Method and application of dual RAA-CRISPR detection of Mycoplasma gallisepticum and Mycoplasma synoviae

Abstract

The present invention relates to the field of poultry farming technology, and in particular to a method and application of dual RAA-CRISPR detection of Mycoplasma gallisepticum and Mycoplasma synoviae. Based on RAA-CRISPR technology, the present invention designs RAA primer pairs for amplifying Mycoplasma gallisepticum and RAA primer pairs for amplifying Mycoplasma synoviae, as well as Cas12a-crRNA and Cas13a-crRNA as crRNA molecules of Mycoplasma gallisepticum and Mycoplasma synoviae, respectively; and optimizes the concentration ratio of crRNA molecules and CAS proteins and the concentration ratio of RAA primers and ssDNA to achieve one-tube nucleic acid detection. The method provided by the present invention can use fluorescence detection or determine the results by naked eye within 20 minutes, with less detection equipment used, low cost, visual detection, and full-process sealing operation, and no aerosol pollution will be generated. Its application may help control the pandemic of Mycoplasma gallisepticum and Mycoplasma synoviae.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry breeding, and in particular to a method and application of dual RAA-CRISPR detection of Mycoplasma gallisepticum and Mycoplasma synoviae. Background Art

[0002] Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the two most harmful mycoplasmas to poultry farming. Mycoplasma gallisepticum infection is a type of respiratory disease in chickens, mainly causing chronic respiratory disease and infraorbital sinusitis. With the increase in the scale of chicken farming, changes in feeding methods and increased feeding density, the incidence of the disease is getting higher and higher, mainly affecting the growth of chicks and reducing the egg production of adult chickens, causing significant economic losses to the poultry industry. The harm is becoming increasingly prominent and has attracted widespread attention from poultry farmers.

[0003] Recombinase-mediated strand replacement nucleic acid amplification (RAA) technology is a constant temperature rapid nucleic acid amplification technology that uses recombinases obtained from bacteria or fungi. At room temperature, the recombinase can tightly bind to the primer DNA to form a polymer of enzyme and primer. When the primer searches for a complementary sequence that completely matches it on the template DNA, it opens the double-stranded structure of the template DNA with the help of single-stranded binding proteins, and forms a new complementary DNA chain under the action of DNA polymerase, and the amplified product grows exponentially.

[0004] In the CRISPR / Cas system, CRISPR is the abbreviation for "Clustered regμlarly interspaced shortpalindromic repeats", which refers to regularly clustered short palindromic repeats. Cas is the abbreviation for "CRISPR associated", which means CRISPR related.

[0005] The CRISPR / Cas system is a prokaryotic immune system used to resist the invasion of foreign genetic material, such as bacteriophage viruses and foreign plasmids. At the same time, it provides bacteria with acquired immunity: this is similar to the secondary immunity of mammals. When bacteria are invaded by viruses or foreign plasmids, they will produce corresponding "memory" to resist their reinvasion. The CRISPR / Cas system can recognize foreign DNA and cut it off.

[0006] Based on the ability of the CRISPR / Cas system to cut off exogenous DNA, after the Cas protein specifically recognizes the target under the guidance of crRNA, it can be activated to perform trans-cleavage activity against ssDNA or ssRNA. By labeling reporter probes such as FAM and biotin with fluorescent groups and quenching groups such as BHQ1, the detection results are converted into visual fluorescent signals or test strip bands. Therefore, the CRISPR / Cas system has the potential to detect nucleic acids.

[0007] In the prior art, detection of Mycoplasma gallisepticum and Mycoplasma synoviae based on RAA and CRISPR / Cas technology has not yet been achieved. Summary of the invention

[0008] The present invention provides a method and application for dual RAA-CRISPR detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

[0009] In a first aspect, the present invention provides a RAA primer combination for Mycoplasma gallisepticum and Mycoplasma synoviae, comprising: a RAA primer pair for amplifying Mycoplasma gallisepticum, whose sequence is shown in SEQ ID NO.1-2, and a RAA primer pair for amplifying Mycoplasma synoviae, whose sequence is shown in SEQ ID NO.4-5.

[0010] In a second aspect, the present invention provides a crRNA molecule for detecting Mycoplasma gallisepticum and Mycoplasma synoviae based on CRISPR / CAS technology, wherein the crRNA molecule of Mycoplasma gallisepticum is Cas12a-crRNA, and its sequence is shown in SEQ ID NO.3; the crRNA molecule of Mycoplasma synoviae is Cas13a-crRNA, and its sequence is shown in SEQ ID NO.6.

[0011] In a third aspect, the present invention provides a dual RAA-CRISPR / CAS reaction system for Mycoplasma gallisepticum and Mycoplasma synoviae, comprising: the above-mentioned RAA primer combination and the above-mentioned crRNA molecule; and also comprising: a Cas12a-ssDNA reporter gene having a sequence as shown in FAM-TTTTTTTTTT-BHQ1, and a Cas13a-ssRNA reporter gene having a sequence as shown in ROX-UUUUUUUU-BHQ2.

[0012] In the dual RAA-CRISPR / CAS reaction system of Mycoplasma gallisepticum and Mycoplasma synoviae provided by the present invention, the concentration ratio of LbCas12a protein to Cas12a-crRNA is (0.6-1): (0.8-2); the concentration ratio of Mycoplasma gallisepticum RAA upstream and downstream primers to Cas12a-ssDNA reporter gene is (2000-6000): (0.1-0.5).

[0013] Preferably, in the dual RAA-CRISPR / CAS reaction system of Mycoplasma gallisepticum and Mycoplasma synoviae provided by the present invention, the concentration ratio of LbCas12a protein to Cas12a-crRNA is 0.8:1.2; the concentration ratio of Mycoplasma gallisepticum RAA upstream and downstream primers to Cas12a-ssDNA reporter gene is 4000:0.2.

[0014] In the dual RAA-CRISPR / CAS reaction system of Mycoplasma gallisepticum and Mycoplasma synoviae provided by the present invention, the concentration ratio of LbCas13a protein to Cas13a-crRNA is 4:0.4; the concentration ratio of RAA upstream and downstream primers of Mycoplasma synoviae to Cas13a-ssRNA reporter gene is 4000:1.

[0015] As a specific embodiment of the present invention, the dual RAA-CRISPR / CAS reaction system of Mycoplasma gallisepticum and Mycoplasma synoviae provided by the present invention includes: 2-10 mmol / L dNTP, 0.2-1.0 μmol / L single-stranded binding protein, 1.0-2.0 μmol / L recombinase protein, 0.2-1.0 U / μL DNA polymerase, 10-50 mmol TrisHCl, 40-80 mmol NaCl, 6-10 mmol / L dithiothreitol, 1.0-5.0 μmol / L Mycoplasma gallisepticum RAA upstream and downstream primers, 1.0-5.0 μmol / L Mycoplasma synoviae RAA upstream and downstream primers, 1-2 μmol / L Cas12a-crRNA, 0.5-1.0 μmol / L LbCas12a, 0.1-0.5 nmol / L Cas12a-ssDNA reporter gene, 0.2-0.8μmol / LCas13a-crRNA, 1-5μmol / L LbCas13a, 0.5-1.5nmol / L Cas13a-ssRNA reporter gene, 0.4-1.0U / μL RNaseInhibitor, 0.4-1.0U / μL T7 RNAPolymerase, 0.1-1U / μL Exo exonuclease.

