Primer, kit and detection method for rapidly detecting staphylococcus clavatus without heating and application of primer, kit and detection method

Abstract

The invention discloses a primer, a kit and a detection method for rapidly detecting staphylococcus aureus without heating and application of the primer, the kit and the detection method, and belongs to the technical field of pathogenic bacterium detection. The kit for rapidly detecting the staphylococcus aureus, provided by the invention, comprises a primer pair of which the sequences are as shown in SEQ ID NO.19 and SEQ ID NO.28, ELbCas12a enzyme, crRNA (Ribonucleic Acid) as shown in SEQ ID NO.1 and RDNA (Ribonucleic Acid) as shown in SEQ ID NO.5. An RAA reaction system and a CRISPR / Cas12a reaction system are respectively prepared on a tube bottom and a tube cover, and the staphylococcus aureus detection method without heating in the same tube is established. Experimental results show that the detection method disclosed by the invention is extremely high in sensitivity, high in specificity, small in background interference and suitable for on-site rapid detection of the staphylococcus aureus, can detect the staphylococcus aureus to a flying mole level, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, and in particular to a primer, a kit, a detection method and an application thereof for rapid detection of Staphylococcus cladocerus without heating. Background Art

[0002] Spiderweb disease is considered one of the four most serious fungal diseases of edible mushrooms, occurring in numerous countries and regions, causing significant economic losses. The pathogen is a member of the genus Cloadobotryum, with common species including C. mycophilum, C. dendroides, C. produsum, and C. varium. Spiderweb disease hyphae first appear in the covering substrate or soil, gradually infecting edible fungi. The pathogen has a wide range of hosts, including shiitake mushrooms, king oyster mushrooms, coprinus comatus, oyster mushrooms, morels, black fungus, and Ganoderma lucidum. Early monitoring and control techniques for spiderweb disease in large-scale cultivated edible and medicinal mushrooms are crucial for improving the yield and quality of these products.

[0003] Traditional detection techniques, such as culture identification and vector culture, are only applicable to culturable microorganisms. Furthermore, the incubation period typically requires at least 2-3 days, resulting in poor effectiveness and accuracy. Modern pathogen detection methods, including enzyme-linked immunosorbent assays (ELISAs), lateral flow immunoassays, and polymerase chain reaction (PCR), are difficult to implement in on-site testing due to their high cost and the need for specialized instrumentation.

[0004] After binding to crRNA, clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) specifically recognize target DNA with a protospacer adjacent motif (PAM), then specifically activate their nuclease activity, hydrolyzing both double-stranded DNA (cis-cleavage) and single-stranded DNA (trans-cleavage). The trans-cleavage activity can cleave large amounts of single-stranded DNA in a short period of time, independent of sequence specificity, thereby amplifying the experimental signal. Combining the specific sequence recognition of crRNA with the trans-cleavage activity of Cas12a, the CRISPR / Cas12 system is widely used in nucleic acid detection. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP), rolling circle nucleic acid amplification (RCA), recombinase polymerase amplification (RPA), and recombinase-mediated isothermal amplification (RAA), can amplify nucleic acid molecules at a constant temperature. Compared to traditional PCR, it does not require a thermal cycler, is simpler to operate, has a faster reaction speed, and is more sensitive. Combining this with CRISPR / Cas12a technology enables highly sensitive and rapid detection of pathogens. However, the combination of isothermal amplification and CRISPR / Cas12a requires a heated instrument to maintain a reaction temperature between 35-42°C to ensure rapid enzyme digestion and a high detection limit. This significantly limits the rapid and sensitive detection technology that can be deployed in the field under limited experimental conditions. Therefore, there is an urgent need for a rapid, highly sensitive pathogen detection method that can complete the amplification reaction at room temperature without the need for heated instruments. Summary of the Invention

[0005] The purpose of the present invention is to provide a primer, a kit, a detection method and an application thereof for rapid detection of C. truncatum without heating, so as to solve the problems existing in the above-mentioned prior art. The present invention is based on specific primers, ELbCas12a enzyme, crRNA and rDNA to develop a rapid detection method of C. truncatum without heating, which has extremely high sensitivity, strong specificity and low background interference, and can be used for on-site detection of C. truncatum.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a primer pair for rapid detection of Staphylococcus cladocerus, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.19 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.28.

[0008] The present invention also provides an application of the primer pair in preparing a kit for rapidly detecting Stenotrophomonas cladocerus.

[0009] The present invention also provides a kit for rapid detection of C. cladocerus, characterized in that it comprises the above-mentioned primer pair, Cas12a enzyme, crRNA and RDNA.

[0010] Furthermore, the Cas12a enzyme is ELbCas12a;

[0011] The nucleotide sequence of the crRNA is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the RDNA is shown in SEQ ID NO.5.

