Kit for rapidly detecting mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method, application and method

Abstract

The invention discloses a kit, application and method for rapidly detecting mycoplasma synoviae based on an ERA-CRISPR / Cas12a one-step method, according to the visual detection kit, on the basis of a new nucleic acid detection target scpA of the mycoplasma synoviae, a sample to be detected is split by a splitting buffer to release genome DNA, then ERA-CRISPR / Cas12a one-pot reaction is carried out in a constant-temperature water bath kettle or a metal bath, and the sample to be detected is subjected to one-pot reaction, so that the ERA-CRISPR / Cas12a one-pot reaction is carried out to rapidly detect the mycoplasma synoviae through the ERA-CRISPR / Cas12a one-pot reaction. Under the mediation of a crRNA guide sequence, after Lb Cas12a enzyme is combined with crRNA and target DNA to form a ternary complex, the trans-shear activity of cas12a is activated, an ssDNA fluorescent probe in a reaction system can be arbitrarily cut to release a fluorescent group on the probe, and an ultraviolet lamp is used for detecting a fluorescent signal or a colloidal gold test strip is used for detecting whether FAM and Biotin groups are cut off for result judgment. According to the ERA-CRISPR / cas12a one-pot method technology for rapidly detecting the mycoplasma synoviae, the DNA target of the mycoplasma synoviae is recognized and cut through ERA amplification and CRISPR / cas12a specificity, the specificity is high, the detection sensitivity is as low as 3.81 pg / mu L, and the ERA-CRISPR / cas12a one-pot method technology can be used for rapidly detecting the mycoplasma synoviae in clinical samples on site.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit, application and method for rapid detection of Mycoplasma synoviae based on the ERA-CRISPR / Cas12a one-step method. Background Art

[0002] Chicken Mycoplasma synoviae (MS) disease is mostly chronic, and sick chickens and latently infected chickens are the main sources of infection, which is endemic. The disease can be transmitted in two ways: horizontally and vertically. It is mainly transmitted through direct contact. The respiratory tract infection rate can reach 100%, but it rarely or cannot produce joint lesions. The poor sanitary conditions of chicken farms are conducive to the spread of MS. It can also be transmitted through dust, feathers, feed, etc. MS can also be transmitted vertically, through breeding eggs to offspring, and the rate of transmission through eggs is higher in the early stage of infection. MS has host specificity and mainly infects chickens and turkeys. In addition, MS has also been isolated from pigeons, ducks, geese, pheasants, quails, sparrows, goldfinches, etc., which may or may not produce obvious symptoms, but may be the source of infection for chickens and turkeys. Rabbits, rats, pigs and other mammals are not susceptible, and it has no effect on humans. Chickens of different breeds and ages are susceptible, and chicks are more susceptible than adult chickens. The resistance increases with age. The incidence rate of imported breeds or strains is higher than that of local breeds.

[0003] At present, the detection standards for Mycoplasma synoviae are based on serological detection, pathogen detection and PCR detection (such as NYT553-2002 Avian Mycoplasma Disease Diagnosis Technology, NYT553-2015 Avian Mycoplasma PCR Detection Method, OIE "Diagnostic Test and Vaccine Standard Manual - Avian Mycoplasma", etc.). For serological and conventional pathogen detection methods, the process involved is very cumbersome and time-consuming, and cannot meet the disease prevention and control requirements of rapid on-site detection of Mycoplasma synoviae, timely control of transmission routes and elimination of pollution sources. The PCR method has relatively low sensitivity, requires a PCR instrument, and needs to be carried out in the laboratory, and the detection time is relatively long. Isothermal amplification technologies, such as LAMP and RPA (or ERA with independent intellectual property rights in China), have the advantages of high detection sensitivity, short detection time, and no need for expensive instruments and equipment. A constant temperature water bath or a constant temperature metal bath is sufficient, which is very suitable for rapid on-site detection, but its high sensitivity also brings non-specific amplification, especially LAMP technology. The CRIPSR / cas12a system is based on the targeted recognition and cutting ability of the cas12a protein. The Cas12a family is a class of nucleases mediated by a single crRNA, which can specifically recognize and cut double-stranded DNA targets with PAM sequences. After cas12a forms a ternary complex with crRNA and target double-stranded DNA, it activates the "trans-cleavage activity" against ssDNA in the reaction system, and can non-specifically cut the reporter ssDNA in the system. However, when it is used alone for molecular diagnosis, a high amount of target DNA is required. Combining the ERA isothermal amplification technology with the CRIPSR / cas12a technology overcomes the shortcomings of these two methods, and retains the advantages of the ERA technology, such as high sensitivity, short detection time, and no need for expensive instruments, and has the advantages of the CRIPSR / cas12a technology, such as good specificity.