[0016] The present invention uses the dual RAA-CRISPR / CAS reaction system containing the above-mentioned Mycoplasma gallisepticum and Mycoplasma synoviae as a lyophilized liquid for lyophilization to obtain a lyophilized powder, or first injects the lyophilized liquid of the dual RAA-CRISPR / CAS reaction system containing the above-mentioned Mycoplasma gallisepticum and Mycoplasma synoviae into a reaction tube, and then lyophilizes to obtain a tubular lyophilized tube.

[0017] More specifically, the lyophilized solution provided by the present invention also includes a lyophilization protectant, which includes: 10-15 mmol / L trehalose, 20-25 mmol / L mannitol, and 5-20% bovine serum albumin.

[0018] Based on this, the fourth aspect of the present invention provides a one-tube freeze-dried tube for simultaneously detecting Mycoplasma gallisepticum and Mycoplasma synoviae, wherein the reaction system for detecting Mycoplasma gallisepticum and Mycoplasma synoviae is fixed in the one-tube freeze-dried tube by freeze-drying, and the reaction system for detecting Mycoplasma gallisepticum and Mycoplasma synoviae contains the above-mentioned dual RAA-CRISPR / CAS reaction system.

[0019] In a fifth aspect of the present invention, a method for preparing the above-mentioned one-tube freeze-drying tube is provided, and the freeze-drying conditions include: freezing the one-tube freeze-drying tube containing the dual RAA-CRISPR / CAS reaction system at -80°C for 1 to 1.5 hours and then performing equipment freeze-drying; the equipment freeze-drying includes: a pre-freezing stage, equipment vacuuming, freeze drying and analytical drying stages; the conditions of the analytical drying stage are: raising the partition temperature to -20 to -25°C and maintaining it for 12 to 16 hours, then raising the partition temperature to 4 to 8°C and maintaining it for 0.5 to 1 hour, and finally lowering the partition temperature to 20 to 25°C and maintaining it for 3 to 5 hours.

[0020] In a fifth aspect, the present invention provides a detection kit for simultaneously detecting Mycoplasma gallisepticum and Mycoplasma synoviae, wherein the detection kit contains the above-mentioned RAA primer combination or the above-mentioned crRNA molecule or the above-mentioned dual RAA-CRISPR / CAS reaction system or the above-mentioned single-tube lyophilized tube.

[0021] In a sixth aspect, the present invention provides a method for detecting Mycoplasma gallisepticum and / or Mycoplasma synoviae for purposes other than disease diagnosis and treatment, wherein the nucleic acid of a test sample is obtained as the test nucleic acid, the test nucleic acid is used as a template, and the above-mentioned detection kit is used to perform real-time PCR on the test nucleic acid, and the amplification process of the real-time PCR is monitored. The appearance of an amplification curve during the amplification process indicates that the test sample is positive for Mycoplasma gallisepticum and / or Mycoplasma synoviae;

[0022] Preferably, the FAM channel generates an amplification curve, which indicates that Mycoplasma gallisepticum is positive; the VIC channel generates an amplification curve, which indicates that Mycoplasma synoviae is positive;

[0023] Or use the above-mentioned detection kit to detect the nucleic acid to be tested to obtain an amplification product; if the amplification product appears as white light under ultraviolet light, it means that the sample to be tested is positive for Mycoplasma gallisepticum and / or Mycoplasma synoviae; if the amplification product appears colorless and transparent under ultraviolet light, it means that the sample to be tested is positive for Mycoplasma gallisepticum and Mycoplasma synoviae.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention provides isothermal amplification. The detection method provided by the present invention can be carried out at room temperature, and its optimum temperature is 37-42°C. Expensive temperature cycle equipment is not required, thus reducing costs.

[0026] (2) The detection method provided by the present invention is time-saving. The entire reaction process can be completed within 10 to 20 minutes, and no denaturation is required, thereby achieving rapid detection of Mycoplasma gallisepticum and Mycoplasma synoviae;

[0027] (3) The detection method provided by the present invention has high sensitivity. The RAA-CRISPR-Cas12a / Cas13a system maintains high sensitivity in a short reaction time and can obtain about 10 12 Order of magnitude of amplification products;

[0028] (4) The detection method provided by the present invention has high specificity. The RAA-CRISPR-Cas12a / Cas13a system only recognizes and cuts the target sequence and does not react to non-target sequences;

[0029] (5) The one-tube nucleic acid detection reagent prepared by the present invention can be stored at room temperature. 2+ In addition, the remaining system components are stored in the reaction tube in a dry powder state, which is stable and easy to store and transport, easy to operate, and reduces the possibility of contamination in the closed reaction system;

[0030] (6) The detection method provided by the present invention has diversified reading results. It can be detected by agarose gel electrophoresis, the amplification process can be monitored in real time by real-time PCR, and it can also be visualized under ultraviolet light.

[0031] The present invention uses the RAA-CRISPR-Cas12a / Cas13a system to perform fluorescence detection equipment and a method for on-site visual detection without power supply on Mycoplasma gallisepticum and Mycoplasma synoviae genes. By combining the sample processing method and RAA technology with the detection based on Cas12a / Cas13a, the results can be determined by fluorescence detection or by naked eye within 20 minutes. With few detection equipment, low cost, visual detection, and full-pipe sealing operation, no aerosol pollution will be generated, and its application may help control the pandemic of Mycoplasma gallisepticum and Mycoplasma synoviae. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is the detection result of Mycoplasma gallisepticum and Mycoplasma synoviae nucleic acid by dual RAA-CRISPR CAS12a / CAS13a. In the figure, 1 is Mycoplasma gallisepticum nucleic acid, 2 is Mycoplasma synoviae nucleic acid, and 3 is the negative control.

[0034] Figure 2 This is the result of ultraviolet light detection of dual RAA-CRISPR CAS12a / CAS13a products of Mycoplasma gallisepticum and Mycoplasma synoviae. In the figure, 1 is Mycoplasma gallisepticum nucleic acid, 2 is Mycoplasma synoviae nucleic acid, and 3 is the negative control.

[0035] Figure 3 This is the specific detection result of dual RAA-CRISPR CAS12a / CAS13a for Mycoplasma gallisepticum and Mycoplasma synoviae. In the figure, 1 and 2 are a mixture of Mycoplasma gallisepticum and Mycoplasma synoviae nucleic acids; 3 is a live infectious bursal disease vaccine; 4 is Escherichia coli; 5 is Salmonella pullorum; 6 is Avibacterium paragallinarum; 7 is avian influenza virus; 8 is a live Newcastle disease vaccine; 9 is chicken infectious anemia virus; and 10 is a negative control.

