template, has good specificity, and the primer set has the advantage of a wide detection range and can detect all strains containing the target sequence amplified by the primer set, including common fungi in the fermentation process. Description of the Drawings

[0028] Figure 1 It is the amplification curve graph for screening and amplifying RPA primers of brewing fungi; Figure 2 It is the RPA amplification curve graph for the feasibility test of the primer and probe combination of the present invention for brewing fungi; Figure 3 It is the optimization of the enzyme amount for the quantitative detection reaction of fungi based on RPA; Figure 4 It is the optimization of the reaction temperature for the quantitative detection reaction of fungi based on RPA; Figures A - E are the amplification curve graphs at temperatures of 33°C, 35°C, 37°C, 39°C, and 41°C respectively; Figure 5 It is the optimization of the primer and probe concentration for the quantitative detection reaction of fungi based on RPA; Figures A - D are the amplification curve graphs under the conditions of System 1, System 2, System 3, and System 4 respectively; Figure 6 It is the RPA amplification curve graph for the universality test of the primer and probe combination of the present invention for brewing fungi; Figure 7RPA amplification curve for the specificity test of the primer and probe combination of the present invention against brewing fungi; among them, the fungal group includes Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces boulardii, Zygosaccharomyces bailii, Kazachstania servazzii, and Aspergillus niger, and the non-fungal group includes Bacillus amyloliquefaciens, Bacillus subtilis, Escherichia coli, Lactobacillus paracasei, Lactobacillus reuteri, human papillomavirus, norovirus, and NC (negative control, nuclease-free pure water as the target). Figure 8 RPA amplification curve for the sensitivity test of the primer and probe combination of the present invention against brewing fungi; Figure 9 It is a linear relationship diagram between time and concentration. Detailed implementation manners

[0029] The embodiments of the present invention are described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0030] In this article, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs.

[0031] In this article, when values are described as ranges, it should be understood that such disclosure includes the disclosure of all possible sub-ranges within the range, as well as specific values falling within the range, regardless of whether specific values or specific sub-ranges are explicitly indicated.

[0032] In this article, when referring to "multiple", etc., unless otherwise specified, it means greater than 2 or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0033] In this article, when referring to "preferred" and "more preferred", they are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation to the protection scope of the present invention.

[0034] In this article, when referring to "further", etc., it is used to describe the purpose and indicates the difference in content, but it should not be construed as a limitation to the protection scope of the present invention.

[0035] In this article, the term "and / or" is a description of the association relationship of an object, indicating that there can be three relationships. For example, A and / or B means: A or B, or A and B these three relationships.

[0036] In this article, the term "about" means + / - 10% of the specified value, preferably + / - 5%, and more preferably + / - 1%.

[0037] In this article, the terms "comprising", "including", "having", "containing", etc. are all open-ended terms, that is, they are meant to include but not limited to.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention.

[0039] The present invention will be described in detail below in conjunction with embodiments.

[0040] Example 1: Design, screening and preparation of primer and probe sequences (1) RPA primer design: In this experiment, the 18S rDNA gene sequences of more than 500 common fungi (such as Saccharomyces cerevisiae, Pichia pastoris, Zygosaccharomyces bailii, Candida utilis, Aspergillus niger, Monascus purpureus, Aspergillus oryzae, Rhizopus nigricans, etc.) during the brewing process were retrieved and downloaded from the NCBI database (National Center for Biotechnology Information). Then, a large number of retrieved sequences were aligned in the software DNAman to screen for conserved sequences with high homology. The nucleotide sequence is shown in SEQ ID NO.8. Three forward primers, three reverse primers and one fluorescent probe were designed in this conserved region. The primer sequences were synthesized by Tsingke Biotechnology Co., Ltd., and the fluorescent probe was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences are shown in Table 1.

[0041] Table 1. Primer combinations for isothermal amplification of brewing fungi

[0042] Wherein: FAM-dT is thymidine nucleotide carrying a fluorescein group, THF is tetrahydrofuran, BHQ1-dT is thymidine nucleotide carrying a fluorescence quenching group BHQ1, and C3-Spacer is to introduce a spacer arm at the 3' end to prevent chain extension.