[0004] The selection of specific DNA targets is the key to establishing a reliable method for detecting Mycoplasma synoviae nucleic acid DNA. The Mycoplasma synoviae detection target scpA of the present invention is a new diagnostic marker screened by the present invention, and the gene only exists in Mycoplasma synoviae.

[0005] Based on the above reasons, the present invention combines ERA isothermal amplification technology with CRIPSR / cas12a technology based on the newly screened and identified target scpA, and establishes a one-pot ERA-CRISPR / cas12a technology, which can be used for rapid and accurate on-site detection of chicken synoviae Mycoplasma in clinical samples, and can report the detection results visually by detecting the fluorescent signal of the reaction or using a test strip. Compared with the existing method for detecting chicken synoviae Mycoplasma, it has the advantages of high sensitivity, strong specificity, rapidity, no need for expensive instruments, and can be used for on-site rapid detection. Summary of the invention

[0006] In view of this, the present invention provides a kit, application and method for rapid detection of Mycoplasma synoviae based on the ERA-CRISPR / Cas12a one-step method.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A kit for rapid detection of Mycoplasma synoviae based on the ERA-CRISPR / Cas12a one-step method, including sample lysis buffer, reaction buffer, ERA primer, crRNA guide sequence, Cas12a enzyme, ssDNA fluorescent probe and colloidal gold test strip;

[0009] The nucleotide sequence of the ERA primer is:

[0010] F:5'-CAATTCAAAGAAATCAAAGACGGCTTTCTA-3',

[0011] R:5'-AAATTAGAGTAGTAATGCTAGAGTGTCCATC-3';

[0012] The crRNA guide sequence is:

[0013] 5'-UAAUUUCUACUAAGUGUAGAUCCGUAGGUUUCUAGCUUUAAA-3'.

[0014] Preferably, the ssDNA fluorescent probe is a single-stranded nucleotide sequence labeled with FAM or FITC at the 5' end and BHQ1 or Biotin at the 3' end.

[0015] Preferably, the nucleotide sequence of the ssDNA fluorescent probe is ssDNA-FQ: 5'-FAM-TTTTTT-BHQ1-3' or 5'-FITC-TTTTTT-BHQ1-3', and ssDNA-FB: 5'-FAM-TTTTTT-BIO-3' or 5'-FITC-TTTTTT-BIO-3'.

[0016] Preferably, the method further comprises a positive control plasmid carrying a Mycoplasma synoviae-specific target DNA, and a negative control Escherichia coli DH5a genomic DNA.

[0017] Application of a one-step rapid detection kit for Mycoplasma synoviae based on ERA-CRISPR / Cas12a in the visual detection of Mycoplasma synoviae.

[0018] The method for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method comprises the following steps:

[0019] S1. Place the sample cotton swab in a lysis buffer to release the mycoplasma genomic DNA in the sample to be tested;

[0020] S2. Add the above-lysed mycoplasma genomic DNA and the reaction system into a new reaction tube, and then place the reaction tube in a constant temperature water bath or a constant temperature metal bath for reaction, so that the ERA isothermal amplification and CRISPR cleavage reaction are carried out simultaneously in the same reaction system, with ERA amplification and CRISPR cleavage at the same time;

[0021] S3. When using the ssDNA-FQ fluorescent probe, after the reaction is completed in a constant temperature water bath or a constant temperature metal bath, use a handheld ultraviolet lamp to detect the fluorescent signal, and determine the test result based on the presence or absence of the fluorescent signal; when using the ssDNA-FB fluorescent probe, after the reaction is completed in a constant temperature water bath or a constant temperature metal bath, insert the test strip into the reaction tube, and determine the result by observing whether a band appears at the test line on the test strip within 5-10 minutes.