[0036] Figure 4 These are the results of dual RAA-CRISPR CAS12a / CAS13a ultraviolet light-specific tests for Mycoplasma gallisepticum and Mycoplasma synoviae. In the figure, 1 is a mixture of Mycoplasma gallisepticum and Mycoplasma synoviae nucleic acids; 2 is a negative control; 3 is an infectious bursal disease live vaccine; 4 is Escherichia coli; 5 is Salmonella pullorum; 6 is Avibacterium paragallinarum; 7 is avian influenza virus; 8 is a live Newcastle disease vaccine; and 9 is an infectious anemia virus.

[0037] Figure 5 This is the sensitivity test result of Mycoplasma gallisepticum and Mycoplasma synoviae by dual RAA-CRISPR CAS12a / CAS13a against Mycoplasma gallisepticum. In the figure, 1 is 1×10 7 copies / mL; 2 is 1×10 6 copies / mL; 3 is 1×10 5 copies / mL; 4 is 1×10 4 copies / mL; 5 is 1×10 3copies / mL; 6 is 500 copies / mL; 7 is 250 copies / mL; 8 is 125 copies / mL; 9 is 62.5 copies / mL; 10 is the negative control.

[0038] Figure 6 is the sensitivity test result of Mycoplasma gallisepticum and Mycoplasma synoviae by dual RAA-CRISPR CAS12a / CAS13a against Mycoplasma synoviae; 1 is 1×10 7 copies / mL; 2 is 1×10 6 copies / mL; 3 is 1×10 5 copies / mL; 4 is 1×10 4 copies / mL; 5 is 1×10 3 copies / mL; 6 is 500 copies / mL; 7 is 250 copies / mL; 8 is 125 copies / mL; 9 is 62.5 copies / mL; 10 is the negative control.

[0039] Figure 7 This is the sensitivity test result of Mycoplasma gallisepticum and Mycoplasma synoviae under ultraviolet light by dual RAA-CRISPR CAS12a / CAS13a. In the figure, 1 is 1×10 7 copies / mL; 2 is 1×10 6 copies / mL; 3 is 1×10 5 copies / mL; 4 is 1×10 4 copies / mL; 5 is 1×10 3 copies / mL; 6 is 500 copies / mL; 7 is 250 copies / mL; 8 is 125 copies / mL; 9 is 62.5 copies / mL; 10 is the negative control.

[0040] Figure 8 This is the sensitivity test result of Mycoplasma gallisepticum and Mycoplasma synoviae under ultraviolet light by dual RAA-CRISPR CAS12a / CAS13a. In the figure, 1 is 1×10 7 copies / mL; 2 is 1×10 6 copies / mL; 3 is 1×10 5 copies / mL; 4 is 1×10 4 copies / mL; 5 is 1×10 3copies / mL; 6 is 500 copies / mL; 7 is 250 copies / mL; 8 is 125 copies / mL; 9 is 62.5 copies / mL; 10 is the negative control.

[0041] Fig. 9 This is the intra-batch reproducibility result of dual RAA-CRISPR CAS12a / CAS13a for Mycoplasma gallisepticum and Mycoplasma synoviae. In the figure, 1-3 are the concentrations of 1×10 7 copies / mL of Mycoplasma gallisepticum positive plasmid, 4-6 is the concentration of 1×10 7 copies / mL of avian synoviae positive plasmid, and 7 copies / mL of negative control.

[0042] Fig.10 It is a dual RAA-CRISPR CAS12a / CAS13a of Mycoplasma gallisepticum and Mycoplasma synoviae under UV light for 1×10 7 The results of mixed positive plasmid detection of Mycoplasma gallisepticum and Mycoplasma synoviae at a concentration of 1×10 7 The positive plasmids of Mycoplasma gallisepticum and Mycoplasma synoviae were mixed at 4 copies / mL, and 4 was the negative control.

[0043] Fig.11 This is the inter-batch reproducibility result of dual RAA-CRISPR CAS12a / CAS13a for Mycoplasma gallisepticum and Mycoplasma synoviae. In the figure, 1-3 are three batches tested at a concentration of 1×10 7 copies / mL of Mycoplasma gallisepticum positive plasmid, 4-6 are three batches tested at a concentration of 1×10 7 copies / mL of avian synoviae positive plasmid, and 7 is nuclease-free water.

[0044] Fig.12 It is a dual RAA-CRISPR CAS12a / CAS13a of Mycoplasma gallisepticum and Mycoplasma synoviae under UV light for 1×10 7 The results of mixed positive plasmid detection of Mycoplasma gallisepticum and Mycoplasma synoviae at a concentration of 1×10 7 The positive plasmids of Mycoplasma gallisepticum and Mycoplasma synoviae were mixed at 4 copies / mL, and 4 was the negative control.

[0045] Fig.13These are the test results of Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a for detecting 11 different genotypes of Mycoplasma synoviae. In the figure, 1 is Mycoplasma synoviae genotype A; 2 is Mycoplasma synoviae genotype B; 3 is Mycoplasma synoviae genotype C; 4 is Mycoplasma synoviae genotype D; 5 is Mycoplasma synoviae E; 6 is Mycoplasma synoviae genotype F; 7 is Mycoplasma synoviae genotype G; 8 is Mycoplasma synoviae genotype H; 9 is Mycoplasma synoviae genotype I; 10 is Mycoplasma synoviae genotype J; 11 is Mycoplasma synoviae genotype K nucleic acid, 12 is Mycoplasma gallisepticum nucleic acid, and 13 is nuclease-free water.

[0046] Fig.14 These are the test results of Mycoplasma gallisepticum and Mycoplasma synoviae using dual RAA-CRISPR CAS12a / CAS13a to detect 11 different genotypes of Mycoplasma synoviae under ultraviolet light. In the figure, 1 is Mycoplasma synoviae genotype A; 2 is Mycoplasma synoviae genotype B; 3 is Mycoplasma synoviae genotype C; 4 is Mycoplasma synoviae genotype D; 5 is Mycoplasma synoviae E; 6 is Mycoplasma synoviae genotype F; 7 is Mycoplasma synoviae genotype G; 8 is Mycoplasma synoviae genotype H; 9 is Mycoplasma synoviae genotype I; 10 is Mycoplasma synoviae genotype J; 11 is Mycoplasma synoviae genotype K nucleic acid, 12 is Mycoplasma gallisepticum nucleic acid, and 13 is nuclease-free water.

[0047] Fig.15 This is the screening result of the best RAA-CRISPR / CAS12a system for Mycoplasma gallisepticum. In the figure, 1 is MG-F, MG-R, and Cas12a-crRNA for detecting Mycoplasma gallisepticum nucleic acid; 2 is MG-F1, MG-R1, and Cas12a-crRNA1 for detecting Mycoplasma gallisepticum nucleic acid; 3 is MG-F2, MG-R2, and Cas12a-crRNA2 for detecting Mycoplasma gallisepticum nucleic acid; 4 is MG-F, MG-R, and Cas12a-crRNA for detecting nuclease-free water; 5 is MG-F1, MG-R1, and Cas12a-crRNA1 for detecting nuclease-free water; 6 is MG-F2, MG-R2, and Cas12a-crRNA2 for detecting nuclease-free water.