[0013] Furthermore, the concentration of ELbCas12a is 100-125 μM;

[0014] The molar ratio of the ELbCas12a to the crRNA is 1:(1.5-2).

[0015] The present invention also provides an application of the primer pair or the kit in detecting Staphylococcus aureus.

[0016] The present invention also provides a method for rapid detection of Staphylococcus aureus, comprising the steps of performing RAA amplification reaction and CRISPR / Cas12a reaction using the above-mentioned kit.

[0017] Further, the following steps are included:

[0018] Take a reaction tube and prepare the RAA system at the bottom of the reaction tube;

[0019] Prepare the CRISPR / Cas12a system on the tube cover of the reaction tube;

[0020] Add the DNA sample to be tested to the reaction tube, close the tube cap, react at 25°C for 40 minutes, centrifuge, react at 25°C for 60 minutes, and read the results;

[0021] Wherein, the RAA system comprises: RAA premixed enzyme, buffer I, the primer pair and a Saccharomyces cerevisiae DNA template;

[0022] The CRISPR / Cas12a system includes: the ELbCas12a, the crRNA, the RDNA and a reaction buffer.

[0023] Furthermore, the RAA premixed enzyme includes a recombinase, a single-strand binding enzyme, and an amplification enzyme;

[0024] The buffer solution I is 25 mM Mg(CH3COO)2 solution;

[0025] The reaction buffer included 40 nM NaCl, 12.5 nM MgCl2, 7.5 nM Tris-HCl, and 75 μg / mL albumin.

[0026] Furthermore, the reading result is performed by ultraviolet irradiation method or lateral flow test strip method;

[0027] When the ultraviolet irradiation method is used, the 5' end of the rDNA is connected to the 6-FAM group, and the 3' end is connected to the BHQ1 group;

[0028] When the lateral flow test strip method is used, the 5' end of the RDNA is connected to a 6-FAM group, and the 3' end is connected to a Biotin group.

[0029] The present invention discloses the following technical effects:

[0030] The present invention provides a kit for rapid detection of Saccharomyces cerevisiae, comprising a specific primer pair, a Cas12a enzyme variant with good trans-cleavage activity at room temperature, and optimized crRNA and rDNA. By preparing the RAA reaction system and the CRISPR / Cas12a reaction system at the bottom of the tube and the tube cover, respectively, the present invention establishes a method for detecting Saccharomyces cerevisiae without heating in the same tube. Experimental results show that the detection method of the present invention is extremely sensitive, can be detected to the femtomolar level, has strong specificity, and has low background interference. It is suitable for on-site rapid detection of Saccharomyces cerevisiae and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Schematic diagram of the RAA-ELbCas12a-RT reaction; A represents the RAA test amplification of DNA at room temperature; B represents CRISPR / Cas12a recognizing the RAA amplification product, and Cas12a activates and cuts the probe DNA to produce a fluorescent signal; C represents the visual detection of RAA-Cas12a-RT combined with lateral flow immunochromatography;

[0033] Figure 2 This is the result of the alignment of ITS sequences of four common Staphylococcus species;

[0034] Figure 3 Statistical graph of the trans-cleavage activity of Cas12a variants at different temperatures; A is 21°C; B is 23°C; C is 25°C; D is 27°C;

[0035] Figure 4 This is a graph showing the results of the crRNA sequence optimization experiment;

[0036] Figure 5Figure 1 shows the results of the CRISPR / ELbCas12a enzyme digestion reaction condition optimization experiment; A shows the ELbCas12a concentration optimization experiment; B shows the ELbCas12a to crRNA1 ratio optimization experiment;

[0037] Figure 6 This is the result of RAA primer screening experiment;

[0038] Figure 7 The figure shows the results of the RAA reaction system optimization experiment at room temperature. A is the result of the reaction system size optimization; B is the result of the reaction time optimization.

[0039] Figure 8 Figure 1 shows the sensitivity test results of the RAA-CRISPR / ELbCas12a one-tube method for detecting S. clavatus at room temperature; A shows the amplification product obtained by amplifying S. clavatus genomic DNA by the RAA method; B shows the fluorescence detection schematic diagram of CRISPR / Cas12a for detecting S. clavatus;

[0040] Figure 9 This is the result diagram of the specificity test experiment;

[0041] Figure 10 This is the experimental result of background interference test;

[0042] Figure 11 This is the result of the immunolateral flow chromatography probe concentration screening experiment;

[0043] Figure 12 This is a visualization result diagram of the detection of Staphylococcus aureus;

[0044] Figure 13 This is the experimental result of real sample test. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] The present invention provides a rapid detection method for edible fungi spider web disease pathogenic fungus Gram-negative fungus without heating, the process is as follows: Figure 1 As shown, the following process is included:

[0051] (1) RAA amplification of specific DNA sequences of S. clavatum at room temperature;

[0052] (2) The low-temperature-tolerant Cas12a variant ELbCas12a cuts specific single-stranded DNA probes to achieve visual detection. Among them, visualization can be achieved by handheld UV light or lateral flow test strips.