[0022] Preferably, in step S2, the reaction system is: 1.0 μM of each F / R primer, 10 μL of solvent, 1.0 μL of activator, 2.5 μL of 10×Reaction buffer, 250 nM LbCas12a enzyme, 250 nM crRNA, 100 nM ssDNA, 2.0 μL of DNA template, ddH2O is supplemented to 25 μL, mixed and reacted at 37°C for 40 minutes.

[0023] Preferably, in step S2, the reaction tube is placed in a 37° C. water bath or metal bath for 40 minutes, and the result is observed under a handheld ultraviolet lamp; or a test strip is inserted into the reaction tube, and the test result can be observed with the naked eye within 10 minutes.

[0024] Compared with the prior art, the present invention has achieved the following technical effects:

[0025] The kit for visually detecting Mycoplasma synoviae of the present invention first performs ERA amplification on the sample to be tested, and then guides the CRISPR / Cas12a system to recognize, bind and cut the target double-stranded DNA of the ERA amplification product under the mediation of the crRNA guide sequence, and activates the non-specific nuclease function, and then arbitrarily cuts the ssDNA fluorescent probe in the system to obtain the sheared product, and finally determines the result by detecting the sheared fluorescent group or the fluorescent signal emitted by it.

[0026] The ERA-CRISPR / Cas12a technology for detecting Mycoplasma synoviae of the present invention detects Mycoplasma synoviae of the present invention in a dual specific manner through ERA amplification and crRNA recognition, has strong specificity, and can be as low as 3.81pg / μL in sensitivity, and can detect whether the chicken is infected with Mycoplasma synoviae of the present invention earlier, so as to prevent and control it. The kit in the present invention has the characteristics of not requiring expensive instruments and equipment, low cost, convenient operation, less time consumption, high sensitivity and strong specificity, and the detection reaction is carried out in a constant temperature water bath or a constant temperature metal bath, which can effectively break away from the dependence on large laboratory instruments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart of the present invention for visual detection of Mycoplasma synoviae based on the ERA-CRISPR / Cas12a system;

[0028] Figure 2 This is a graph showing the specificity experimental results of the ERA detection of Mycoplasma synoviae of the present invention;

[0029] Figure 3 This is a crRNA screening diagram of the Mycoplasma synoviae scpA gene of the present invention;

[0030] Figure 4 This is a sensitivity test result diagram of the ERA-CRISPR / Cas12a one-step method for detecting Mycoplasma synoviae in chicken;

[0031] Figure 5 This is a specific analysis result diagram of the ERA-CRISPR / Cas12a one-step method for detecting Mycoplasma synoviae in chicken;

[0032] Figure 6 The positive control results of the ERA-CRISPR / Cas12a one-step method for detecting Mycoplasma synoviae in chicken are shown in (A) the results are observed under ultraviolet light; (B) the detection results are displayed using a test strip for color development. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0034] The present invention discloses a kit for rapid detection of chicken synoviae Mycoplasma based on the ERA-CRISPR / Cas12a one-step method, comprising a sample lysis buffer, a reaction buffer, an ERA primer, a crRNA guide sequence, a Cas12a enzyme, a ssDNA fluorescent probe and a colloidal gold test strip;

[0035] The nucleotide sequence of the ERA primer is:

[0036] F:5'-CAATTCAAAGAAATCAAAGACGGCTTTCTA-3',

[0037] R:5'-AAATTAGAGTAGTAATGCTAGAGTGTCCATC-3';

[0038] The crRNA guide sequence is:

[0039] 5'-UAAUUUCUACUAAGUGUAGAUCCGUAGGUUUCUAGCUUUAAA-3'.