[0048] Fig.16These are the test results of the optimal RAA-CRISPR / CAS12a system screening for Mycoplasma gallisepticum under ultraviolet light. In the figure, 1 is the detection of Mycoplasma gallisepticum nucleic acid by MG-F, MG-R, and Cas12a-crRNA; 2 is the detection of Mycoplasma gallisepticum nucleic acid by MG-F1, MG-R1, and Cas12a-crRNA1; 3 is the detection of Mycoplasma gallisepticum nucleic acid by MG-F2, MG-R2, and Cas12a-crRNA2; 4 is the detection of nuclease-free water by MG-F, MG-R, and Cas12a-crRNA; 5 is the detection of nuclease-free water by MG-F1, MG-R1, and Cas12a-crRNA1; and 6 is the detection of nuclease-free water by MG-F2, MG-R2, and Cas12a-crRNA2.

[0049] Fig.17 This is the screening result of the best RAA-CRISPR / CAS13a system for avian synoviae Mycoplasma. In the figure, 1 is the detection of avian synoviae Mycoplasma nucleic acid by MS-F, MS-R, and Cas13a-crRNA; 2 is the detection of avian synoviae Mycoplasma nucleic acid by MS-F1, MS-R1, and Cas13a-crRNA1; 3 is the detection of avian synoviae Mycoplasma nucleic acid by MS-F2, MS-R2, and Cas13a-crRNA2; 4 is the detection of nuclease-free water by MS-F, MS-R, and Cas12a-crRNA; 5 is the detection of nuclease-free water by MS-F1, MS-R1, and Cas13a-crRNA1; 6 is the detection of nuclease-free water by MS-F2, MS-R2, and Cas13a-crRNA2.

[0050] Fig.18 These are the test results of the optimal RAA-CRISPR / CAS13a system screening for avian synoviae Mycoplasma under ultraviolet light. In the figure, 1 is the detection of avian synoviae Mycoplasma nucleic acid by MS-F, MS-R, and Cas13a-crRNA; 2 is the detection of avian synoviae Mycoplasma nucleic acid by MS-F1, MS-R1, and Cas13a-crRNA1; 3 is the detection of avian synoviae Mycoplasma nucleic acid by MS-F2, MS-R2, and Cas13a-crRNA2; 4 is the detection of nuclease-free water by MS-F, MS-R, and Cas12a-crRNA; 5 is the detection of nuclease-free water by MS-F1, MS-R1, and Cas13a-crRNA1; and 6 is the detection of nuclease-free water by MS-F2, MS-R2, and Cas13a-crRNA2. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are 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.

[0052] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0053] The components of the tube reaction dry powder in the present invention are as follows: 2-10 mmol / L dNTP, 0.2-1.0 μmol / L single-stranded binding protein, 1.0-2.0 μmol / L recombinase protein, 0.2-1.0 U / μL DNA polymerase, 10-50 mmol TrisHCl, 40-80 mmol NaCl, 6-10 mmol / L dithiothreitol, 1.0-5.0 μmol / L Mycoplasma gallisepticum dual RAA-CRISPR CAS12a / CAS13a upstream and downstream primers, 1.0-5.0 μmol / L Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a upstream and downstream primers, 1-2 μmol / LCas12a-crRNA, 0.5-1.0 μmol / L LbCas12a, 0.1-0.5 nmol / L ssDNA (FAM-BHQ1), 0.2-0.8 μmol / LCas13a-crRNA, 1-5 μmol / L LbCas13a, 0.5-1.5 nmol / L ssRNA reporter gene (ROX-BHQ2), 0.4-1.0 U / μL RNase Inhibitor, 0.4-1.0 U / μL T7 RNA Polymerase, 0.1-1 U / μL Exo exonuclease, 10-15 mmol / L trehalose, 20-25 mmol / L mannitol, and 5-20% bovine serum albumin.

[0054] 41.5 μL premixed Buffer A (components: 20-50% polyethylene glycol), 2.5 μL premixed Buffer B (components: 250-320 mmol magnesium acetate, 80-120 mmol magnesium chloride), 6 μL template, the total reaction system is 50 μL.

[0055] Example 1 Primer Design for Mycoplasma Gallisepticum and Mycoplasma Synovium

[0056] According to JN102624.1 and CP034544.1 of Mycoplasma gallisepticum and Mycoplasma synoviae registered in Genbank, RAA primers, crRNA primers, ssDNA and ssRNA reporter genes were designed. The designed primer sequences are shown in Table 1 and were synthesized by a gene company.

[0057] Table 1

[0058]

[0059] Example 2 Establishment of a dual RAA-CRISPR CAS12a / CAS13a detection method for Mycoplasma gallisepticum and Mycoplasma synoviae

[0060] The present invention provides a one-tube type Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a detection method.

[0061] (1) Preparation of a single-tube freeze-dried reaction solution of dual RAA-CRISPR CAS12a / CAS13a of Mycoplasma gallisepticum and Mycoplasma synoviae

[0062] The one-tube Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a freeze-dried reaction solution comprises 8mmol / L dNTP, 0.8μmol / L single-stranded binding protein, 1.2μmol / L recombinase protein, 0.6U / μL DNA polymerase, 30mmol TrisHCl, 50mmol NaCl, 8mmol / L dithiothreitol, 4.0μmol / L Mycoplasma gallisepticum dual RAA-CRISPR CAS12a / CAS13a upstream and downstream primers, 4.0μmol / L Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a upstream and downstream primers, 1.2μmol / L Cas12a-crRNA, 0.8μmol / L LbCas12a, 0.2nmol / L ssDNA (FAM-BHQ1), 0.4μmol / L Cas13a-crRNA, 4μmol / L LbCas13a, 1.0 nmol / L ssRNA reporter gene (ROX-BHQ2), 0.8 U / μL RNase Inhibitor, 0.6 U / μL T7 RNA Polymerase, 0.5 U / μL Exo exonuclease, 12 mmol / L trehalose, 22 mmol / L mannitol, 10% bovine serum albumin and nuclease-free water were added to 50 μL in a 0.2 mL eight-row PCR tube. First freeze at -80℃ for 1h, then perform freeze-drying on the equipment, and the freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the partition temperature drops to -55℃, and the pre-freezing holding time is 0.6h, then the equipment is vacuumed and freeze-dried for 1.2h; in the analytical drying stage, the partition temperature is raised to -25℃ and maintained for 16h, then the partition temperature is raised to 5℃ and maintained for 0.5h, and finally the partition is lowered to 25℃ and maintained for 3h; a tubular Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPRCAS12a / CAS13a freeze-dried tube is obtained.

[0063] (2) Genome extraction: The samples to be tested are extracted with a commercial nucleic acid extraction kit to obtain nucleic acid to be tested. The samples to be tested are derived from clinical lesion tissue samples;

[0064] (3) Testing using a tube of Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a lyophilized powder or a tube of Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a lyophilized tube:

[0065] To the single-tube Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPRCAS12a / CAS13a freeze-dried tube prepared in step (1), 41.5 μL of premixed buffer A (30% polyethylene glycol) was added to reconstitute, and then 6 μL of the nucleic acid to be tested obtained in step (2) was added to prepare a reaction tube.