[0053] The present invention provides a kind of rapid detection primer for edible and medicinal fungi spider web disease pathogenic fungus branch grape mold, detection method and application.This method utilizes the Cas12a enzyme variant with good trans-cutting activity at room temperature to realize enzyme digestion reaction without any heating operation, combines the RAA reaction system optimized at room temperature, realizes the pathogen detection method without any heating operation in the same tube, can realize pathogen detection result visualization within 1h, simplifies the operation steps, breaks through the limitation that CRISPR / Cas12a system needs heating instrument for pathogen detection.The method provided by the present invention has high sensitivity, can detect to femtomolar level and background interference is small, and the signal-to-noise ratio of detecting fungal pathogen branch grape mold in the host sample of fungus is greater than 9.0, and has huge application prospects in resource-limited on-site rapid detection.

[0054] Buffer I of the present invention is a 25mM Mg(CH3COO)2 solution.

[0055] The reaction buffer of the CRISPR / Cas12a reaction system of the present invention comprises 40 nM NaCl, 12.5 nM MgCl2, 7.5 nM Tris-HCl and 75 μg / mL albumin.

[0056] The RAA premixed enzyme lyophilized powder of the present invention comprises recombinase, single-chain binding enzyme and amplification enzyme.

[0057] Example 1 Establishment of a method for detecting CRISPR / Cas12a with low temperature tolerance

[0058] 1. Experimental Methods

[0059] 1.1 crRNA Design

[0060] The ITS sequences of four common species of C. loadobotryum, including C. dendroides, C. mycophilum, C. produsum, and C. varium, were compared, and the 597 bp ITS sequence of C. mycophilum was used as a numbering reference. The four species of C. varium were consistent in the sequence of 216-375 bp, such as Figure 2 Four different crRNAs were designed based on the ITS sequence, and their sequence information is shown in Table 1.

[0061] Table 1 Nucleotide sequence information

[0062]

[0063] Note: In actual application, if the UV irradiation method is selected to read the results, the fluorescent detection probe F-RDNA is used, and the 5' end of F-RDNA is connected to the 6-FAM group and the 3' end is connected to the BHQ1 group;

[0064] If the lateral flow test strip method is chosen to read the results, the lateral flow chromatography probe L-RDNA is used, with the 5' end of the L-RDNA connected to the 6-FAM group and the 3' end connected to the Biotin group.

[0065] 1.2 Screening of Cas12a enzyme species with low temperature tolerance

[0066] Wild-type Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a, purchased from New England Biotechnology (Beijing) Co., Ltd., catalog number #M0653T), engineered double-site mutant Acidaminococcus sp. Cas12a (EAsCas12a, purchased from Synthetic DNA Technology, catalog number 10001272), and LbaCas12a (ELbCas12a, purchased from Synthetic DNA Technology, catalog number 10007922) were selected as the Cas12a enzymes to be tested, and the above three Cas12a enzymes were subjected to CRISPR / Cas12a enzyme digestion reactions at 21, 23, 25, and 27 ° C, respectively.

[0067] The CRISPR / Cas12a digestion system (25 μL) mainly includes: Cas12a (100 nM), crRNA1 (150 nM), 2.5 μL NEBuffer r2.1 (10×), 1 μL of Saccharomyces cerevisiae template (10 nM), 1 μL of F-rDNA (10 μM), and enzyme-free sterile water to make up the volume. Enzyme-free sterile water was used as a negative control. After rapid mixing, centrifugation at 3000 rpm for 10 seconds was performed, and the cells were placed in a real-time fluorescence quantitative PCR instrument and reacted at different temperatures for 90 minutes. The fluorescence value was recorded every minute.

[0068] 1.3 CRISPR / Cas12a reaction optimization

[0069] Optimize crRNA: Four strains of Saccharomyces cerevisiae were selected as templates, and crRNAs 1-4 were used to test crRNA specificity for Saccharomyces cerevisiae. ElbCas12a was used for the experiment, and the CRISPR / Cas12a enzyme digestion system was the same as above. Incubate in a real-time fluorescence PCR instrument at 25°C for 60 minutes, recording fluorescence every minute.

[0070] ELbCas12a concentration optimization: The reaction system was set to 25 μL, with a fixed ELbCas12a to crRNA ratio of 1:1.5. Different concentrations of ELbCas12a (50, 75, 100, 125, and 150 nM), 2.5 μL NEBuffer r2.1 (10×), 1 μL of cleavage of the ...

