On-site rapid detection method for mycoplasma ovipneumoniae based on MIRA technology and application of on-site rapid detection method

Abstract

The invention relates to the technical field of sheep contagious pleuropneumonia diagnosis, and discloses a method for detecting mycoplasma ovipneumoniae based on a multi-enzyme constant-temperature nucleic acid rapid amplification technology (MIRA), which comprises the following steps: extracting DNA of a sample to be detected; taking the DNA as a template, and carrying out MIRA amplification reaction by using a specific primer pair and a probe; an upstream primer sequence of the primer pair is as shown in SEQ ID NO: 1, and a downstream primer sequence is as shown in SEQ ID NO: 2; the probe sequence is shown as SEQ ID NO: 3, the 5'end of the probe is marked with an FAM fluorophore, the 3 'end of the probe is marked with a BHQ1 quenching group, and a tetrahydrofuran (THF) site is inserted into the position 30-35 nt away from the 5' end; reacting for 20-25 minutes at the constant temperature of 41 DEG C, and monitoring a fluorescence signal in real time. According to the invention, a multi-enzyme constant-temperature nucleic acid rapid amplification technology (MIRA) is applied to on-site rapid detection of mycoplasma ovipneumoniae (Mo) for the first time, and the technical bottlenecks that traditional PCR depends on expensive instruments, is complex to operate and consumes long time are broken through.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of diagnosis of contagious pleuropneumonia of sheep, and in particular to a method for rapid detection of mycoplasma pneumonia of sheep based on MIRA technology and application thereof. BACKGROUND

[0002] With the rapid expansion of sheep breeding scale in Yunnan, contagious pleuropneumonia of sheep (MPSG) has become the primary respiratory disease restricting the healthy development of the industry. The disease is caused by mycoplasma pneumonia of sheep (Mo) and other mycoplasma, and is characterized by high fever, cough, and fibrous pleuropneumonia. It is highly contagious and has a high mortality rate. Due to the lack of scientific prevention and detection capabilities of grass-roots breeders, the epidemic has been endemic and persistent for a long time, causing serious economic losses.

[0003] There are currently three types of diagnostic methods:

[0004] Morphological diagnosis requires isolation and culture, which is time-consuming and has low sensitivity;

[0005] Serological diagnosis (ELISA, IHA, etc.) has cross-reactions and is prone to miss early infections;

[0006] Molecular detection, mainly PCR and fluorescent quantitative PCR, relies on laboratory precision instruments and cold chains, and has poor adaptability in the field.

[0007] MIRA (Multienzyme Isothermal Rapid Amplification) is a constant-temperature nucleic acid amplification technology developed on the basis of RPA, which can complete exponential amplification of target genes within 20 minutes at 37-42℃, and the results can be read in real time through a fluorescent probe. This technology does not require a thermal cycler and has been widely used in the field of foodborne pathogens, viruses, etc., but it is still a blank in the field of rapid detection of mycoplasma pneumonia of sheep.

[0008] Therefore, there is an urgent need for a new method to detect Mo quickly and accurately in the field, such as on a farm or at a border port, without complex equipment, to make up for the limitations of traditional PCR technology and provide technical support for early warning and precise prevention and control of contagious pleuropneumonia of sheep. SUMMARY

[0009] To solve the technical problems raised in the background art, the present application provides a method for detecting mycoplasma pneumonia of sheep based on multienzyme constant-temperature nucleic acid rapid amplification technology (MIRA), a primer probe combination for MIRA detection of mycoplasma pneumonia of sheep, and the application of a method or kit for detecting mycoplasma pneumonia of sheep in the preparation of a diagnostic product for mycoplasma pneumonia of sheep, and the application of a method or kit for detecting mycoplasma pneumonia of sheep in rapid detection of contagious pleuropneumonia of sheep in the field.

[0010] The application adopts the following technical scheme to realize the method for detecting mycoplasma pneumoniae of sheep based on multi-enzyme constant temperature nucleic acid rapid amplification technology (MIRA), which comprises the following steps:

[0011] extracting DNA of the sample to be detected;

[0012] using specific primer pairs and probes for MIRA amplification reaction with the DNA as a template;

[0013] the sequence of the upstream primer of the primer pairs is shown in SEQ ID NO:1, and the sequence of the downstream primer is shown in SEQ ID NO:2;

[0014] the sequence of the probe is shown in SEQ ID NO:3, the 5' end of which is labeled with a FAM fluorescent group, the 3' end is labeled with a BHQ1 quenching group, and a tetrahydrofuran (THF) site is inserted at a position 30-35 nt away from the 5' end;

[0015] reacting for 20-25 minutes under constant temperature conditions at 41 DEG C, and monitoring the fluorescence signal in real time;

[0016] determining the result according to the fluorescence signal growth curve: the appearance of an "S" curve is positive, and otherwise is negative.

[0017] Further, the MIRA amplification reaction system comprises:

[0018] A buffer solution;

[0019] an upstream primer;

[0020] a downstream primer;

[0021] a probe;

[0022] a DNA template and a ddH2O mixture;

[0023] a B buffer solution.

[0024] Further, premixing treatment is required before the reaction, and the premixing time is 3 minutes.

[0025] The second purpose of the application is to provide a primer probe combination for MIRA detection of mycoplasma pneumoniae of sheep,

[0026] the sequence of the upstream primer is 5'-CGTTAAATGATCCGCCTGAGTAGTATGCTCGCAAG-3' (SEQ ID NO:1);

[0027] the sequence of the downstream primer is 5'-GCCATTGTAGCACGTGTGTTGCCCCACTCGTAAGA-3' (SEQ ID NO:2);

[0028] The probe sequence is:

[0029] 5'-GTCGTCAGCTCGTGTCGTGAGATGTTAGGT[FAM]AA[THF][BHQ1]CCTGCAACGAGCGCA-3'(SEQ ID NO:3).