[0043] (2) RPA reaction system and conditions RPA reaction system: Operate according to the instructions of the DNA isothermal amplification reaction kit (fluorescent type). Add the following components to the reaction tube containing RPA freeze-dried powder in sequence: 32.9 μL of Buffer A buffer, 0.5 μL of forward primer BF-F1 / F2 / F3 (20 μM), 0.5 μL of reverse primer BF-R1 / R2 / R3 (20 μM), 0.3 μL of fluorescent probe BF-P1 (10 μM), 9.8 μL of ultrapure water. After vortex mixing, use a pipette to divide the prepared solution in the tube into two equal parts and transfer them to an eight-strip row. Then, add 2 μL of DNA template to the eight-strip row in sequence. Finally, add 1 μL of magnesium acetate to the wall of the eight-strip row reaction tube and add it to the system by centrifugation to catalyze the reaction. Note that the preparation of the system needs to be carried out on ice throughout the process.

[0044] RPA reaction conditions: Place the RPA reaction tube added with various components into a fluorescence quantitative PCR instrument and react at 37 °C for 20 min, collecting fluorescence signals every 30 s. Analyze the fluorescence signal intensities collected at different time points through the instrument software to construct an amplification curve and judge the results according to the curve situation.

[0045] The results are as Figure 1 shown. According to the principle of the cross-pairing method, a set of RPA primers with the best comprehensive performance was successfully screened out: BF-F1 and BF-R1, and the probe BF-P1.

[0046] It can be seen from Figure 1 the amplification curves corresponding to the negative controls of BF-F1 and BF-R1 are flatter than those of the other two groups in the RPA primer screening experiment, and the fluorescence value of the corresponding positive control group is the highest. In this way, the negative can show an obvious difference from the positive group. Therefore, BF-F1 and BF-R1 and the probe BF-P1 will be used for subsequent experiments.

[0047] Example 2: Feasibility detection test and condition optimization of RPA primers for brewing fungi (1) Plasmid synthesis and DNA template preparation: Using the BamH I and EcoR I double enzyme digestion sites, insert the synthesized target gene into the puc57 vector as the target for feasibility detection.

[0048] (2)RPA reaction system and conditions The DNA template was the above synthetic plasmid and the control group NC (the control group NC was the pUC57 empty vector without the target sequence).

[0049] RPA reaction system: Operate according to the instructions of the DNA isothermal amplification reaction kit (fluorescent type). Add the following components successively to the reaction tube containing the RPA lyophilized powder: 32.9 μL of Buffer A buffer, 0.5 μL of forward primer BF-F1 (20 μM), 0.5 μL of reverse primer BF-R1 (20 μM), 0.3 μL of fluorescent probe BF-P1 (10 μM), 9.8 μL of ultrapure water. After vortex mixing, divide the solution prepared in the tube into two equal parts with a pipette gun into an eight-well strip, and then add 2 μL of DNA template to the eight-well strip in sequence. Finally, add 1 μL of magnesium acetate to the wall of the eight-well strip reaction tube in sequence, and add it to the system by centrifugation to catalyze the reaction. Note that the preparation of the system needs to be carried out on ice throughout the process.

[0050] RPA reaction conditions: Place the RPA reaction tube containing various components in a real-time fluorescence quantitative PCR instrument, react at 37 °C for 15 min, and collect fluorescence signals every 30 s. Analyze the fluorescence signal intensity collected at different time points through the instrument software to construct an amplification curve, and judge the results according to the curve situation.

[0051] The results are as Figure 2 shown. It can be seen from the figure that the fluorescence signal of the negative control NC group was extremely low throughout the experiment, and there was no obvious change in the detection line, ensuring the reliability of the results, excluding the risk of false positives, and ensuring the credibility of positive results. When the synthetic plasmid was used as the DNA template, the fluorescence signal climbed rapidly at the initial stage of the reaction and quickly reached the saturation state. This result proved the feasibility of this method for detecting brewing fungi.

[0052] (3)Optimization of reaction conditions for quantitative detection of fungi based on RPA By optimizing the enzyme amount, temperature, and primer-probe concentration respectively, an efficient, sensitive, and stable detection system and detection method were established.

[0053] 1) Enzyme amount optimization: By setting experiments (100% enzyme amount, 50% enzyme amount, 30% enzyme amount, 25 μL reaction system), evaluate the effects of different enzyme amounts on the amplification efficiency and signal intensity to determine the optimal enzyme dosage.