[0071] Optimization of the ratio of ELbCas12a to crRNA: The reaction system was set to 25 μL, the ELbCas12a concentration was fixed at 100 nM, and the ratios of ELbCas12a to crRNA were set to 1:0.75, 1:1, 1:1.5, 1:1.75, and 1:2, respectively, as well as 2.5 μL NEBufferr2.1 (10×), 1 μL of cleavage mold template (1 nM), 1 μL F-RDNA (10 μM), and enzyme-free sterile water to make up the volume. The reaction tube was mixed and centrifuged, and incubated at 25 ° C for 90 min in a real-time fluorescence quantitative PCR instrument, and the fluorescence value was recorded every minute.

[0072] 2. Experimental Results

[0073] 2.1 Selection of Cas12a enzymes with low temperature tolerance

[0074] Three Cas12a enzymes including LbaCas12a, EAsCas12a, and ELbCas12a were tested for trans-cleavage at different temperatures below 30°C. The results are as follows Figure 3 As shown. The ELbCas12a enzyme showed the highest fluorescence value at 21, 23, 25, and 27 ° C. The end point fluorescence value at 21-25 ° C was similar, all around 4000 ( Figure 3 AC), and when the temperature was raised to 27℃, the reaction was significantly enhanced ( Figure 3 (D) shows that the ELbCas12a enzyme activity is relatively stable at 21-25°C, which is suitable for maintaining stable detection performance without heating the instrument.

[0075] The cleavage effects of the three enzymes were observed at room temperature (25°C). Figure 3 In Figure C), ELbCas12a>LbaCas12a, and the fluorescence signal increased by more than 35%. However, when there was no target present, EAsCas12a showed nonspecific cleavage over time and was not suitable for pathogen detection. ELbCas12a was ultimately selected as the Cas12a with low temperature tolerance for subsequent studies.

[0076] 2.2 crRNA Selection

[0077] Four crRNAs were used as templates to detect common cladoplasts including cladoplasts, bacteriotrophic cladoplasts, convex cladoplasts and heteromorphic cladoplasts. The results are as follows Figure 4 As shown in the figure, four crRNAs were used for rapid detection of four common species of the genus Saccharomyces. The fluorescence value after 60 minutes of reaction was used as the evaluation indicator. crRNA1 recognized all four species of Saccharomyces with a significant fluorescence signal enhancement, while crRNAs 2-4 only recognized one or two of the species. crRNA1 was selected as the universal crRNA for detecting Saccharomyces.

[0078] 2.3 CRISPR / ELbCas12a reaction system optimization

[0079] The results of ELbCas12a concentration optimization are as follows Figure 5 As shown in Figure A, ELbCas12a can effectively achieve fluorescence detection of S. truncatula in the concentration range of 50-125 nM. As the concentration increases, the fluorescence value increases, while the enzyme digestion efficiency decreases under the condition of 150 nM. Among them, the enzyme digestion effect is similar at a concentration of 100-125 nM, and the fluorescence value is the highest.

[0080] The ratio of ELbCas12a to crRNA1 affects the efficiency of CRISPR / ELbCas12a enzyme digestion reaction, that is, the speed of generating fluorescent signal. Figure 5 As shown in B, the optimization experiment results show that fluorescence signals can be generated under the conditions of 1:0.75-1:2, but the effect is better when the ratio is between 1:1.5-1:2, among which 1:1.75 has the fastest reaction.

[0081] Example 2 One-tube detection of RAA binding to CRISPR / ELbCas12a at room temperature

[0082] 1. Experimental Methods

[0083] The optimal reaction temperature of RAA is 39-43 ° C. The present invention needs to meet the requirement of no instrument heating throughout the process. It is necessary to perform RAA testing and optimization at room temperature (taking 25 ° C as an example) to improve the sensitivity of reaction detection. In order to reduce the operating steps and the risk of aerosol contamination, a one-tube test method for rapid detection of RAA-ELbCas12a-RT-based branched mold at room temperature was developed. Based on the characteristics of the RAA reaction, the RAA system (25 μL) was used as the main reaction at the bottom of the tube, and the ELbCas12a system (5 μL) was used as the side reaction of the tube cover.

[0084] Room temperature RAA system (25 μL) in the reaction tube: According to the instructions of the RAA kit (purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd., catalog number T00000, 48T), each 50 μL system contains 25 μL buffer V, 5 μL buffer I (Mg 2+ ), 15 μL enzyme-free sterile water, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM) and 1 μL DNA template.

[0085] The room temperature ELbCas12a system (5 μL) in the reaction tube cap contained 100 nM ELbCas12a, 175 nM crRNA1, 400 nM F-RDNA, reaction buffer (1×), and enzyme-free sterile water to make up the volume.

[0086] Procedure: Add 2.5 μL of the DNA solution to be tested to the bottom of the reaction tube and gently close the cap. After reacting at 25°C for 40 minutes in a real-time fluorescence quantitative PCR instrument, centrifuge (shake the reaction tube) to allow the ELbCas12a system in the tube cap to mix with the RAA system in the tube. React at 25°C for 60 minutes and record the fluorescence value.