[0030] The third object of the present application is to provide a Mycoplasma ovinum MIRA detection kit, characterized in that it comprises the primer probe combination of claim 4.

[0031] Further, the kit is a freeze-dried preparation, comprising recombinant enzymes, single-stranded DNA binding protein (SSB), DNA polymerase and dNTPs.

[0032] Further, the kit is used with a portable fluorescence detection device, and the detection is completed within 25 minutes at a constant temperature of 41℃.

[0033] The fourth object of the present application is to provide the use of the method or kit for detecting Mycoplasma ovinum in the preparation of a diagnostic product for Mycoplasma ovinum.

[0034] The fifth object of the present application is to provide the use of the method or kit for detecting Mycoplasma ovinum in the rapid on-site detection of contagious pleuropneumonia of sheep.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The present application first applies the multi-enzyme constant-temperature nucleic acid rapid amplification technology (MIRA) to the on-site rapid detection of Mycoplasma ovinum (Mo), breaking through the technical bottleneck of traditional PCR which relies on expensive instruments, complex operation and long time consumption. By precisely designing the Mo 16S rRNA gene specific primer probe combination (SEQ ID NO: 1-3), optimizing the reaction system and procedure (41℃ constant temperature, 3 minutes pre-mixing, 20-25 minutes for completing amplification), a high sensitivity of 0.12fg / μL is achieved, and there is no cross reaction with 6 kinds of pathogens such as bovine mycoplasma and orf virus, with a specificity of 100%. The portable fluorescence detection device is matched, and the detection can be completed on-site at the border, pasture and the like without laboratory environment, with a positive detection rate of clinical samples of 59.8%, which is significantly better than that of PCR method (46.3%). The kit adopts freeze-dried preparation, and can be transported and stored at room temperature, and is easy to operate, which provides an efficient, economical and reliable technical means for the epidemic monitoring and cross-border animal disease prevention and control of sheep mycoplasma pneumonia in remote areas such as Yunnan. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The test sample collection area schematic diagram provided for the embodiments of the present application is shown in the figure;

[0038] Figure 2 Respiratory symptom chart for embodiments of the present application;

[0039] Figure 3 Sheep nasal swab Mo PCR detection results for embodiments of the present application; Wherein: M: DNA marker; P: positive control; N: negative control; 1-20: nasal swab;

[0040] Figure 4 Sheep nasal swab Mycoplasma filamentous cluster PCR detection results for embodiments of the present application; Wherein: M: DNA marker; P: positive control; N: negative control; 1-20: nasal swab;

[0041] Figure 5 Sheep nasal swab Ma PCR detection results for embodiments of the present application; Wherein: M: DNA marker; P: positive control; N: negative control; 1-22: nasal swab;

[0042] Figure 6 Statistical column chart of PCR detection results of sheep nasal swabs under different breeding methods for embodiments of the present application;

[0043] Figure 7 Statistical column chart of PCR detection results of sheep nasal swabs of different breeds for embodiments of the present application;

[0044] Figure 8 Statistical column chart of mixed infection PCR detection results for embodiments of the present application;

[0045] Figure 9 Mo phylogenetic tree for embodiments of the present application; Wherein: Tengchong(1): Tengchong(Zhonghe Town); Tengchong(2): Tengchong(Gudong Town); Shidian(1): Shidian(Dianyang Town); Shidian(3): Shidian(Renhen);

[0046] Figure 10 Homology comparison chart of Mo for embodiments of the present application;

[0047] Figure 11 Ma pathogen phylogenetic tree for embodiments of the present application; Wherein: Tengchong(2): Tengchong(Gudong Town); Shidian(1): Shidian(Dianyang Town); Shidian(3): Shidian(Renhen) Note: Tengchong(2): Tengchong(Gudong Town); Shidian(1): Shidian(Dianyang Town); Shidian(3): Shidian(Renhen);

[0048] Figure 12 Ma homology comparison chart proposed for the embodiments of the present application;

[0049] Figure 13 Probe 1 and primer screening comparison chart proposed for the embodiments of the present application;

[0050] Figure 14 Probe 2 and primer screening comparison chart proposed for the embodiments of the present application;

[0051] Figure 15 Probe 1 and probe 2 screening comparison chart proposed for the embodiments of the present application;

[0052] Figure 16 MIRA reaction temperature optimization comparison chart proposed for the embodiments of the present application;

[0053] Figure 17 MIRA preheating time optimization comparison chart proposed for the embodiments of the present application;

[0054] Figure 18 Positive plasmid PCR result schematic diagram proposed for the embodiments of the present application;

[0055] Figure 19 pMD-19T-Movi bacterial liquid PCR result chart proposed for the embodiments of the present application;

[0056] Figure 20 Mo plasmid result peak chart proposed for the embodiments of the present application;

[0057] Figure 21 Mo plasmid fragment sequencing result and reference sequence comparison result proposed for the embodiments of the present application;

[0058] Figure 22 Mo MIRA sensitivity test result comparison chart proposed for the embodiments of the present application;

[0059] Figure 23 Mo MIRA repeatability test result comparison chart proposed for the embodiments of the present application;

[0060] Instrument name Mo MIRA specificity test result comparison chart proposed for the embodiments of the present application. DETAILED DESCRIPTION

[0061] The present application will be further described below in conjunction with the drawings and specific embodiments, and it should be noted that the following described embodiments or technical features can be combined in any manner to form new embodiments without conflict.