[0040] The ssDNA fluorescent probe is a single-stranded nucleotide sequence labeled with FAM or FITC at the 5' end and BHQ1 or Biotin at the 3' end.

[0041] The nucleotide sequences of the ssDNA fluorescent probes are ssDNA-FQ: 5'-FAM-TTTTTT-BHQ1-3' or 5'-FITC-TTTTTT-BHQ1-3', and ssDNA-FB: 5'-FAM-TTTTTT-BIO-3' or 5'-FITC-TTTTTT-BIO-3'.

[0042] Also included are a positive control plasmid carrying a target DNA specific for Mycoplasma synoviae, and a negative control, E. coli DH5a genomic DNA.

[0043] Application of a one-step rapid detection kit for Mycoplasma synoviae based on ERA-CRISPR / Cas12a in the visual detection of Mycoplasma synoviae.

[0044] The method for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method comprises the following steps:

[0045] S1. Place the sample cotton swab in a lysis buffer to release the mycoplasma genomic DNA in the sample to be tested;

[0046] S2. Add the above-lysed mycoplasma genomic DNA and the reaction system into a new reaction tube, and then place the reaction tube in a constant temperature water bath or a constant temperature metal bath for reaction, so that the ERA isothermal amplification and CRISPR cleavage reaction are carried out simultaneously in the same reaction system, with ERA amplification and CRISPR cleavage at the same time;

[0047] S3. When using the ssDNA-FQ fluorescent probe, after the reaction is completed in a constant temperature water bath or a constant temperature metal bath, use a handheld ultraviolet lamp to detect the fluorescent signal, and determine the test result based on the presence or absence of the fluorescent signal; when using the ssDNA-FB fluorescent probe, after the reaction is completed in a constant temperature water bath or a constant temperature metal bath, insert the test strip into the reaction tube, and determine the result by observing whether a band appears at the test line on the test strip within 5-10 minutes.

[0048] In step S2, the reaction system is: 1.0 μM each of F / R primers, 10 μL of solvent, 1.0 μL of activator, 2.5 μL of 10×Reaction buffer, 250 nM LbCas12a enzyme, 250 nM crRNA, 100 nM ssDNA, 2.0 μL of DNA template, ddH2O is added to 25 μL, mixed and reacted at 37°C for 40 minutes.

[0049] In step S2, the reaction tube is set to simultaneously perform constant temperature reaction and fluorescence signal detection; or react in a 37°C water bath or metal bath for 40 minutes, and then observe the results under a handheld ultraviolet lamp; or insert a test strip into the reaction tube and observe the test results with the naked eye within 10 minutes.

[0050] Results of the ERA primer screening experiment for Mycoplasma synoviae: Figure 2 shown.

[0051] Screening of crRNA for scpA gene of Mycoplasma synoviae: Figure 3 shown.

[0052] The sensitivity test results of ERA-CRISPR / Cas12a one-step detection of Mycoplasma synoviae: Figure 4 As shown, A is the fluorescence signal detected on the real-time fluorescence PCR instrument; B is the observation result under ultraviolet light. The concentration of the extracted chicken synoviae Mycoplasma genome was measured to be 381ng / μL. 3 , 10 4 , 10 5 A total of three different concentrations of genomic DNA extracted from Mycoplasma synoviae were used as detection templates and detected using the ERA-CRISPR / Cas12a one-step method. The experimental results showed that the minimum detection limit of Mycoplasma synoviae by the ERA-CRISPR one-step method based on the scpA target gene was 3.81 pg / μL.

[0053] Plasmids carrying Mycoplasma synoviae-specific target DNA and Escherichia coli DH5a genomic DNA as negative control.