[0087] 1.1 RAA primer screening

[0088] Using the conserved region of the ITS sequence as the target region, forward and reverse primers were designed and combined to screen RAA primer pairs for detecting S. clavatus. Sequence information is shown in Table 2. The forward primer has the optimal PAM sequence (TTTG) of Cas12a added to the 5′ end. The room temperature RAA reaction was performed using the above-mentioned "room temperature RAA system in the reaction tube (25 μL)", and the product was examined by 2% agarose gel electrophoresis to examine the production of RAA products.

[0089] 2% agarose gel electrophoresis method: Weigh agarose powder and add it to TBE buffer (1×) and mix thoroughly to prepare a 2% (w / v) agarose gel. Heat in a microwave oven three times to fully dissolve the agarose. After cooling, add 5% (v / v) SYBR Green I (10000×), mix thoroughly, pour into a gel mold, and cool to room temperature to solidify. Take 2.5μL of RAA product and add 0.5μL of 6× DNA loading buffer and mix thoroughly. Take 2μL and 2μL of DNA molecular weight marker (100-2000bp) and perform electrophoresis at 135V for 60 minutes. Image the gel using a gel imager.

[0090] Table 2 RAA primer sequence information

[0091]

[0092]

[0093] 1.2 RAA reaction volume optimization

[0094] The one-tube method for rapid detection of S. clavatus based on RAA-ELbCas12a-RT was optimized, and 25 and 50 μL of the RAA system were tested. The room temperature RAA system (25 μL) in the reaction tube contained 1 / 2 RAA premixed enzyme lyophilized powder, 12.5 μL buffer V, 2.5 μL buffer I, 7.5 μL enzyme-free sterile water, 1 μL F4b (10 μM) and 1 μL R3 (10 μM), and 0.5 μL S. clavatus template (100 fM).

[0095] The specific operation is as follows: First, add buffer V, enzyme-free sterile water, and buffer I to the lyophilized premixed enzyme. Use your fingers to gently shake and mix to fully dissolve the lyophilized enzyme. According to the experiment, divide it into 25 and 50 μL, add the corresponding volume of primers and DNA amplification template respectively, react at 25°C for 30 minutes, and then add the ELbCas12a enzyme digestion system to react, and record the fluorescence value.

[0096] 1.3RAA response time

[0097] The room temperature RAA reaction system was set to 25 μL, and the DNA template solution was selected from different concentrations of S. clavatum (100 fM, 1 pM, 10 pM, and 100 pM), and the reaction was carried out at 25° C. for 20, 40, and 60 min, respectively. The room temperature RAA product was analyzed by 2% agarose gel electrophoresis.

[0098] 2. Experimental Results

[0099] 2.1 RAA primer optimization

[0100] This study designed 20 forward primers and 4 reverse primers, using different pairing combinations to perform RAA amplification at room temperature. Primers numbered ad represent identical primers with the same initiation site, differing in length. RAA primers between 30 and 35 bp are effective for amplification, but with increasing length, differences in the resulting RAA products are observed. Optimizing the site design and length of RAA primers is essential.

[0101] According to the gel map ( Figure 6 The results showed that, when using reverse primers R2-R4 with the forward primer, most amplicons did not produce a single band of the target molecular weight, indicating nonspecific amplification. The products of the combinations F1a-F1d, F2a-F2d, and F4a-F4d with R1 each produced a single band with high yields. Furthermore, the combination F4b + R3 also produced a clear single band. Considering the product length and the length and location of the conserved ITS sequence of S. clavatum, the optimal primer selection was F4b + R3.

[0102] 2.2 Optimization of room temperature RAA reaction system

[0103] The RAA reaction system is generally 50 μL. In order to achieve the reaction of RAA and CRISPR / ELbCas12a in one tube at room temperature, the ELbCas12a system is reduced to 5 μL and shaken to the bottom of the tube by centrifugation. The effective enzyme concentration is increased while the reaction system volume remains unchanged. Excessive product amount can easily lead to the ineffective binding of the ELbCas12a-crRNA complex and the RAA product and inhibit enzyme activity, thereby reducing the rate of fluorescence signal generation. Figure 7As shown in Figure A, the fluorescence signal value of the 25 μL RAA reaction system is much higher than that of the 50 μL RAA system at the same reaction time. Therefore, the final optimization is to first perform a 25 μL RAA reaction at room temperature, and then shake the 5 μL ELbCas12a system to the bottom of the tube by centrifugal shaking to achieve fluorescence signal detection.