[0062] Embodiments:

[0063] This embodiment is directed to a method for rapid detection of Mycoplasma ovipneumoniae in the field based on MIRA technology, which is illustrated by the following examples.

[0064] Step 2.1.1: From July 2024 to February 2025, 384 sheep nasal swab samples and 182 sheep serum samples were randomly collected from 15 farms in 8 prefectures of Yunnan Province, including Chuxiong Yi Autonomous Prefecture, Kunming City, Baoshan City, Nujiang Lisu Autonomous Prefecture, Lijiang City, and Zhaotong City. See Table 1 for detailed information.

[0065] Table 1: Information of sheep nasal swab samples

[0066]

[0067] Step 2.1.2 Test Strains

[0068] Mycoplasma ovipneumoniae was donated by the Animal Disease Prevention and Control Center of Yunnan Province; Mycoplasma bovis, Mycoplasma synoviae, and pseudorabies virus were donated by the Basic Laboratory of Yunnan Agricultural University; Orf virus, fowlpox virus, and Clostridium perfringens were preserved in the laboratory.

[0069] Step 2.1.3 Main Instruments and Equipment

[0070] The main instruments used in this test are shown in Table 2.

[0071] Table 2: Main Instruments and Manufacturers

[0072] Manufacturer PCR instrument Hangzhou Biheng Technology Co., Ltd. Autoclave Zhisu (Xiamen) Instrument Co., Ltd. Snowflake ice maker Tuohe Electromechanical Technology (Shanghai) Co., Ltd. Reagent

[0073] Table 2-2 (continued)

[0074]

[0075]

[0076] Step 2.1.4 Main Reagent Materials

[0077] The main reagent materials used in this test are shown in Table 3.

[0078] Table 3: Main Reagent Materials

[0079]

[0080] Step 2.1.5 Preparation of Culture Medium and Reagents

[0081] The main culture medium preparation in this test is shown in Table 4.

[0082] Table 4: Preparation of Main Culture Medium and Reagents

[0083]

[0084]

[0085] Step 2.2.1 Sample collection

[0086] When collecting the sheep nasal swab, a sterile cotton swab was inserted into the left and right nasal cavities of the sheep, respectively, to collect the nasal secretions, which were placed in a 2 mL cryogenic tube. The cryogenic tube was transported back to the Basic Laboratory of Yunnan Agricultural University in a liquid nitrogen tank for processing.

[0087] Step 2.2.2 Extraction of sample DNA:

[0088] Add 500 μL of PBS solution to the cryogenic tube containing the nasal swab, vortex or repeatedly press the cotton swab to ensure that the sample is fully dissolved in physiological saline. Discard the cotton swab and use TaKaRa to extract DNA. The specific steps are as follows:

[0089] Step (1), take 200 μL of the above solution in a 2 mL centrifuge tube; add 200 μL of Buffer VGB, 20 μL of Proteinase K and 1 μL of Carrier RNA, and mix well in a 56°C water bath for 10 min.

[0090] Step (2), add 200 μL of 100% anhydrous ethanol to the lysis solution.

[0091] Step (3), place a Spin Column on the Collection Tube; transfer the above solution to the Spin Column, centrifuge at 12 000 rpm for 2 min, and discard the filtrate.

[0092] Step (4), add 500 μL of Buffer RWA, centrifuge at 12 000 rpm for 1 min, and discard the filtrate.

[0093] Step (5), add 700 μL of Buffer RWB, centrifuge at 12 000 rpm for 1 min, and discard the filtrate.

[0094] Step (6), repeat step (5).

[0095] Step (7), place the Spin Column on the Collection Tube, centrifuge at 12 000 rpm for 2 min.

[0096] Step (8), place the Spin Column on a new 1.5 mL RNase free collection tube, add 50 μL of RNase free ddH2O at the center membrane, and stand for 5 min.

[0097] Step (9), after measuring the DNA concentration using micro-spectrophotometer, store at -20℃.

[0098] Step 2.2.3 PCR detection

[0099] Step 2.2.3.1 Primer synthesis

[0100] All were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. Primer information is shown in Table 5.

[0101] Table 5: PCR detection primer

[0102]

[0103]

[0104] Step 2.2.3.2 PCR amplification

[0105] Using the extracted nasal swab DNA as template, PCR detection was performed using the above primers, and the amplification system is shown in Table 6, and the reaction program is shown in Table 7.

[0106] Table 6: PCR reaction system

[0107] Volume (μL) 2×Taq Master Mix Forward primer 10.0 Reverse primer 1.0 DNA template 1.0 ddH2O 7.0 Total system 1.0 Composition 20.0

[0108] Table 7: PCR reaction program

[0109]

[0110] Note: The annealing temperature of Mycoplasma pneumoniae and Mycoplasma arginini is 55℃, and the annealing temperature of Mycoplasma filamentous cluster is 61℃.

[0111] Step 2.2.3.3 Agarose gel electrophoresis detection

[0112] Prepare 1% ordinary agarose gel and 1x TAE electrophoresis liquid, take 10 μL of PCR product for spotting in turn, take 8 μL of Marker as a marker, connect the positive and negative poles, adjust the voltage to 100 V, the current to 400 mA, and electrophorese for 40 min. After the end, place the agarose gel under the ultraviolet gel imager to observe, record and save.

[0113] Step 2.2.4 TA cloning

[0114] Step 2.2.4.1 PCR and agarose gel electrophoresis

[0115] Using the primers of 2.2.3.1, select part of the samples for PCR, and the steps are the same as 2.2.3.2 and 2.2.3.3.