[0054] 100% enzyme amount reaction system: 2 μL of BF-F1 (5 μM), 2 μL of BF-R1 (5 μM), 3 μL of BF-P1 (1 μM), 4.1 μL of ddH2O, 32.9 μL of buffer A (after mixing the above solutions, take 22 μL for each reaction tube on average), 2 μL of the template to be detected, 1 μL of magnesium acetate.

[0055] 50% enzyme amount reaction system: 4 μL of BF-F1 (5 μM), 4 μL of BF-R1 (5 μM), 6 μL of BF -P1 (1 μM), 8.2 μL of ddH2O, 65.8 μL of buffer A (after mixing the above solutions, take 22 μL for each reaction tube on average), 2 μL of the template to be detected, 1 μL of magnesium acetate.

[0056] 30% enzyme amount reaction system: 6 μL of BF-F1 (5 μM), 6 μL of BF-R1 (5 μM), 9 μL of BF-P1 (1 μM), 12.3 μL of ddH2O, 98.7 μL of buffer A (after mixing the above solutions, take 22 μL for each reaction tube on average), 2 μL of the template to be detected, 1 μL of magnesium acetate.

[0057] Reaction conditions: React at 37 °C for 20 min, and collect fluorescence signals every 30 s. Analyze the fluorescence signal intensities collected at different time points through the instrument software to construct an amplification curve, and judge the results according to the curve situation.

[0058] The results are as Figure 3 shown. When the enzyme amount is 100%, the signal differences between the positive and negative groups are significant. There is an obvious amplification curve and fluorescence enhancement in the positive group, and no obvious amplification signal in the negative group, indicating that this enzyme amount supports the specific amplification of the target DNA and avoids non-specific amplification and background interference. When the enzyme amount is reduced, the amplification curve of the high-concentration (such as 10 8 ) positive target rises slowly and the final intensity is low, indicating that the reduction of the enzyme amount leads to a decrease in the amplification efficiency. In the low-concentration (such as 10 2 ) positive target group, there is no obvious amplification curve at low enzyme amounts. Therefore, 100% enzyme amount is the optimal dosage for this detection system, which can balance high amplification efficiency, detection specificity and signal-to-noise ratio.

[0059] 2) Optimization of reaction temperature: By setting temperature gradients (33 °C, 35 °C, 37 °C, 39 °C and 41 °C), analyze the kinetic curves and amplification efficiencies of the amplification reactions at different temperatures to determine the optimal reaction temperature.

[0060] The results are as Figure 4As shown, at 37°C, the fluorescence signal of the positive target group has a fast rising rate and a high fluorescence intensity at the plateau phase. Meanwhile, no obvious non-specific amplification signal appears in the negative control group, indicating that the RPA reaction has high efficiency and specificity at this temperature. In contrast, when the temperature is lower than 37°C (such as 33°C, 35°C), the amplification efficiency is relatively low, and the fluorescence signal of the negative control group shows a tendency to peak after 15 minutes of amplification. When the temperature is higher than 37°C (such as 39°C, 41°C), although the amplification speed increases, the risk of non-specific amplification increases, and the reaction stability decreases. Therefore, 37°C is determined as the optimal reaction temperature for this RPA detection system, which can minimize non-specific reactions while ensuring efficient amplification, providing reliable temperature conditions for the quantitative detection of fungi.

[0061] 3) Optimization of primer and probe concentrations: By adjusting the concentration ratio of primers and probes, their effects on amplification specificity, sensitivity, and signal-to-noise ratio were evaluated to determine the optimal concentration combination. Four different reaction systems (25 μL) were designed for the experiment, as follows: System 1: 5 μM BF-F1: 1 μL, 5 μM BF-R1: 1 μL, 1 μM BF-P1: 1.5 μL, ddH2O: 2.05 μL, Buffer A: 16.45 μL, target (NC\10 2 \10 8 ): 2 μL, magnesium acetate: 1 μL.

[0062] System 2: 5 μM BF-F1: 0.5 μL, 5 μM BF-R1: 0.5 μL, 1 μM BF-P1: 1.5 μL, ddH2O: 3.05 μL, Buffer A: 16.45 μL, target (NC\10 2 \10 8 ): 2 μL, magnesium acetate: 1 μL.

[0063] System 3: 5 μM BF-F1: 1 μL, 5 μM BF-R1: 1 μL, 1 μM BF-P1: 0.75 μL, ddH2O: 2.8 μL, Buffer A: 16.45 μL, target (NC\10 2 \10 8 ): 2 μL, magnesium acetate: 1 μL.