[0104] 2.3 Optimization of RAA reaction time in the room temperature one-tube method

[0105] Different concentrations of S. clavatum DNA template (100 fM, 1 pM, 10 pM and 100 pM) were used to carry out RAA reaction at room temperature, and the products were observed by agarose gel electrophoresis. Figure 7 As shown in Figure B, a 60-minute reaction time allows detection down to 100 fM. For rapid detection, a reaction time between 20 and 40 minutes is generally chosen. When the reaction time is set to 40 minutes, a band is observed at a DNA concentration of 1 pM. Considering both reaction time and detection limit, a 40-minute RAA reaction time at room temperature was ultimately selected.

[0106] Example 3 Performance test of the rapid detection method (RAA-ELbCas12a-RT) of Stem Cells without heating

[0107] 1. Experimental Methods

[0108] The RAA-ELbCas12a-RT-based one-tube rapid detection method for Staphylococcus aureus at room temperature includes:

[0109] A room temperature RAA system (25 μL) was set up at the bottom of a 200 μL reaction tube, including 1 / 2 RAA premixed enzyme lyophilized powder, 12.5 μL buffer V, 2.5 μL buffer I, 7.5 μL enzyme-free sterile water, 1 μL F4b (10 μM) and 1 μL R3 (10 μM) and 0.5 μL of Saccharomyces cerevisiae DNA template (100 fM).

[0110] A room temperature ELbCas12a system (5 μL) was set up in the tube cap, containing 100 nM ELbCas12a, 175 nM crRNA1, 400 nM F-RDNA, reaction buffer (1×), and enzyme-free sterile water to make up the volume.

[0111] Procedure: Add 2.5 μL of the DNA solution to be tested to the bottom of the reaction tube and gently close the cap. After reacting at 25°C for 40 minutes in a real-time fluorescence quantitative PCR instrument, centrifuge (shake the reaction tube) to allow the ELbCas12a system in the tube cap to mix with the RAA system in the tube. React at 25°C for 60 minutes and record the fluorescence value.

[0112] 1.1 Sensitivity test

[0113] RAA-ELbCas12a-RT assays were performed using LbaCas12a and ELbCas12a as Cas12a enzymes. DNA concentrations were set to 0, 0.1, 1, 10, and 100 fM. After reacting at 25°C for 40 minutes, the room-temperature RAA was mixed into the CRISPR / ELbCas12a system and the reaction continued for 90 minutes. The fluorescence signal was recorded.

[0114] Similarly, the DNA concentrations were set to 0, 1aM, 10aM, 100aM, 1fM, 10fM, and 100fM, respectively. Room temperature RAA was reacted at 25°C for 40 minutes, then mixed into the CRISPR / ELbCas12a system and continued to react for 90 minutes, and the fluorescence signal value was recorded.

[0115] 1.2 Specificity test

[0116] Specificity testing was performed using DNA extracts from seven strains not tested, including Trichoderma viren, T. lixii, T. harzianum, T. pleuroticola, Paecilomyces variotii, Alternaria alternata, and T. variabilis. Enzyme-free sterile water was used as the NTC, and a DNA extract from Staphylococcus aureus served as a positive control. The RAA was reacted at room temperature at 25°C for 40 minutes, then mixed with the CRISPR / ELbCas12a system and continued to react for 90 minutes. Fluorescence signal was recorded.

[0117] 1.3 Background interference test

[0118] Crude DNA extracts from Lentinula edodes, Morchella esculenta, and Ganoderma tsugae were used as potential host backgrounds in the Cladosporium test. To 90 μL of crude DNA extracts (10 ng / μL) from Lentinula edodes, Morchella esculenta, and Ganoderma tsugae, 10 μL of a Cladosporium extract (10 ng / μL) was added as the test sample for background interference testing. Enzyme-free sterile water was used as the negative control (NTC), and a DNA extract from a Cladosporium extract was used as a positive control. After reacting the room-temperature RAA at 25°C for 40 minutes, the CRISPR / ELbCas12a system was added and the reaction continued for 90 minutes. The fluorescence signal was recorded.

[0119] 2. Experimental Results

[0120] 2.1 Sensitivity test

[0121] The detection sensitivity results of ELbCas12a and wild-type LbaCas12a binding to RAA at room temperature are as follows Figure 8 As shown in A. The fluorescence signal detected by ELbCas12a is higher than that of LbaCas12a. LbaCas12a and ELbCas12a respectively detected the 90min fluorescence endpoint value of Bacillus subtilis. The results showed that compared with the negative control (0fM), ELbCas12a can detect as low as 0.1fM, while LbaCas12a detects at least 1fM. Compared with the two, ELbCas12a has a greater change in relative fluorescence intensity and a lower detection limit, indicating better low-temperature tolerance.

[0122] The sensitivity results of the rapid detection method of C. truncatula at room temperature using RAA-ELbCas12a-RT are as follows: Figure 8 As shown in Figure B. The detection limit was further analyzed using 0, 1aM, 10aM, 100aM, 1fM, 10fM, and 100fM solutions of S. clavatum. Fluorescence-time curves showed that the enzyme digestion reaction accelerated with increasing DNA concentration in the S. clavatum solution, with the lowest detectable concentration being as low as 1fM.