[0116] Step 2.2.4.2 PCR product gel DNA recovery

[0117] The PCR product was recovered by gel, and the specific steps were as follows:

[0118] (1) When the electrophoresis was finished and the DNA fragments were separated, the agarose was placed under the ultraviolet gel imager, the gel containing the target DNA fragments was quickly cut off using a gel cutter, and the excess gel was removed as much as possible and placed in a 1.5 mL centrifuge tube.

[0119] (2) According to the weight and concentration of the gel block, 100 μL of Buffer GDP was added to the centrifuge tube containing the gel block.

[0120] (3) The centrifuge tube was placed in a 50℃ water bath for 15 min to completely dissolve the gel block. During the water bath, it was inverted and mixed 3 times to accelerate the dissolution.

[0121] (4) The liquid droplets on the wall of the collection tube were quickly collected. The HiPure DNA Mini Column I was placed in the collection tube. The melted solution was completely transferred to the adsorption column, and centrifuged at 12 000 r / min for 60 s, and the filtrate was discarded.

[0122] (5) The centrifugal column was placed back into the collection tube, 150 μL Buffer GDP was added to the adsorption column, and it was placed for 1 min, and centrifuged at 12 000 r / min for 60 s, and the filtrate was discarded.

[0123] (6) The centrifugal column was placed back into the collection tube, 600 μL Buffer DW2 (diluted with anhydrous ethanol) was added to the adsorption column, and centrifuged at 12 000 r / min for 60 s, and the filtrate was discarded.

[0124] (7) The centrifugal column was placed back into the collection tube, 300 μL Buffer DW2 (diluted with anhydrous ethanol) was added to the adsorption column, and centrifuged at 12 000 r / min for 2 min, and the filtrate was discarded.

[0125] (8) The lid of the centrifugal column was opened, and air-dried for 10 min to completely remove the ethanol.

[0126] (9) The adsorption column was placed in a new 1.5 mL centrifuge tube, and 15 μL Elution Buffer (preheated to 55℃) was added to the center of the adsorption column membrane. It was placed for 2 min, and centrifuged at 12 000 r / min for 1 min.

[0127] (10) After measuring the concentration of the gel-recovered DNA using a microspectrophotometer, it was stored at -20℃.

[0128] Step 2.2.4.3 Ligation of the target gene and pMD-19T cloning vector

[0129] pMD-19T Vector is a special vector for cloning PCR products (TACloning) with the features of blunt-end ligation, containing a multiple cloning site, with ampicillin selection marker. The connection system is prepared according to Table 8, and the gel-recovered target fragment is connected to the cloning vector pMD-19T. The PCR tube is centrifuged at 16°C for 2h.

[0130] Table 8: pMD-19T vector connection system

[0131] Volume (μL) pMD-19T vector Gel recovery 0.3 Solution I 2.2 Total 2.5 Component 5.0

[0132] Step 2.2.4.4 Connection product transformation

[0133] The connection product is transformed into E. coli DH5a competent cells by heat shock method, and the specific operation steps are as follows:

[0134] (1) Prepare Amp-containing agar plates, Amp-containing liquid LB, and ice box.

[0135] (2) Take the previously prepared DH5a competent cells from the -80°C refrigerator and place them in the ice box.

[0136] (3) Open the clean bench, and add 5mL of connection product to 50mL of DH5a competent cells.

[0137] (4) Ice bath for 30min, quickly transfer to 42°C metal bath heat shock for 90s, and then ice bath for 3min.

[0138] (5) Add 800mL of blank LB broth medium in the clean bench, and place it in a 37°C, 250rpm shaker for 1h.

[0139] (6) Take 150μL of bacteria-containing LB broth in step (5) and evenly spread it on the Amp-containing LB agar plate using a spreader.

[0140] (7) Invert in a 37°C constant temperature incubator for 12h overnight culture.

[0141] Step 2.2.4.5 Pick bacteria and send for testing

[0142] Pick a single colony from the overnight culture in 2.2.4.4 in 500mL of LB broth medium with Amp, and place it in a 37°C, 250rpm shaker for 1h. Then send it to GenScript Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results are saved and analyzed.

[0143] Step 2.2.5 Genetic evolution analysis

[0144] The sequencing results obtained in 2.2.4 were spliced, and after splicing, the sequence results were subjected to sequence alignment in BLAST of NCBI (https: / / www.ncbi.nlm.nih.gov / ). MEGA11 was used to make a phylogenetic tree for data analysis.

[0145] Step 2.2.6 ELISA

[0146] After the collected serum was placed for 30 min, it was centrifuged at 3000 r / min for 5 min, and the supernatant was collected and stored at -20℃ for standby. The Mo ELISA antibody detection kit was used, and the steps were as follows:

[0147] (1) Warm the serum and all reagents of the ELISA kit.

[0148] (2) Number the samples according to the microwell numbers, and set up 2 wells of negative control, 2 wells of positive control, and 1 well of blank control for each plate.

[0149] (3) Add 50 μL of negative control and positive control to the negative and positive control wells, respectively. In the sample wells, first add 40 μL of sample diluent, and then add 10 μL of serum to be tested (try to add the serum to the bottom of the enzyme-labeled plate, and do not touch the wall of the well).

[0150] (4) After sealing the plate with a sealing film, incubate at 37℃ for 30 min.

[0151] (5) Dilute the concentrated solution with distilled water to 1-fold dilution concentration.

[0152] (6) Remove the sealing film, discard the liquid, and dry, then add enough washing solution to each well, stand for 30 s, and discard the liquid. Repeat 5 times and pat dry.