[0066] Then add 2.5 μL of Buffer B (300 mM magnesium acetate and 120 mM magnesium chloride) solution to the lid of the reaction tube. After closing the lid of the reaction tube, briefly rotate and centrifuge Buffer B to the bottom of the reaction tube, a total of 50 μL system, mix evenly, and perform amplification reaction. The amplification program used is: 38°C, 100 s preheating; 40°C, 30 s amplification, 40 cycles, to obtain nucleic acid amplification products;

[0067] Results of dual RAA-CRISPR CAS12a / CAS13a nucleic acid tests for Mycoplasma gallisepticum and Mycoplasma synoviae are shown in Figure 1 , Mycoplasma gallisepticum nucleic acid and Mycoplasma synoviae nucleic acid both have amplification curves, while the negative control has no amplification curve, indicating that the method provided by the present invention can detect whether Mycoplasma gallisepticum and Mycoplasma synoviae exist in poultry samples.

[0068] The reaction tubes that have completed the amplification reaction were excited by a UV lamp at 302nM using a gel imager and the results were read by naked eyes and photographed. The results were as follows: Figure 2 As shown, when the nucleic acid products of Mycoplasma gallisepticum and Mycoplasma synoviae are present in the reaction tube, white light appears, while the negative control is colorless.

[0069] Example 3 Specificity test of the dual RAA-CRISPR CAS12a / CAS13a detection method for Mycoplasma gallisepticum and Mycoplasma synoviae

[0070] In this example, infectious bursal disease live vaccine, Escherichia coli, Salmonella pullorum, Avibacterium paragallinarum, avian influenza virus, Newcastle disease live vaccine, and chicken infectious anemia virus were used together with positive and negative controls to verify the specificity of the dual RAA-CRISPR CAS12a / CAS13a method for Mycoplasma gallisepticum and Mycoplasma synoviae, and the steps were as follows:

[0071] (1) Extract DNA / RNA as a template and perform RAA-CRISPR CAS12a / CAS13a amplification detection according to the method in Example 2.

[0072] (2) The reaction system is as follows: add 41.5 μL of premixed buffer A and 6 μL of nucleic acid to be tested to a tube of Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPRCAS12a / CAS13a freeze-dried tube, and then add 0.5 μL of buffer B solution on the tube cap. After closing the tube cap of the reaction tube, briefly rotate and centrifuge buffer B to the bottom of the freeze-dried tube, a total of 50 μL system, mix well, and perform amplification reaction. The amplification program is as follows: 38°C, 60s preheating; 38°C, 30s amplification, 40 cycles; to obtain nucleic acid amplification products.

[0073] (3) The nucleic acid amplification product obtained in step (2) is tested. The results are shown in Figure 3 , Figure 3 It can be seen that the detection method of Mycoplasma gallisepticum and Mycoplasma synoviae by dual RAA-CRISPR CAS12a / CAS13a can detect Mycoplasma gallisepticum nucleic acid in the mixture of Mycoplasma gallisepticum and Mycoplasma synoviae nucleic acid through the FAM channel, and generate an amplification curve. The VIC channel can detect Mycoplasma gallisepticum nucleic acid in the mixture of Mycoplasma gallisepticum and Mycoplasma synoviae nucleic acid, and generate an amplification curve. However, there is no amplification curve for other types of pathogens (live vaccine for infectious bursal disease, Escherichia coli, Salmonella pullorum, Avibacterium paragallinarum, avian influenza virus, live vaccine for Newcastle disease, and infectious anemia virus of chickens), indicating that there is no nonspecific amplification. The detection method has better detection specificity for Mycoplasma gallisepticum and Mycoplasma synoviae.

[0074] Figure 4 The results of the dual RAA-CRISPR CAS12a / CAS13a UV-specific test for Mycoplasma gallisepticum and Mycoplasma synoviae were obtained by Figure 4 It can be seen that the dual RAA-CRISPR CAS12a / CAS13a detection method detects a mixture of Mycoplasma gallisepticum and Mycoplasma synoviae as white light, and has no amplification for other types of pathogens, which is colorless and transparent, indicating that in the method provided by the present invention, each primer will not undergo non-specific amplification. The detection method has good specificity for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

[0075] Example 4 Sensitivity test of the dual RAA-CRISPR CAS12a / CAS13a detection method for Mycoplasma gallisepticum and Mycoplasma synoviae

[0076] This example provides a sensitivity test experiment of the dual RAA-CRISPR CAS12a / CAS13a detection method for Mycoplasma gallisepticum and Mycoplasma synoviae, and the steps are as follows:

[0077] (1) The concentration is 1×10 7The positive plasmid with 10 copies / mL was diluted 10-fold. 3 The plasmid with 1×10 copies / mL was diluted 2-fold. 7 copies / mL~10 3 The sensitivity of the RAA-CRISPR-Cas12a / Cas13a reagent was tested using plasmids of various gradient concentrations of 500 copies / mL, 250 copies / mL, 125 copies / mL, and 62.5 copies / mL as templates and nuclease-free water as a negative control. The reaction conditions and reaction system were the same as in Example 2.

[0078] (2) Experimental results are shown in Figure 5 and Figure 6 , Figure 5 These are the results of the sensitivity test of Mycoplasma gallisepticum and Mycoplasma synoviae using dual RAA-CRISPRCAS12a / CAS13a to Mycoplasma gallisepticum. Figure 6 These are the results of the sensitivity test of Mycoplasma gallisepticum and Mycoplasma synoviae using dual RAA-CRISPR CAS12a / CAS13a against Mycoplasma synoviae.

[0079] The experimental results show that when the method of the present invention is used for detection, the minimum detection limit of Mycoplasma gallisepticum is 125 copies / mL, and the minimum detection limit of Mycoplasma synoviae is 125 copies / mL. The dual RAA-CRISPR CAS12a / CAS13a provided by the present invention has high sensitivity to Mycoplasma gallisepticum and Mycoplasma synoviae.

[0080] Figure 7 These are the results of the sensitivity test of Mycoplasma gallisepticum and Mycoplasma synoviae by dual RAA-CRISPR CAS12a / CAS13a under ultraviolet light. Figure 8 These are the results of the sensitivity test of Mycoplasma gallisepticum and Mycoplasma synoviae using dual RAA-CRISPRCAS12a / CAS13a under ultraviolet light.

[0081] Depend on Figure 7 It can be seen that the detection method of RAA-CRISPR CAS12a / CAS13a can detect white light for samples with a concentration of Mycoplasma gallisepticum positive plasmid above 125 copies / mL, but cannot detect white light at 62.5 copies / mL, indicating that the minimum detection limit of the present invention is 125 copies / mL.

[0082] Depend on Figure 8It can be seen that the detection method of RAA-CRISPR CAS12a / CAS13a can detect white light for samples with a concentration of 125 copies / mL or above for avian Mycoplasma synoviae positive plasmid, but cannot detect white light at 62.5 copies / mL, indicating that the minimum detection limit of the present invention is 125 copies / mL.

[0083] In summary, the detection method provided by the present invention has the characteristic of high sensitivity.