[0123] 2.2 Specificity test

[0124] Seven common non-target fungal DNA extracts were used to detect S. clavatus by RAA-ELbCas12a-RT, with enzyme-free sterile water as NTC and S. clavatus DNA extract as positive control. The results are shown in Figure 9 Compared with NTC, the relative fluorescence signal of C. cladodes was significantly enhanced, 8.7 times that of NTC, while no significant increase in the fluorescence intensity of non-target DNA extracts was observed. The fluorescence signal of C. cladodes was more than 5.5 times that of non-target DNA, indicating that RAA-ELbCas12a-RT has extremely high specificity in detecting C. cladodes.

[0125] 2.3 Background interference test

[0126] The ability of RAA-ELbCas12a-RT to detect Sclerotium was tested using DNA extracts from three common edible and medicinal mushroom hosts, Lentinus edodes, Morchella oleracea, and Ganoderma lucidum, as interference background in the Sclerotium test. Compared with the negative control, the fluorescence intensity of the DNA extract of Sclerotium oleracea was 13.9 times that of the negative control, and the fluorescence signal increased significantly. Figure 10 shown.

[0127] The fluorescence signal values ​​of DNA extracts from Lentinus edodes, Morchella oleracea, and Ganoderma lucidum containing the fungus S. cladocercum were 9.3, 13.7, and 9.9 times higher than those of their negative controls without the target, respectively. The results showed that in samples of host edible fungi mixed with S. cladocercum, RAA-ELbCas12a-RT was less susceptible to background interference in detecting S. cladocercum signals, making it easier to distinguish the fluorescence signals of positive samples from those of negative samples.

[0128] Example 4 Visualization of RAA-ELbCas12a-RT

[0129] 1. Experimental Methods

[0130] 1.1 Lateral flow immunochromatography (LFA)

[0131] When there is DNA from the branched fungus, the ELbCas12a trans-cleavage activity is activated, cutting the ssDNA probe (L-RDNA, 5′-FAM-ssDNA-Biotin-3′, Table 1), that is, the FAM group is separated from the Biotin group. The solution passes through the sample absorption area FAM binds to the anti-FAM antibody to form a FAM+FAM antibody-AuNPs complex and Biotin. After the C line area, Biotin is captured by streptavidin, and the FAM+FAM antibody-AuNPs complex continues forward. In the T line area, the anti-FAM antibody is captured by the IgG antibody, and AuNPs aggregate in the T line area, showing a purple-red line, indicating that the sample is positive. When RDNA2 is not completely cut, there is a FAM+FAM antibody-AuNPs complex, Biotin, and Biotin-ssDNA-FAM+FAM antibody-AuNPs complex, so that AuNPs are aggregated in both the C line and the T line, showing a purple-red line, indicating that the sample is positive. The concentration of probe RDNA2 in the ELbCas12a enzyme digestion system was optimized. The final concentrations of RDNA2 in the ELbCas12a enzyme digestion system were set to 100pM, 1nM, 10nM, 20nM, 40nM, 60nM, 80nM, 100nM, 200nM, 400nM, 600nM, 800nM, 1μM, 10μM and 20μM, respectively. After the reaction was completed, 30μL of enzyme-free sterile water was added to the total reaction system (30μL), mixed and inserted into the LFA test strip, and the test results were observed with the naked eye after 5min and photographed.

[0132] 1.2 Fluorescence detection visualization

[0133] Fluorescence detection was performed based on CRISPR / Cas12a using double-end modified ssDNA (F-RDNA, 5′-FAM-ssDNA-BHQ1-3′, Table 1). The released FAM fluorescent group has a high absorption peak in the ultraviolet light region. Rapid visualization of the reaction tube can be achieved using a gel imager or a handheld UV lamp (365nm). The DNA concentrations of R. clavatum were set to 0, 100aM, 1fM, 10fM, 100fM, 1pM, and 10pM, respectively. After reacting at room temperature for 40 minutes at 25°C, RAA was mixed into the CRISPR / Cas12a system and the reaction continued for 60 minutes. The reaction tube was illuminated with a handheld UV lamp under natural light and photographed.

[0134] 1.3 Real Sample Testing

[0135] To simulate real samples, crude DNA extracts of Lentinus edodes, Morchella oleracea, and Ganoderma lucidum were spiked with either Staphylococcus aureus or Trichoderma harzianum, respectively, for a total of 14 samples. The specific simulated infection conditions are shown in Table 3. Visual detection was performed using LFA. The reaction tubes were photographed and read using LFA test strips.