[0153] (7) Add 50 μL of enzyme-labeled reagent to the sample wells except the blank wells.

[0154] (8) Repeat step (4).

[0155] (9) Repeat step (6).

[0156] (10) Add 50 μL of color developing agent A to each well, then add 50 μL of color developing agent B, shake well, and develop color at 37℃ for 15 min.

[0157] (11) Add 50 μL of stop solution to each well to stop the reaction.

[0158] (12) Adjust the zero with the blank well, and measure the absorbance (OD value) of each well at 450 nm.

[0159] (13) Result determination: when the average OD value of the positive control hole is ≥1.0, and the negative control is ≤0.2, the test is valid. The average OD value of the negative control hole +0.15 is the critical value. The OD value of the sample hole < the critical value is negative, and ≥ the critical value is positive.

[0160] Step 2.2.7 Design and synthesis of Mo MIRA primer probe

[0161] The conserved gene of Mycoplasma ovipneumoniae is 16S rRNA, so this fragment is selected for the design of probes and primers. Ten 16S rRNA of Mo pathogens were screened and downloaded from the NCBI GenBank database. After aligning the 10 sequences using Geneious Prime software, according to the probe design principle of MIRA: select a segment of 46nt-52nt in the middle of the upstream and downstream, which does not overlap with the specific primer recognition site, avoid palindromic sequences, internal secondary structure and continuous repeated bases. As the recognition site of specific exonuclease, one base is replaced with tetrahydrofuran (THF) in the middle and back of 30nt-35nt from the 5' end, and the base is any one of the four bases. The T base upstream of the THE site is labeled with a fluorescent group (FAM), and the T base downstream is labeled with a quenching group (BHO1). The distance between the two modified T bases is 1nt-3nt. A modified group C3-spacer is labeled at the 3' end. The designed Mo probe primer information is shown in Table 9.

[0162] Table 2-9 Primer probe information table

[0163]

[0164]

[0165] Step 2.2.8 Optimization of Mo MIRA reaction system

[0166] Step 2.2.8.1 Primer screening

[0167] Match the 3 upstream and 3 downstream primers corresponding to probe 1, a total of 9 pairs of primers are obtained, and the primer matching information is shown in Table 10. Match the 3 upstream and 3 downstream primers corresponding to probe 2, a total of 9 pairs of primers are obtained, and the primer matching information is shown in Table 11. Use the MIRA detection kit (fluorescent type), refer to the reaction system table 12 and reaction procedure table 13 provided by the kit to screen primers, and arrange and record the test results. Select one pair of primers with the smallest Ct value and the highest fluorescence value from the 9 pairs of primers for subsequent experiments.

[0168] Table 10: MIRA probe 1 primer matching information

[0169]

[0170] Table 11: Probe 2 primer matching information

[0171]

[0172] Table 12: MIRA reaction system

[0173] Volume (μL) A buffer Upstream primer (10 μM) 29.4 Downstream primer (10 μM) 2.0 Probe (10 μM) 2.0 B buffer 0.6 DNA template and ddH2O 13.5 Total volume 2.5 Temperature 50.0

[0174] Note: Add 5 μL of nucleic acid template and 8.5 μL of ddH2O. The volume of template added can be adjusted according to actual needs, and the added ddH2O is adjusted accordingly, so that the total volume of template and ddH2O is 13.5 μL.

[0175] Table 13: MIRA reaction program

[0176] Time Cycle number Sample source 39℃ 30s 40

[0177] Step 2.2.8.2 Optimization of the optimal reaction program of Mo MIRA

[0178] (1) Reaction temperature optimization

[0179] While keeping other conditions unchanged, set the reaction temperature to 39°C, 40°C, 41°C and 42°C. After the reaction is completed, select the temperature with the smallest Ct value and the highest peak value as the optimal reaction temperature of the MIRA detection method.

[0180] (2) Reaction premixing time optimization

[0181] Premixing before the original reaction program can accelerate the reaction. While keeping other conditions unchanged, set the premixing time to 1 min, 2 min, 3 min, 4 min and 5 min at the temperature selected in 2.2.8.2(1). After the reaction is completed, select the temperature with the smallest Ct value and the highest peak value as the optimal reaction temperature of the MIRA detection method.

[0182] Step 2.2.9 Preparation of template plasmid

[0183] Step 2.2.9.1 Fragment amplification

[0184] The preparation of Mycoplasma pneumoniae positive plasmid can provide detection materials with higher purity for the subsequent determination of the sensitivity of the detection method. The primers selected in 2.2.8.1 are used for PCR amplification of the target fragment, and the reaction system is shown in Table 2-6 and the reaction program is shown in Table 2-7. After the PCR reaction is completed, 1% agarose gel electrophoresis is performed.

[0185] Step 2.2.9.2 Product recovery and purification

[0186] The steps are the same as 2.2.4.2.

[0187] Step 2.2.9.3 Recombination of the fragment with the vector

[0188] Step same as 2.2.4.3.

[0189] Step 2.2.9.4 Transformation of the ligated vector into competent cells

[0190] Step same as 2.2.4.4.

[0191] Step 2.2.9.5 Shake culture and PCR identification of the bacterial solution

[0192] Step same as 2.2.4.5.

[0193] Step 2.2.9.6 Plasmid extraction

[0194] The identified bacterial solution of 2.2.9.5 is subjected to plasmid extraction, and the specific steps are as follows:

[0195] (1) The above bacterial solution is subjected to overnight culture in LB broth, and the bacterial solution is collected by centrifugation at 4200 x g for 10 min at room temperature.