[0084] Example 5 Repeatability test of the dual RAA-CRISPR CAS12a / CAS13a detection method for Mycoplasma gallisepticum and Mycoplasma synoviae

[0085] This example provides a repeatability verification experiment of a one-tube Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a detection method, the steps of which are as follows:

[0086] (1) Repeatability analysis

[0087] Select 1×10 7 The positive plasmids of Mycoplasma gallisepticum and Mycoplasma synoviae at 100 copies / mL were tested for intra-batch repeat detection and inter-batch repeat detection respectively. Intra-batch repeat detection: the positive plasmids were repeated 3 times in the same batch experiment. Inter-batch repeat experiment: the positive plasmids were tested in 3 batches. The reaction conditions and reaction system were the same as those in Example 2.

[0088] Fig. 9 The results of the dual RAA-CRISPR CAS12a / CAS13a assays for Mycoplasma gallisepticum and Mycoplasma synoviae were obtained by Fig. 9 It can be seen that the RAA-CRISPR CAS12a / CAS13a detection method is effective for 1×10 7 1×10 copies / mL of positive plasmid and Mycoplasma synoviae 7 The positive plasmids with 100 copies / mL all had amplification curves, and the fluorescence signals and CT values ​​within the batch were basically consistent. The detection method had good intra-batch repeatability.

[0089] Fig.10 It is a dual RAA-CRISPR CAS12a / CAS13a for Mycoplasma gallisepticum and Mycoplasma synoviae under ultraviolet light 1×10 7 The positive plasmid test results were 100 copies / mL. Fig.10 It can be seen that the same batch of Mycoplasma gallisepticum and Mycoplasma synoviae dual RAA-CRISPR CAS12a / CAS13a positive plasmid 1×10 7White light can be detected for all copies / mL, indicating that the detection method has good intra-batch repeatability.

[0090] Fig.11 The results of the dual RAA-CRISPR CAS12a / CAS13a assays for Mycoplasma gallisepticum and Mycoplasma synoviae were obtained by Fig.11 It can be seen that the RAA-CRISPR CAS12a / CAS13a detection method is effective for 1×10 7 The positive plasmids with 100 copies / mL all had amplification curves, and the fluorescence signals and CT values ​​between batches were basically consistent. The detection method had good reproducibility between batches.

[0091] Fig.12 It is a dual RAA-CRISPR CAS12a / CAS13a for Mycoplasma gallisepticum and Mycoplasma synoviae under ultraviolet light 1×10 7 The positive plasmid test results were 100 copies / mL. Fig.12 It can be seen that the dual RAA-CRISPR CAS12a / CAS13a of three batches of Mycoplasma gallisepticum and Mycoplasma synoviae was 1×10 7 The white light can be detected for all positive plasmids with 100 copies / mL, indicating that the detection method has good batch reproducibility. From the above results, it can be seen that the method provided by the present invention has good reproducibility.

[0092] Example 6 Detection effect of the dual RAA-CRISPR CAS12a / CAS13a detection method for Mycoplasma gallisepticum and Mycoplasma synoviae on different types of Mycoplasma synoviae

[0093] According to the 5' terminal sequence of the vlhA gene of avian synoviae from different sources, 11 different genotypes (A to K) have been found in the present invention, among which the A to J genotypes are all found by foreign research, and the K genotype is first found in my country. No other genotypes of gallisepticum have been found so far.

[0094] The present invention uses the RAA-CRISPR / CAS detection method provided by the present invention to detect the lon gene synthetic plasmids of avian synoviae Mycoplasma genotypes A to J and the K genotype sample nucleic acid, and the results are shown in Fig.13 ; RAA-CRISPR CAS12a / CAS13a amplification reaction products were excited by UV lamp 302nM using gel imaging instrument, and the results were interpreted by naked eyes and photographed. Fig.14The results show that the dual RAA-CRISPR CAS12a / CAS13a detection method for Mycoplasma gallisepticum and Mycoplasma synoviae provided by the present invention can detect the lon genes of 11 genotypes (A to K) of Mycoplasma synoviae, and there is no nonspecific amplification in the Mycoplasma gallisepticum detection channel; it can detect Mycoplasma gallisepticum nucleic acid, and there is no nonspecific amplification in the Mycoplasma synoviae detection channel.

[0095] Comparative Example 1 Screening of the best RAA-CRISPR / CAS12a primers for Mycoplasma gallisepticum

[0096] Using Mycoplasma gallisepticum nucleic acid as a template, the three pairs of designed RAA-CRISPR / CAS12a primers (Table 2) were combined with ssDNA probes for RAA-CRISPR / CAS12a fluorescence detection. The specific reaction system is shown in Table 3 below. 2.5 μL of 300 mM magnesium acetate and 120 mM magnesium chloride reagents were added to the tube cap, and all reaction reagents were mixed to form the CRISPR / Cas12a initial detection system for centrifugation. After centrifugation, fluorescence amplification detection was performed immediately, and the FAM channel was selected. The amplification program was: 38°C, 100s preheating; 40°C, 30s amplification, 40 cycles; after completion, the data was saved for fluorescence signal analysis. The results are shown in the following table. Fig.15 shown.

[0097] The RAA-CRISPR-Cas12a amplification reaction was excited by a gel imager at 302nM UV light, and the results were interpreted by naked eyes and photographed. Fig.16 By comparing the Ct value and fluorescence intensity, the best RAA primers were screened as MS-F and MS-R, and the best Cas12a-crRNA was the Cas12a-crRNA shown in SEQ ID NO.3.

[0098] Table 2 RAA primer list

[0099]

[0100] Table 3 RAA-CRISPR CAS12a system preparation table

[0101]

[0102]

[0103] Comparative Example 2 Screening of the best RAA-CRISPR / CAS13a primers for avian Mycoplasma synoviae

[0104] Using the nucleic acid of avian synoviae as a template, the three pairs of designed RAA-CRISPR / CAS13a primers (Table 4) were combined with ssRNA probes for RAA-CRISPR / CAS13a fluorescence detection. The specific reaction system is shown in Table 5 below. 2.5 μL of 300 mM magnesium acetate and 120 mM magnesium chloride reagents were added to the tube cap, and all the reaction reagents were mixed to form the CRISPR / Cas13a initial detection system for centrifugation. After centrifugation, fluorescence amplification detection was performed immediately, and the FAM channel was selected. The amplification program was: 38°C, 100s preheating; 40°C, 30s amplification, 40 cycles; after the end, the data was saved for fluorescence signal analysis. The results are shown in the figure. Fig.17 shown.

[0105] The RAA-CRISPR-Cas13a amplification reaction was excited by a gel imager at 302nM UV light, and the results were read and photographed by naked eyes. Fig.18 By comparing the Ct value and fluorescence intensity, the best RAA primers were screened as MS-F and MS-R, and the best Cas13a-crRNA was the Cas13a-crRNA shown in SEQ ID NO.6.