[0136] Table 3 Simulated real sample infection

[0137]

[0138] 2. Experimental Results

[0139] 2.1 LFA Visualization Method

[0140] By introducing LFA to visualize RAA-ELbCas12a-RT, a fully instrument-free visualization detection method was developed. The results of the probe L-RDNA concentration optimization are shown in Figure 2. Figure 11 As shown. Optimizing the concentration of the double-end modified ssDNA probe (5′-FAM-ssDNA-Biotin-3′) can avoid unclear bands and false positive double-line results. If the RDNA2 concentration is too low, it cannot completely bind to the FAM antibody-AuNPs on the sample pad. The excess FAM antibody-AuNPs will be captured by the IgG antibody on the T line, resulting in a false positive; if the RDNA2 concentration is too high, the ELbCas12a enzyme digestion is incomplete, which will also cause false positive results. When the concentration is in the range of 20nM-400nM, a clear C line will be formed in the negative sample. As the concentration increases, the line width increases. When the concentration exceeds 100nM, the C line is wide and diffuses. Finally, considering the cost and visualization effect, the final concentration of L-RDNA was selected to be 20-100nM for subsequent experiments.

[0141] When the DNA concentration of S. clavatus reaches 10 fM, a clear C line and a fuzzy T line can be observed, indicating a positive result. As the DNA concentration of S. clavatus increases, the T line becomes clearer and a positive result can be identified by the naked eye ( Figure 12 ).

[0142] 2.2 Fluorescence visualization

[0143] To simplify the requirements of the detection instrument, a handheld UV lamp was used to irradiate the reaction tube under natural light and under light-proof conditions, and a smartphone was used to take pictures. The results are as follows: Figure 12 Without the use of commercial instruments or light protection measures, a handheld UV lamp can be used to illuminate the reaction tube directly, and DNA samples above 10 fM can be resolved.

[0144] 2.3 Real Sample Testing

[0145] The simulated real samples were tested by RAA-ELbCas12a-RT, and the LFA test results were as follows Figure 13 As shown. Samples 4-10 simulated real samples in which crude DNA extracts from Lentinus edodes, Morchella oleracea, and Ganoderma lucidum were mixed with Staphylococcus brevicornis. Clear T lines (positive signals) were observed in samples 4-10. Samples 11-14 simulated real samples in which crude DNA extracts from Lentinus edodes, Morchella oleracea, and Ganoderma lucidum were mixed with Trichoderma harzianum. No clear positive signals were observed in any of these samples, demonstrating good specificity when testing real samples.

[0146] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A primer pair for rapid detection of Staphylococcus aureus, characterized in that: It includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.19 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

28.

2. Use of the primer pair according to claim 1 in preparing a kit for rapid detection of Staphylococcus aureus.

3. A kit for rapid detection of Staphylococcus aureus, characterized in that Comprise the primer pair of claim 1, Cas12a enzyme, crRNA and rDNA.

4. The kit according to claim 3, wherein The Cas12a enzyme is ELbCas12a; The nucleotide sequence of the crRNA is shown in SEQ ID NO.1; The nucleotide sequence of the RDNA is shown in SEQ ID NO.

5.

5. The kit according to claim 4, wherein The concentration of ELbCas12a is 100-125 μM; The molar ratio of the ELbCas12a to the crRNA is 1:(1.5-2).

6. Use of the primer pair according to claim 1 or the kit according to any one of claims 3 to 5 in detecting Staphylococcus aureus.

7. A method for rapid detection of Staphylococcus aureus, characterized in that The method comprises the steps of performing RAA amplification reaction and CRISPR / Cas12a reaction using the kit according to any one of claims 3 to 5.

8. The method according to claim 7, wherein The following steps are involved: Take a reaction tube and prepare the RAA system at the bottom of the reaction tube; Prepare the CRISPR / Cas12a system on the tube cover of the reaction tube; Add the DNA sample to be tested to the reaction tube, close the tube cap, react at 25°C for 40 minutes, centrifuge, react at 25°C for 60 minutes, and read the results; Wherein, the RAA system comprises: RAA premixed enzyme, buffer I, the primer pair and a Saccharomyces cerevisiae DNA template; The CRISPR / Cas12a system includes: the ELbCas12a, the crRNA, the RDNA and a reaction buffer.

9. The method according to claim 8, wherein The RAA premixed enzyme includes a recombinase, a single-chain binding enzyme and an amplification enzyme; The buffer solution I is 25 mM Mg(CH3COO)2 solution; The reaction buffer included 40 nM NaCl, 12.5 nM MgCl2, 7.5 nM Tris-HCl, and 75 μg / mL albumin.

10. The method according to claim 8, wherein The reading result is a UV irradiation method or a lateral flow test strip method; When the ultraviolet irradiation method is used, the 5' end of the rDNA is connected to the 6-FAM group, and the 3' end is connected to the BHQ1 group; When the lateral flow test strip method is used, the 5' end of the RDNA is connected to a 6-FAM group, and the 3' end is connected to a Biotin group.

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