[0196] (2) The culture solution is discarded, 500 μL of Solution I / RNaes A mixed solution is added, and the cells are fully suspended by vortex oscillation.

[0197] (3) 500 μL of Solution II is added, and the centrifuge tube is gently inverted up and down for 8-10 times, and is allowed to stand at room temperature for 2 min (the standing time is not more than 5 min, and vigorous shaking is avoided, otherwise the chromosomal DNA will be broken and the purity of the plasmid will be reduced).

[0198] (4) 250 μL of pre-cooled N3 Buffer is added, and the centrifuge tube is gently inverted up and down until a white flocculent precipitate appears, and is centrifuged at 13 000 x g for 10 min at room temperature.

[0199] (5) The supernatant is transferred to a new 1.5 mL centrifuge tube, the volume of the supernatant is estimated, and 0.1 times the volume of the supernatant is added to the lysate ETR Solution, which is mixed by inverting the tube up and down for 10 times, and is placed in ice bath for 10 min (after ice bath, the solution gradually becomes clear).

[0200] (6) The lysate is placed in a 42°C water bath for 5 min, and the lysate becomes turbid again, and is centrifuged at 12 000 x g for 3 min at 25°C, and the ETR Solution forms a blue layer at the bottom of the test tube.

[0201] (7) The supernatant is transferred to a new 2 mL centrifuge tube, 0.5 times the volume of anhydrous ethanol is added, and the test tube is gently inverted up and down for 6-7 times, and is allowed to stand at room temperature for 1-2 min.

[0202] (8) Insert DNAMini Column II into a 2mL collection tube, add 250μL of 3M sodium hydroxide to the empty equilibration chamber, centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0203] (9) Transfer no more than 700 μL of the mixture to Centrifuge DNAMini Column II at 12,000 × g for 1 min at room temperature, and discard the filtrate. Repeat until all the mixture obtained in step 7 is transferred.

[0204] (10) Insert the DNAMini Column II into the same collection tube, and add 500 μL of HBC Buffer (with isopropanol added). In DNAMini Column II, centrifuge at 12,000×g for 1 min at room temperature and discard the filtrate.

[0205] (11) will Insert DNA Mini Column II into the same collection tube, add 700 μL of DNA Wash Buffer (with anhydrous ethanol added), centrifuge at 12,000 × g for 1 min at room temperature, and discard the filtrate.

[0206] (12) Repeat step (11).

[0207] (13) will DNA Mini Column II was inserted into the same collection tube and centrifuged at room temperature at 12,000 × g for 2 min to dry the binding column matrix.

[0208] (14) will Insert the DNAMini Column II into a new, clean 1.5 mL centrifuge tube, add 100 μL Endo-Free Elution Buffer to the column substrate, incubate at room temperature for 1 mL, and centrifuge at 12,000 × g for 1 min to elute the DNA. Note: The first elution may yield 70% plasmid DNA. The Endo-Free Elution Buffer from the first elution can be added back to the column for a second elution, increasing the elution concentration without increasing the elution volume.

[0209] (15) Discard the binding column and store the DNA product at -20°C.

[0210] Step 2.2.9.7 Plasmid Identification

[0211] (1) Template plasmid concentration determination: Use ddH2O to repeatedly scrub the microspectrophotometer, and take 1 μL ultrapure water to zero. After adjusting the wavelength to 260 nm, take 1 μL plasmid to test, repeatedly determine 3 times, and take the average value.

[0212] (2) Template plasmid sequencing: The constructed positive plasmid is sent to Beijing Qikang Biotechnology Co., Ltd. for sequencing, and the sequencing result is compared with the reference sequence. If the gene sequence is consistent with the reference sequence, it indicates that the positive plasmid construction is successful, and can be used for subsequent experiments.

[0213] Step 2.2.10 Sensitivity test

[0214] The positive plasmid prepared in 2.2.10 with an initial concentration of 120 ng / μL (copy number 2.78 x 10 11 copies / μL) is diluted for 10 dilutions. The positive plasmid of different dilutions is used as a template, and the MIRA detection kit (fluorescent type) is used to test the sensitivity, and the reaction program is optimized. The CFX96 TM Real-Time System is used for amplification, and the fluorescence curve and Ct value are recorded.

[0215] Step 2.2.11 Reproducibility test

[0216] Select three different dilutions of Mo positive plasmid, divided into high concentration group 1.2 x 10 1 ng / μL, medium concentration group 1.2 x 10 -1 ng / μL, low concentration group 1.2 x 10 -3 ng / μL, multiple and repeated determination of three different dilutions, record the fluorescence curve and Ct value. The results are statistically analyzed to evaluate the reproducibility of the established Mo MIRA detection method.

[0217] Step 2.2.12 Specificity test

[0218] Extract nucleic acid from M. bovis, M. S, OrfV, FPV, PRV, and CP, as shown in 2.2.2. Use the Mo MIRA detection method established in this test to test the specificity, and record the fluorescence curve and Ct value.

[0219] Step 2.2.13 Clinical sample detection

[0220] Randomly selected 82 samples of sheep nasal swab nucleic acid from Ninglang, Renhe and Lancang areas in Yunnan Province, and used the Mo MIRA detection method established in this test to detect the detection of Mo positive samples in clinical samples. The test sample information is shown in Table 14.

[0221] Table 14: Detection sample information

[0222] Random sampling portion (parts) Ninglang Renzhe 20 Lancang 30 Luquan 12 Total 20 Figure 1 82

[0223] Step 3.1 PCR detection results

[0224] Step 3.1.1 Sampling area

[0225] This study was conducted from July 2024 to February 2025 in 15 farms in 8 prefectures of Yunnan Province, and the sampling information is shown in Table 13. Figure 2 .