[0106] Table 4 RAA primer list

[0107]

[0108] Table 5 RAA system preparation table

[0109]

[0110] Comparative Example 3 Selection of the optimal concentration of Cas12a-crRNA and CAS12a protein in the dual RAA-CRISPR CAS12a / CAS13a system of Mycoplasma gallisepticum and Mycoplasma synoviae

[0111] Using Mycoplasma gallisepticum nucleic acid as a template, nuclease-free water as a negative control, dNTPs, single-stranded binding proteins, recombinase proteins, DNA polymerases, TrisHCl, NaCl, dithiothreitol, RNase Inhibitor, T7 RNA Polymerase, and Exo exonucleases within the optimal range were used for dual RAA-CRISPR CAS12a / CAS13a fluorescence detection, and the specific reaction system is shown in Table 6 below. Among them, the final concentration of Cas12a-crRNA is X and the final concentration of Cas12a is Y as variables. The volume of LbCas12a protein and Cas12a-crRNA required to be added at different concentrations needs to be calculated with a total reaction volume of 50 μL, and the final concentrations of other components remain unchanged.

[0112] The selected Cas12a-crRNA concentrations were 0.8μM, 1.0μM, 1.2μM, 1.5μM, and 2.0μM, respectively; the selected LbCas12a protein concentrations were 0.6μM, 0.7μM, 0.8μM, 0.9μM, and 1.0μM, respectively, for cross-reaction. 2.5μL of 300mM magnesium acetate and 120mM magnesium chloride reagents were added to the tube cap, mixed evenly, centrifuged, and amplified immediately. The reporter molecule selected was a fluorescent reporter molecule, and the amplification program was 38°C, 60s preheating for 1 cycle; 38°C, 30s amplification, 40 cycles. Nucleic acid amplification products were obtained, and the Ct value data after the reaction was completed was saved and analyzed. The results are shown in Table 7. The experimental results were analyzed and the final concentration of Cas12a protein in the optimal reaction system was determined to be 0.8μmol / L, and the final concentration of Cas12a-crRNA was 1.2μmol / L.

[0113] Table 6 RAA-CRISPR CAS12a / CAS13a system preparation table

[0114]

[0115] Table 7 Screening results of the ratio of Cas12a to Cas12a-crRNA

[0116]

[0117] Comparative Example 4 Selection of the optimal concentration of Cas13a-crRNA and CAS13a protein in the dual RAA-CRISPR CAS12a / CAS13a system of Mycoplasma gallisepticum and Mycoplasma synoviae

[0118] Using avian synoviae Mycoplasma nucleic acid as a template, nuclease-free water as a negative control, dNTPs within the optimal range, single-stranded binding protein, recombinase protein, DNA polymerase, TrisHCl, NaCl, dithiothreitol, RNase Inhibitor, T7RNA Polymerase, Exo exonuclease and optimal concentrations of Cas12a-crRna and cas12a protein were used for dual RAA-CRISPR CAS12a / CAS13a fluorescence detection, and the specific reaction system is shown in Table 8 below. Among them, the final concentration of Cas13a-crRNA is X and the final concentration of Cas13a is Y as variables. The volume of LbCas13a protein and Cas13a-crRNA required to be added at different concentrations needs to be calculated with a total reaction volume of 50 μL, and the final concentrations of other components remain unchanged.

[0119] The selected Cas13a-crRNA concentrations were 0.2μM, 0.3μM, 0.4μM, 0.5μM, and 0.6μM, respectively; the selected LbCas13a protein concentrations were 2.0μM, 3.0μM, 4.0μM, 5.0μM, and 6.0μM for cross-reaction. 2.5μL of 300mM magnesium acetate and 120mM magnesium chloride reagent were added to the tube cap, mixed evenly, centrifuged, and amplified immediately. The reporter molecule selected was a fluorescent reporter molecule, and the amplification program was 38°C, 60s preheating for 1 cycle; 38°C, 30s amplification, 40 cycles. The nucleic acid amplification product was obtained, and the Ct value data after the reaction was completed was saved and analyzed. The results are shown in Table 9. The experimental results were analyzed and the final concentration of Cas13a protein in the optimal reaction system was determined to be 4μmol / L, and the final concentration of Cas13a-crRNA was 0.4μmol / L.

[0120] Table 8 RAA-CRISPR CAS12a / CAS13a system preparation table

[0121]

[0122] Table 9 Screening results of the ratio of Cas13a to Cas13a-crRNA

[0123]

[0124]

[0125] Comparative Example 5 Selection of the Optimal Concentrations of Mycoplasma gallisepticum RAA Primers and ssDNA in the Dual RAA-CRISPR CAS12a / CAS13a System of Mycoplasma gallisepticum and Mycoplasma synoviae

[0126] Using Mycoplasma gallisepticum nucleic acid as a template, nuclease-free water as a negative control, dNTPs within the optimal range, single-stranded binding protein, recombinase protein, DNA polymerase, TrisHCl, NaCl, dithiothreitol, RNase Inhibitor, T7 RNA Polymerase, Exo exonuclease and optimal concentrations of Cas12a-crRNA, cas12a protein, Cas13a-crRNA, cas13a protein were used for dual RAA-CRISPR CAS12a / CAS13a fluorescence detection, and the specific reaction system is shown in Table 10 below. Among them, the final concentration of Mycoplasma gallisepticum RAA upstream and downstream primers is X and the final concentration of ssDNA is Y as variables. The volume of Mycoplasma gallisepticum RAA upstream and downstream primers and ssDNA required to be added at different concentrations needs to be calculated with a total reaction volume of 50 μL, and the final concentrations of other components remain unchanged.

[0127] The concentrations of the upstream and downstream primers of Mycoplasma gallisepticum RAA were selected to be 2.0μM, 3.0μM, 4.0μM, 5.0μM, and 6.0μM, respectively; the concentrations of the ssDNA selected were 0.1nM, 0.2nM, 0.3nM, 0.4nM, and 0.5nM, respectively, for cross-reaction. 2.5μL of 300mM magnesium acetate and 120mM magnesium chloride reagent were added to the tube cap, mixed evenly, centrifuged, and amplified immediately. The reporter molecule selected was a fluorescent reporter molecule, and the amplification program was 38℃, 60s preheating for 1 cycle; 38℃, 30s amplification, 40 cycles. The nucleic acid amplification product was obtained, and the Ct value data after the reaction was completed was saved and analyzed. The results are shown in Table 11. The experimental results were analyzed and the final concentrations of the upstream and downstream primers of Mycoplasma gallisepticum RAA in the optimal reaction system were determined to be 4.0μmol / L, and the final concentration of ssDNA was 0.2nmol / L.

[0128] Table 10 RAA-CRISPR CAS12a / CAS13a system preparation table

[0129]

[0130] Table 11 Screening results of the ratio of Mycoplasma gallisepticum RAA primers to ssDNA

[0131]

[0132] Comparative Example 6 Selection of the Optimal Concentrations of RAA Primers and ssRNA for Mycoplasma gallisepticum and Mycoplasma synoviae in the Dual RAA-CRISPR CAS12a / CAS13a System

[0133] Using avian synoviae Mycoplasma nucleic acid as a template, nuclease-free water as a negative control, dNTPs within the optimal range, single-stranded binding protein, recombinase protein, DNA polymerase, TrisHCl, NaCl, dithiothreitol, RNase Inhibitor, T7 RNA Polymerase, Exo exonuclease and optimal concentrations of Cas12a-crRNA, cas12a protein, Cas13a-crRNA, and cas13a protein were used for dual RAA-CRISPR CAS12a / CAS13a fluorescence detection. The specific reaction system is shown in Table 12 below. Among them, the final concentration of avian synoviae Mycoplasma RAA upstream and downstream primers is X and the final concentration of ssRNA is Y as variables. The volume of avian synoviae Mycoplasma RAA upstream and downstream primers and ssRNA required to be added at different concentrations needs to be calculated with a total reaction volume of 50 μL, and the final concentrations of other components remain unchanged.