[0064] System 4: 5 μM BF-F1: 0.5 μL, 5 μM BF – R1: 0.5 μL, 1 μM BF-P1: 0.75 μL, ddH2O: 3.8 μL, Buffer A: 16.45 μL, target (NC\102 \10 8 ): 2 μL, magnesium acetate: 1 μL.

[0065] Reaction conditions: React at 37 °C for 20 min, and collect fluorescence signals every 30 s. Analyze the fluorescence signal intensities collected at different time points through the instrument software to construct an amplification curve, and judge the results according to the curve conditions.

[0066] The experimental results are as Figure 5 shown. When the final concentration of the primer is 200 nM and the probe concentration is 60 nM, the amplification reaction shows the best performance. Under this concentration combination, the fluorescence signal of the positive target group has a fast rising rate and a high fluorescence intensity at the plateau stage. At the same time, no obvious non-specific amplification signal appears in the negative control group, indicating that this condition has excellent selectivity and sensitivity. In addition, the signal-to-noise ratio under this concentration combination is significantly higher than that of other experimental groups, further verifying its reliability.

[0067] Example 3: Broad-spectrum and specific detection test of RPA primers for brewing fungi in the present invention (1) Genome extraction The strains used in the present invention include Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces boulardii, Zygosaccharomyces bailii, Kazachstania servazzii, Torulaspora delbrueckii, Rhizopus nigricans, Rhizopus oryzae, Aspergillus niger, Acetobacter, Lactobacillus paracasei, Limosilactobacillus reuteri, Bacillus amyloliquefaciens, Streptococcus thermophilus, Bacillus subtilis, and Escherichia coli.

[0068] Before using the strains, first streak and isolate them on the corresponding solid medium, culture them overnight at 37 °C for activation, and then pick single colonies and inoculate them into the liquid medium for overnight culture at 37 °C for secondary activation.

[0069] Extract the genomes used in the experiment using the reagent method. The steps are as follows: A. Add about 200 μL of quartz sand to a centrifuge tube, and then add 200 μL of cell lysis buffer, 200 μL of TE buffer, and 200 μL of DNA extraction solution (the ratio of phenol:chloroform:isoamyl alcohol is 25:24:1, product number: p1012). Scrape a tip of bacterial sludge into the above centrifuge tube in a laminar flow hood, cover the centrifuge tube, vortex at high speed for 5 min, and then centrifuge at 12000 r / min for 10 min in a centrifuge; B. Carefully aspirate the supernatant (pay attention to aspirating gently to prevent sucking in proteins), transfer it to a new 1.5 mL EP tube, add 1 mL of absolute ethanol, and invert and mix evenly; C. Centrifuge at 12,000 r / min for 5 min, discard the supernatant to obtain a small amount of precipitate, and leave the centrifuge tube open at room temperature or in a 60 °C oven for 15 - 20 min until there is no ethanol smell, i.e., it is completely dried. D. Add an appropriate volume of ultrapure water to the DNA precipitate extracted from the centrifuge tube (to make the nucleic acid reach a suitable concentration for PCR, generally add 100 μL of ultrapure water) to dissolve it.

[0070] (2) RPA reaction system and conditions The RPA reaction system is the same as in step (2) of Example 2.

[0071] Reaction conditions: Place the RPA reaction tube with all components added into a fluorescence quantitative PCR instrument, react at 37 °C for 20 min, and collect fluorescence signals every 30 s. Analyze the fluorescence signal intensities collected at different time points through the instrument software to construct an amplification curve, and judge the results according to the curve situation.

[0072] The positive group consists of fungal strains from different species, including Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces boulardii, Kazachstania servazzii, and Zygoascus hellenicus. The negative group is a control group with pure water as the target.

[0073] The experimental results are as Figure 6 shown. It can be seen that when the sample to be detected is a fungal strain, even if these strains are from different ecological environments and different species, this method can still stably detect the target fungus. Through the fluorescence quantitative PCR instrument, it can be clearly observed that as time goes by, the fluorescence signal intensity representing the nucleic acid amplification of the target fungus gradually increases. Although there are certain differences in the fluorescence signal intensities corresponding to fungal samples of different genera, they are all in a clearly distinguishable state. Based on these experimental results, the reagents and methods of the present invention are expected to become a general and efficient fungal screening means.