[0226] The clinical symptoms of sheep infected with Mycoplasma ovis disease mainly showed respiratory symptoms, mostly with nasal discharge (see Figure 3 ), cough (more continuous dry cough, and severe sheep can see continuous convulsive cough), depression, loss of appetite, and some with secretions on the eyelids.

[0227] Step 3.1.2 PCR detection results

[0228] Mo primer Umo / Lmo, filamentous mycoplasma cluster primer MmF / MmR, Ma primer Marg, PCR detection of 384 samples from 15 farms in 8 prefectures of Yunnan Province. The results show that Mo appears at the 563bp position of the target band, such as Figure 4 ; Mmc appears at the 548bp position of the target band, such as Figure 5 ; Ma appears at the 546bp position of the target band, such as Figure 6 .

[0229] According to the PCR results, 174 positive samples of Mo were detected in 384 nasal swabs, with a positive rate of 45.3%; the positive number of filamentous mycoplasma cluster was 117, with a positive rate of 30.5%; the positive number of Ma was 176, with a positive rate of 45.8%. See Table 15.

[0230] Table 15: PCR detection results of sheep nasal swab

[0231]

[0232] Step 3.1.3 PCR detection results of different breeding methods

[0233] The PCR results of the nasal swab samples collected from some areas in Yunnan Province were classified according to different breeding methods (free-range and cage) and further analysis found that the positive rates of each pathogen were not significantly different between different breeding methods. The positive detection rate of Mo in free-range sheep was 44.0%, the positive detection rate of Mycoplasma capripneumoniae cluster was 30.6%, and the positive detection rate of Ma was 46.9%; the positive detection rate of Mo in cage sheep was 46.9%, the positive detection rate of Mycoplasma capripneumoniae cluster was 30.3%, and the positive detection rate of Ma was 44.6%. The specific PCR detection results are shown in Table 16, and the statistical analysis results are shown in Figure 7 .

[0234] Table 16: PCR detection results of sheep nasal swabs under different breeding methods

[0235]

[0236] Step 3.1.4 PCR detection results of different breeds of sheep

[0237] The PCR results of the nasal swab samples collected from some areas in Yunnan Province were classified according to different breeds of sheep and further analysis found that the positive rates of the three pathogens in goats were higher than in sheep. The average positive rate of Mo in black goats was 40.9%, the average positive rate of Mo in yellow goats was 49.6%, and the average positive rate of Mo in sheep was 23.3%; the average positive rate of Mycoplasma capricolum cluster in black goats was 23.4%, the average positive rate of Mycoplasma capricolum cluster in yellow goats was 36.4%, and the average positive rate of Mycoplasma capricolum cluster in sheep was 21.7%; the average positive rate of Ma in black goats was 47.4%, the average positive rate of Ma in yellow goats was 47.9%, and the average positive rate of Ma in sheep was 43.3%. The specific PCR detection results are shown in Table 17, and the statistical analysis results are shown in Figure 8 .

[0238] Table 17: PCR detection results of sheep nasal swabs of different breeds

[0239]

[0240] Note: The statistics of Anning and Lanping areas are not included.

[0241] Step 3.1.5 PCR detection results of mixed infection

[0242] The PCR results of the nasal swab samples collected from some areas in Yunnan Province were classified according to the mixed infection of different pathogens. Further analysis found that the positive rate of mixed infection of Mo, Ma and filamentous mycoplasma cluster was 10.2% (39 / 384), the positive rate of mixed infection of Mo and filamentous mycoplasma cluster was 16.7% (64 / 384), the positive rate of mixed infection of Mo and Ma was 21.6% (83 / 384), and the positive rate of mixed infection of filamentous mycoplasma cluster and Ma was 17.5% (67 / 384). The specific PCR detection results are shown in Table 18, and the statistical analysis results are shown in Figure 3-9 .

[0243] Table 18: PCR detection results of mixed infection

[0244]

[0245] Step 3.2 Genetic evolution analysis

[0246] Step 3.2.1 Mo sequencing results and genetic evolution analysis

[0247] The results of TA cloning of part of the samples of each city obtained in 2.2.4 were compared in the NCBI database, and the phylogenetic tree was constructed using MEGA 11. The phylogenetic tree of Mo pathogen is shown in Figure 3-10 , the nasal swab sample of Luqian is in the same branch as the Mo sample AH2205 (accession number: OR964952) detected in Lanzhou, with a homology of 98.4% ( Figure 3-10 A); the nasal swab samples of Zianyi and Shidian (Dianyang Town) are in the same branch as the Mo sample 004_Julie (accession number: MN028333) isolated from a goat in the United States, with a homology of 99.6% and above ( Figure 3-10 B); the nasal swab samples of Tengchong (Gudong Town), Lancang, Mojiang and Shidian (Moluo Yuan Town) and the Mo sample FJ-FZ (accession number: KU870647) isolated from a goat in Fujian are in the same large branch, with a homology of 99.5% and above ( Figure 3-10 C); the nasal swab samples of Tengchong (Zhonghe Town) and Lanping are in the same branch as the MYCO1 sample (accession number: MK789475) isolated in Turkey, with a homology of 99.8% and above ( Figure 3-11 D); the samples of Weixin and Shidian (Renhe Town) are in the same branch and have a close genetic relationship. The samples of Anning, Ninglang and Wuding are each in a separate branch.