[0134] The concentrations of the upstream and downstream primers of avian synoviae Mycoplasma RAA were selected to be 2.0μM, 3.0μM, 4.0μM, 5.0μM, and 6.0μM, respectively; the concentrations of the ssRNA selected were 1nM, 2nM, 3nM, 4nM, and 5nM for cross-reaction. 2.5μL of 300mM magnesium acetate and 120mM magnesium chloride reagent were added to the tube cap, mixed evenly, centrifuged, and amplification reaction was performed immediately. The reporter molecule selected was a fluorescent reporter molecule, and the amplification program was 38°C, 60s preheating for 1 cycle; 38°C, 30s amplification, 40 cycles. The nucleic acid amplification product was obtained, and the Ct value data after the reaction was completed was saved and analyzed. The results are shown in Table 13. The experimental results were analyzed and the final concentrations of the upstream and downstream primers of avian synoviae Mycoplasma RAA in the optimal reaction system were determined to be 4.0μmol / L, and the final concentration of ssRNA was 1.0nmol / L.

[0135] Table 12 RAA-CRISPR CAS12a / CAS13a system preparation table

[0136]

[0137] Table 13 Screening results of the ratio of RAA primers to ssRNA of avian mycoplasma synoviae

[0138]

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual RAA-CRISPR / CAS reaction system for Mycoplasma gallisepticum and Mycoplasma synoviae, characterized in that: include: RAA primer combinations for Mycoplasma gallisepticum and Mycoplasma synoviae, crRNA molecules for detecting Mycoplasma gallisepticum and Mycoplasma synoviae based on CRISP R / CAS technology, Cas12a-ssDNA reporter genes with a sequence such as FAM-TT TTTTTTTT-BHQ1, and Cas13a-ssRNA reporter genes with a sequence such as ROX-UUUUUUUU-BHQ2; The RAA primer combination includes a RAA primer pair for amplifying Mycoplasma gallisepticum, whose sequence is shown in SEQ ID NO.1-2, and a RAA primer pair for amplifying Mycoplasma synoviae, whose sequence is shown in SEQ ID NO.4-5; The crRNA molecule of Mycoplasma gallisepticum is Cas12a-crRNA, and its sequence is shown in SEQ ID NO.3; the crRNA molecule of Mycoplasma synoviae is Cas13a-crRNA, and its sequence is shown in SEQ ID NO.6; In the dual RAA-CRISPR / CAS reaction system, the concentration ratio of LbCas12a protein to Cas12a-crRNA is (0.6-1): (0.8-2); the concentration ratio of Mycoplasma gallisepticum RAA upstream and downstream primers to Cas12a-ssDNA reporter gene is (2000-6000): (0.1-0.5).

2. The dual RAA-CRISPR / CAS reaction system according to claim 1, characterized in that: In the dual RAA-CRISPR / CAS reaction system, the concentration ratio of LbCas13a protein to Cas13a-crRNA is (2-6): (0.2-0.6); the concentration ratio of avian synoviae Mycoplasma RAA upstream and downstream primers to Cas13a-ssRNA reporter gene is (2000-6000): (1-5).

3. The dual RAA-CRISPR / CAS reaction system according to any one of claims 1 to 2, characterized in that: The dual RAA-CRISPR / CAS reaction system includes: 2-10 mmol / L dNTP, 0.2-1.0 μmol / L single-stranded binding protein, 1.0-2.0 μmol / L recombinase protein, 0.2-1.0 U / μL DNA polymerase, 10-50 mmol TrisHCl, 40-80 mmol NaCl, 6-10 mmol / L dithiothreitol, 1.0-5.0 μmol / L Mycoplasma gallisepticum RAA upstream and downstream primers, 1.0-5.0 μmol / L Mycoplasma synoviae RAA upstream and downstream primers, 1-2 μmol / L Cas12a-crRNA, 0.5-1.0 μmol / L LbCas12a, 0.1-0.5 nmol / L Cas12a-ssDNA reporter gene, 0.2-0.8μmol / LCas13a-crRNA, 1-5μmol / LLbCas13a, 0.5-1.5nmol / L Cas13a-ssRNA reporter gene, 0.4-1.0U / μL RNase Inhibitor, 0.4-1.0U / μL T7 RNA Polymerase, 0.1-1U / μL Exo exonuclease.

4. A single-tube freeze-dried tube for simultaneously detecting Mycoplasma gallisepticum and Mycoplasma synoviae, characterized in that: The reaction system for detecting Mycoplasma gallisepticum and Mycoplasma synoviae is fixed in the one-tube freeze-drying tube by freeze-drying, and the reaction system for detecting Mycoplasma gallisepticum and Mycoplasma synoviae contains the dual RAA-CRISPR / CAS reaction system according to any one of claims 1 to 3.

5. The method for preparing a tubular freeze-drying tube according to claim 4, characterized in that: The freeze-drying conditions include: freezing a tubular freeze-drying tube containing the dual RAA-CRISPR / CAS reaction system at -80°C for 1 to 1.5 hours and then performing device freeze-drying; the device freeze-drying includes: a pre-freezing stage, equipment vacuuming, freeze drying and analytical drying stages; the conditions of the analytical drying stage are: raising the partition temperature to -20 to -25°C and maintaining it for 12 to 16 hours, then raising the partition temperature to 4 to 8°C and maintaining it for 0.5 to 1 hour, and finally lowering the partition temperature to 20 to 25°C and maintaining it for 3 to 5 hours.

6. A detection kit for simultaneously detecting Mycoplasma gallisepticum and Mycoplasma synoviae, characterized in that: The detection kit contains the dual RAA-CRISPR / CAS reaction system according to any one of claims 1 to 3 or the one-tube lyophilized tube according to claim 4.

7. A method for detecting Mycoplasma gallisepticum and / or Mycoplasma synoviae for non-disease diagnosis and treatment purposes, characterized in that: The method comprises the steps of obtaining a nucleic acid of a test sample as the test nucleic acid, taking the test nucleic acid as a template, performing real-time PCR on the test nucleic acid using the detection kit according to claim 6, monitoring the amplification process of the real-time PCR, and generating an amplification curve during the amplification process, indicating that the test sample is positive for Mycoplasma gallisepticum and / or Mycoplasma synoviae; generating an amplification curve in the FAM channel, indicating that Mycoplasma gallisepticum is positive; and generating an amplification curve in the VIC channel, indicating that Mycoplasma synoviae is positive; Or use the detection kit described in claim 6 to detect the nucleic acid to be tested to obtain an amplification product; if the amplification product appears as white light under ultraviolet light, it means that the sample to be tested is positive for Mycoplasma gallisepticum and / or Mycoplasma synoviae; if the amplification product appears colorless and transparent under ultraviolet light, it means that the sample to be tested is positive for Mycoplasma gallisepticum and Mycoplasma synoviae.

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