[0074] Additionally, common fungi (Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces boulardii, Zygoascus hellenicus, Kazachstania servazzii, Aspergillus niger), common bacteria (Lactobacillus, Bacillus, Escherichia coli), and viruses (HPV, NoV) during the brewing process were used as test samples for specificity testing. Amplification was carried out according to the fluorescence-based RPA reaction system, with fungi as the positive control to verify the specificity of the fungal universal primer combination. Analyze the peak time and peak value of the fluorescence quantitative PCR instrument program. As Figure 7 shown, the primers only had a positive reaction with fungi, and there was no amplification reaction with Lactobacillus, Bacillus, Escherichia coli, HPV, NoV, or the blank control, proving that the fungal universal primer combination has good specificity.

[0075] Example 4: Sensitivity detection test of the RPA primer pair of the present invention for brewing fungi (1)DNA Preparation: After preparing the DNA template (i.e., the plasmid containing the target) using a plasmid miniprep kit, the template DNA was serially diluted 10-fold with TE buffer to obtain ten gradient samples with concentrations of 10 0 、10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 , accurate to copies / μL. Then, 2 μL of the sample at each dilution was accurately taken and added to the fluorescence RPA amplification system to verify the method sensitivity; the plasmid empty vector was used as the negative control NC group.

[0076] (2)RPA Reaction System and Conditions The RPA reaction system was the same as that in step (2) of Example 2 above.

[0077] RPA Reaction Conditions: The RPA reaction tube containing various components was placed in a fluorescence quantitative PCR instrument and reacted at 37°C for 20 min, and the fluorescence signal was collected every 30 s. The fluorescence signal intensity collected at different time points was analyzed by the instrument software to construct an amplification curve, and the results were judged according to the curve situation.

[0078] (3)The results were as Figure 8 shown. When the concentration of the template DNA changed in a gradient from high to low, the fluorescence signal intensity changed regularly, and there was no amplification in the negative control. At high concentrations such as 10 6 、10 8 、10 10 copies / μL, the fluorescence signal increased rapidly to a relatively high peak value. When the concentration decreased to 10 2 、10 0 copies / μL, although the fluorescence signal weakened, it was still recognizable, indicating that this method could efficiently capture nucleic acid amplification information in this concentration range, with high sensitivity and a wide detection concentration range.

[0079] Further exploring the experimental data, a linear relationship model between time and concentration was constructed. The fluorescence intensity data of each gradient sample at different reaction times were fitted, and a linear regression curve ( Figure 9 )was drawn. Its linear regression equation was y = 23.19048 - 2.02143x, R 2= 0.980 (where y is time, x is the template concentration, and the correlation coefficient R² = 0.980). During subsequent actual detections, based on the real-time fluorescence intensity and the corresponding time, the initial concentration of the template DNA can be quickly calculated, achieving the leap from qualitative to quantitative analysis.

[0080] Example 5: Experiment on detecting different fermented grains samples using the primer set of the present invention (1) Sample pretreatment: Use a sterile sampling tool to collect the fermented grains samples from the fourth round. Immediately place the collected samples into sterile sealed bags and store them in a -20°C refrigerator. Weigh 20 g of each fermented grains sample into a 100 mL centrifuge tube, add 35 mL of sterilized 1×PBS buffer (136 mM NaCl, 2.6 mM KCl, 2 mM KH2PO4, and 8 mM Na2HPO4, pH = 7.2) for suspension and 3 - 5 glass beads, shake vigorously for 7 min, centrifuge at 400 r / min for 5 min, and aspirate the supernatant.

[0081] Wash the precipitate with 1×PBS, vortex for 4 min, centrifuge at 400 r / min for 5 min, and collect the supernatant.

[0082] Repeat the above operation to collect a total of 40 - 50 mL of supernatant. After balancing, centrifuge at 12000 r / min for 5 min, discard the supernatant, and store the precipitate in a -20°C refrigerator.

[0083] (2) Sample DNA extraction: The DNA in the sample is extracted using the silica sand-assisted phenol-chloroform method, and the specific steps are the same as those in step (1) of Example 3.

[0084] (3) The RPA reaction system is the same as that in step (2) of Example 2.