[0054] The present invention provides a method for visually detecting Mycoplasma synoviae based on the ERA-CRISPR / Cas12a system, the process is as follows: Figure 1 As shown, the sample to be tested is first lysed with lysis buffer to release genomic DNA, then all the reaction systems are added to a new EP tube, and then the ERA-CRISPR / Cas12a one-pot reaction is carried out in a constant temperature water bath or a constant temperature metal bath. The system recognizes, binds and cuts the target double-stranded DNA of the ERA amplification product, and activates the non-specific nuclease function. Finally, a handheld UV lamp or colloidal gold test strip is used to develop the color and report the test results.

[0055] Embodiment 1:

[0056] ERA primer design, screening and amplification system determination:

[0057] The scpA gene (locus tag: DEH79_00545) DNA sequence (5'--3') in the genome of Mycoplasma synoviae 86079-7NS strain (CP029258.1) was used to design a series of primer pairs that met the ERA primer design principles. The primers were synthesized by Beijing Qingke Biotechnology (PAGE purification); after a series of preliminary tests and screening, the primer screening results are as follows:

[0058] F:5'-CAATTCAAAGAAATCAAAGACGGCTTTCTA-3',

[0059] B:5'-AAATTAGAGTAGTAATGCTAGAGTGTCCATC-3';

[0060] The amplified fragment size was 148 bp;

[0061] After determining the best primer set, the reaction system including primer concentration, amplification temperature, and amplification time was optimized.

[0062] The optimized ERA amplification system was as follows: solvent 10 μL, activator 1.0 μL, F (10 μM) 1.5 μL, R (10 μM) 1.5 μL, sample DNA 2.0 μL, and ddH2O to 25.0 μL.

[0063] Embodiment 2:

[0064] crRNA screening:

[0065] Based on the 148 bp DNA sequence amplified by ERA, five possible Cas12a cleavage PAM sites (5'-TTN-3') and their 3' end 20-23 bp fragments were selected as targets for potential crRNA guide sequences. A plasmid carrying a T7 promoter and a Cas12a DRDNA fragment (constructed in the applicant's laboratory) was used as a template and HiScribe TM RNA was obtained by reverse transcription using the T7 Rapid and Efficient RNA Synthesis Kit (NEB) and then Each crRNA was purified using an RNA purification kit (NEB).

[0066] The designed crRNAs were verified and screened using the ERA-CRIPSR / Cas12a one-step method. The crRNA sequences that can be used in the ERA-CRIPSR / Cas12a one-step detection system are:

[0067] 5'-UAAUUUCUACUAAGUGUAGAUCCGUAGGUUUCUAGCUUUAAA-3'.

[0068] Embodiment 3:

[0069] Specificity and sensitivity determination of the ERA-CRISPR / Cas12a method:

[0070] 1. Methods

[0071] (1) Based on the previously optimized ERA reaction system based on the scpA target gene, the reaction temperature, Cas12a and crRNA concentrations of ERA-CRISPR / Cas12a for detecting Mycoplasma synoviae were further optimized, as well as the stability of the reaction detected by collecting fluorescence signals or colorimetric analysis of test strips.

[0072] (2) The extracted genomic DNA of Mycoplasma synoviae, Mycoplasma gallisepticum, Haemophilus paragallinarum, avian pathogenic Escherichia coli, Salmonella, Staphylococcus aureus and Riemerella anatipestifer and ultrapure sterile water were used as templates, and ultrapure sterile water was used as the blank control group. The specificity of the detection system was analyzed by the ERA-CRISPR / Cas12a one-step method. When the fluorescent probe used was ssDNA-FQ, a fluorescent quantitative PCR instrument was used to select the FAM fluorescent channel to collect the fluorescent signal. The reaction temperature was set to 37°C, and the fluorescent signal was measured once every 1 minute, for a total of 40 times for 40 minutes, and then the test results were determined by the fluorescent signal curve; or after the reaction was completed, the measured PCR tube was placed under ultraviolet light for observation. The positive reaction tube should show a green fluorescent reaction, while the negative reaction tube and the blank control group should not show obvious changes.

[0073] (3) The concentration of the extracted Mycoplasma synoviae genome was 381 ng / μL. 3 , 10 4 , 10 5 A total of three different concentrations of genomic DNA extracted from Mycoplasma synoviae were used as detection templates, and the sensitivity of the ERA-CRISPR / Cas12a one-step detection system was then determined.

[0074] 2. Results

[0075] 1. Specificity analysis of the detection system:

[0076] The specificity of the established ERA-CRISPR / Cas12a one-step detection method for Mycoplasma synoviae was analyzed using the extracted genomic DNA of Mycoplasma gallisepticum, Mycoplasma gallisepticum, Haemophilus paragallinarum, avian pathogenic Escherichia coli, Salmonella, Staphylococcus aureus and Riemerella anatipestifer as well as ultrapure sterile water as detection templates. The results showed that only when the genomic DNA of Mycoplasma synoviae was used as the detection template, an amplification curve appeared in the real-time fluorescence detection. When the genomic DNA of the other six foodborne pathogens and ultrapure sterile water were used as templates, no obvious fluorescence signal appeared. The reaction tube was placed under ultraviolet light for observation. Only the reaction tube with the genomic DNA of Mycoplasma synoviae as the template showed obvious green fluorescence, and no fluorescence reaction appeared in other groups. Therefore, the established ERA-CRISPR / Cas12a one-step detection system for Mycoplasma synoviae has good specificity, such as Figure 5 As shown, A is the fluorescence signal curve on the real-time fluorescence PCR instrument; B is the observation result under 365nm ultraviolet light.

[0077] Embodiment 4:

[0078] A kit for visually detecting Mycoplasma synoviae:

[0079] A kit for visually detecting Mycoplasma synoviae based on the ERA-CRISPR / Cas12a integrated detection system, comprising all reagents such as sample lysis buffer, reaction buffer, ERA primers, crRNA guide sequence, ssDNA fluorescent probe, dNTPs, a plasmid carrying Mycoplasma synoviae-specific target DNA as a positive control, and Escherichia coli DH5a genomic DNA as a negative control.

[0080] ERA Primers:

[0081] F:5'-CAATTCAAAGAAATCAAAGACGGCTTTCTA-3',

[0082] R:5'-AAATTAGAGTAGTAATGCTAGAGTGTCCATC-3';

[0083] crRNA guide sequence:

[0084] 5'-UAAUUUCUACUAAGUGUAGAUCCGUAGGUUUCUAGCUUUAA A-3';

[0085] ssDNA fluorescent probe:

[0086] 5'-FAM-TTTTTT-BHQ1-3', this fluorescent probe is used to detect the presence of Mycoplasma synoviae in the target system under the excitation of blue-green light or with the naked eye.

[0087] The method for visually detecting Mycoplasma synoviae using a kit comprises the following steps:

[0088] (1) Place the sample cotton swab in a lysis buffer to release the mycoplasma genomic DNA in the sample to be tested;

[0089] (2) Add the above-mentioned lysed mycoplasma genomic DNA and reaction system (including reaction buffer, ERA primers, LbCas12a enzyme, crRNA and ssDNA fluorescent probes and dNTPs, etc.) to a new reaction tube, and then place the reaction tube in a constant temperature water bath or a constant temperature metal bath for reaction, so that ERA isothermal amplification and CRISPR cleavage reaction are carried out simultaneously in one reaction system;

[0090] (3) When using the ssDNA-FQ fluorescent probe, place the reaction tube in a 37°C water bath for 40 minutes, and then observe the results under a handheld ultraviolet lamp. If the reaction product has no fluorescence brightness under ultraviolet light or no brightness when observed with the naked eye, it means that Mycoplasma synoviae is not detected in the sample to be tested; if the reaction product has fluorescence brightness under ultraviolet light or has brightness when observed with the naked eye, it means that Mycoplasma synoviae is detected in the sample to be tested.