[0085] RPA reaction conditions: Place the RPA reaction tube with all components added into a fluorescence quantitative PCR instrument, react at 37°C for 20 min, and collect the fluorescence signal every 30 s. Analyze the fluorescence signal intensity collected at different time points through the instrument software to construct an amplification curve, and judge the results according to the curve situation.

[0086] (4) After the reaction, draw an amplification curve based on the fluorescence signal data. And according to the standard curve equation y = 23.19048 - 2.02143x fitted in step (3) of Example 4 (where y is time, x is the template concentration, and the correlation coefficient R 2= 0.980), the fungal content in each fermented grains sample was calculated. At the same time, it was compared with the gold standard q-PCR method for detecting fungal content. Specifically, the q-PCR method used the general brewing fungal primers 5'-CAAATTTCTGCCCTATCAACTT-3' (SEQ ID NO:9) and 5'-AACTGCAACAACTTTAATATAC -3' (SEQ ID NO:10) to amplify the DNA of the above-mentioned extracted fermented grains sample. The reaction system was 25 μL, including 10 μL SYBR Premix Ex Taq™ (TaKaRa), 0.8 μL forward / reverse primer (10 μM), 2 μL template DNA, and 11.4 μL nuclease-free water. The real-time fluorescence quantitative PCR system was used: pre-denaturation at 95°C for 5 min, after denaturation for 10 s, annealing at 60°C and extension for 50 s as the conditions, and 40 cycles of amplification detection were carried out. The comparison results are shown in Table 2. It can be concluded that the fungal content calculated by the method of the present invention has good consistency with the q-PCR method and can more accurately reflect the actual fungal content in the fermented grains sample.

[0087] Table 2. Comparison table of quantitative results of q-PCR and q-RPA nucleic acid detections

[0088] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer-probe combination for detecting fungi, characterized in that: It includes primers with nucleotide sequences shown in SEQ ID NO: 1-2 and a single-stranded fluorescent probe with nucleotide sequence shown in SEQ ID NO:

3.

2. The primer-probe combination for detecting fungi according to claim 1, characterized in that: The primer-probe combination is designed based on the conservative region of the 18S rDNA gene of fungi, and the nucleotide sequence of the conservative region is shown in SEQ ID NO:

8.

3. The primer-probe combination for detecting fungi according to claim 1, characterized in that: Tetrahydrofuran is inserted into the single-stranded fluorescent probe sequence as a cleavage site, and a fluorescent group and a quenching group are labeled on both sides of the tetrahydrofuran, with a spacing of 1-10 nt between the fluorescent group and the quenching group, and a modification group is labeled at the 3' end of the single-stranded fluorescent probe; The fluorescent group is selected from FAM, TET, NED, ROX, CY3, CY5, VIC, JOE, HEX, Texas RED, and LC RED460; the quenching group is selected from MGB, TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1, and BHQ2; and the modifying group is selected from an amine group, a phosphate group, and a C3-Spacer.

4. The primer-probe combination for detecting fungi according to claim 3, characterized in that: The fluorescent group is FAM, the quenching group is BHQ, the interval between the fluorescent group and the quenching group is 3 nt, and the modifying group is C3-Spacer.

5. A kit for fungal detection, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that: Primer pair and single-stranded fluorescent probe: The sequence of the primer pair is shown in SEQ ID NO: 1-2; the sequence of the single-stranded fluorescent probe is shown in SEQ ID NO:

3.

7. The kit according to claim 5, characterized in that: Tetrahydrofuran is inserted into the sequence of the single-stranded fluorescent probe as a cleavage site, and fluorescent groups and quenching groups are labeled on both sides of tetrahydrofuran, with a spacing of 1-10 nt between the fluorescent groups and the quenching groups, and a modification group is labeled at the 3' end of the single-stranded fluorescent probe.

8. The kit according to claim 5, characterized in that: The final concentrations of the primers are 0.001 μM to 30 μM; the final concentration of the single-stranded fluorescent probe is 0.001 μM to 20 μM.

9. A method for detecting fungi, characterized in that: The primer-probe combination according to any one of claims 1 to 4 is used to perform RPA detection on the sample to be tested.

10. The method for detecting fungi according to claim 9, characterized in that: The method for detecting fungi comprises the following steps: S1: extracting DNA from the test substance containing fungi to obtain a DNA template; S2: Using the extracted DNA as a template, perform RPA amplification using a primer-probe combination, interpret the results of RPA amplification, and identify the fungi in the sample to be tested.

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