[0091] When using the ssDNA-FB fluorescent probe, during on-site testing, after the reaction tube is placed in a 37-degree water bath or metal bath for 40 minutes, the test strip is inserted into the reaction system. Within 5-10 minutes, the result is determined based on whether the required bands appear at the test line and control line on the test strip: if bands appear at both the test line and the control line, or only at the test line, the test result is positive; if only at the control line, the test result is negative; if there are no bands at both the test line and the control line, the test reaction is invalid and needs to be repeated. The test results of the positive control are shown as follows: Figure 6 As shown, (A) observe the results under ultraviolet light; (B) use test strips to display the test results.

[0092] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention, as well as other detection techniques or detection kits of Mycoplasma synoviae based on the new nucleic acid detection marker scpA of the present invention, all belong to the scope of protection of the present invention.

Claims

1. A kit for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method, characterized in that: Includes sample lysis buffer, reaction buffer, ERA primer, crRNA guide sequence, Cas12a enzyme, ssDNA fluorescent probe and colloidal gold test strip; The nucleotide sequence of the ERA primer is: F:5'-CAATTCAAAGAAATCAAAGACGGCTTTCTA-3', R:5'-AAATTAGAGTAGTAATGCTAGAGTGTCCATC-3'; The crRNA guide sequence is: 5'-UAAUUUCUACUAAGUGUAGAUCCGUAGGUUUCUAGCUUUAAA-3'.

2. The kit for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method according to claim 1, characterized in that: The ssDNA fluorescent probe is a single-stranded nucleotide sequence with FAM or FITC labeled at the 5' end and BHQ1 or Biotin labeled at the 3' end.

3. The kit for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method according to claim 1, characterized in that: The nucleotide sequence of the ssDNA fluorescent probe is ssDNA-FQ: 5'-FAM-TTTTTT-BHQ1-3' or 5'-FITC-TTTTTT-BHQ1-3', and ssDNA-FB: 5'-FAM-TTTTTT-BIO-3' or 5'-FITC-TTTTTT-BIO-3'.

4. The kit for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method according to claim 1, characterized in that Also included are a positive control plasmid carrying a target DNA specific for Mycoplasma synoviae, and a negative control, E. coli DH5a genomic DNA.

5. Use of the kit for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method according to any one of claims 1 to 4 in visual detection of Mycoplasma synoviae.

6. A method for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method, characterized in that: The steps include: S1. Place the sample cotton swab in a lysis buffer to release the mycoplasma genomic DNA in the sample to be tested; S2. Add the above-lysed mycoplasma genomic DNA and the reaction system into a new reaction tube, and then place the reaction tube in a constant temperature water bath or a constant temperature metal bath for reaction, so that the ERA isothermal amplification and CRISPR cleavage reaction are carried out simultaneously in the same reaction system, with ERA amplification and CRISPR cleavage at the same time; S3. When using the ssDNA-FQ fluorescent probe, after the reaction is completed in a constant temperature water bath or a constant temperature metal bath, use a handheld ultraviolet lamp to detect the fluorescent signal, and determine the test result based on the presence or absence of the fluorescent signal; when using the ssDNA-FB fluorescent probe, after the reaction is completed in a constant temperature water bath or a constant temperature metal bath, insert the test strip into the reaction tube, and determine the result by observing whether a band appears at the test line on the test strip within 5-10 minutes.

7. The method for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method according to claim 6, characterized in that: In the step S2, the reaction system is: 1.0 μM of each F / R primer, 10 μL of solvent, 1.0 μL of activator, 2.5 μL of 10×Reaction buffer, 250 nM LbCas12a enzyme, 250 nM crRNA, 100 nM ssDNA, 2.0 μL of DNA template, ddH2O is added to 25 μL, mixed and reacted at 37°C for 40 minutes.

8. The method for rapid detection of Mycoplasma synoviae based on ERA-CRISPR / Cas12a one-step method according to claim 6, characterized in that: In step S2, the reaction tube is placed in a 37° C. water bath or metal bath for 40 minutes, and the result is observed under a handheld ultraviolet lamp; or a test strip is inserted into the reaction tube, and the test result can be observed with the naked eye within 10 minutes.

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