[0248] Step 3.2.2 Ma sequencing results and genetic evolution analysis

[0249] The results of TA cloning of each city part sample obtained in step 2.2.4 were aligned in the NCBI database, and a phylogenetic tree was constructed using MEGA11. The phylogenetic tree of Ma pathogen is shown in Figure 3-12 Figure 1. The nasal swab sample of Ninglang is in the same branch as the Ma sample strain A (accession number: MT740482) isolated in Turkey, with a homology of 96.7% (A); the nasal swab sample of Tengchong (Gudong Town) is in the same large branch as the Ma sample Ma strain EGL3 (accession number: MW493226) isolated in Egypt, with a homology of 99.1% (B); the nasal swab samples of Shidian (Dianyang Town), Anning, and Shidian (Renhe Town) are in the same branch and have a close genetic relationship. Figure 3-12 District / county

[0250] Step 3.3 ELISA results

[0251] ELISA test was performed on 182 serum samples collected from some areas of Yunnan Province, and the number of positive samples was 72, with a positive rate of 39.6%. The test results are shown in Table 19.

[0252] Table 19: ELISA detection results of sheep serum

[0253] Detection number (parts) Positive number (parts) Positive rate Wuding Anning 26 10 38.5％ Lanping 18 8 44.4％ Shidian 19 10 52.6％ Tengchong 20 7 35.0％ Luquan 25 16 64.0％ Zhaotong 10 3 30.0％ Zhenyi 28 8 28.6％ Lancang 24 9 37.5％ Total 12 1 8.3％ Figure 13 182 72 39.6％

[0254] Step 3.4 MIRA results

[0255] Step 3.4.1 Screening of MIRA primers

[0256] The probes 1 and 2 and the corresponding 18 pairs of primers designed in step 2.2.8.1 were screened, and the probes and corresponding primers with the smallest Ct value and the highest peak value were selected as the most suitable probes and primers. The results are shown in Figure 14 、 Upstream and downstream primer matching Table 20 and Table 21. F1-3 and R1-1 of probe 1, F1-3 and R1-2 of probe 1, and F2-1 and R2-2 of probe 2 were selected for further comparison and screening of probes.

[0257] Table 20: Ct value corresponding to the screening results of probe 1 primers

[0258] Ct F1-1+R1-1 F1-1+R1-2 21.69 F1-1+R1-3 19.33 F1-2+R1-1 20.31 F1-2+R1-2 20.78 F1-2+R1-3 17.03 F1-3+R1-1 21.82 F1-3+R1-2 11.94 F1-3+R1-3 15.51 Negative control 18.53 Upstream and downstream primer matching -

[0259] Table 21: Ct value corresponding to the screening results of probe 2 primers

[0260] Ct F2-1+R2-1 F2-1+R2-2 13.87 F2-1+R2-3 11.85 F2-2+R2-1 19.00 F2-2+R2-2 24.23 F2-2+R2-3 16.32 F2-3+R2-1 38.96 F2-3+R2-2 15.85 F2-3+R2-3 14.43 Negative control 26.13 Figure 15 -

[0261] Step 3.4.2 Screening of MIRA probes

[0262] ​​The probes and primers with high peak values ​​and low Ct values ​​selected in step 3.4.1 were then compared and screened again. The results are shown below. Upstream and downstream primer matching Table 22 shows that the primers F2-1 and R2-2 corresponding to probe 2 have the highest peak values ​​and the lowest Ct values ​​(6.19), with an amplified fragment size of 393 bp. Therefore, they are selected as the best probes and primers for subsequent experiments.

[0263] Table 22: Ct Values ​​Corresponding to Screening Results of Probe 1 and Probe 2

[0264] Ct F1-3+R1-1 F1-3+R1-2 6.47 F2-1+R2-2 12.47 Negative control 6.19 Figure 16 -

[0265] Step 3.4.3 Optimization of the Mo MIRA optimal reaction program

[0266] Step 3.4.3.1 Optimization of the optimal reaction temperature for Mo MIRA

[0267] With the reaction system unchanged, the reaction temperatures are set to 39℃, 40℃, 41℃, and 42℃, as follows: Reaction temperature / ℃ The corresponding Ct values ​​are shown in Table 23; that is, the optimal reaction temperature is 41℃.

[0268] Table 23: Optimized Ct values ​​for MIRA reaction temperature

[0269] Ct Figure 17 39 12.18 40 12.21 41 5.05 42 5.10

[0270] Step 3.4.3.2 Screening for the optimal premixing time of Mo MIRA

[0271] With the reaction system unchanged, at the temperature selected in 3.3.3.1, the premixing time was set to 1 min, 2 min, 3 min, 4 min, and 5 min, as follows: Premixing time / min The corresponding Ct values ​​are shown in Table 24. The optimal premixing time is 3 minutes.

[0272] Table 24: Optimized Ct values ​​for MIRA preheating time

[0273] Ct Figure 18 1 5.05 2 6.05 3 5.01 4 5.37 5 9.82

[0274] Step 3.4.4 Construction of template plasmid

[0275] Step 3.4.4.1 Amplification of the target fragment

[0276] PCR amplification was performed using the selected primers for Mo, and the fragment size matched the expected size (393 bp). Figure 19 It was initially identified as a positive plasmid and designated as pMD-19T-Movi.

[0277] Step 3.4.4.2 Identification of plasmids

[0278] (1) Bacterial PCR

[0279] The positive plasmid of pMD-19T-Movi was subjected to TA cloning, and the single colony was subjected to ordinary PCR and agarose gel electrophoresis identification, which was consistent with the expected fragment size (393 bp), as shown in Figure 20 .

[0280] (2) Sequencing of positive plasmid

[0281] The sequencing results of the obtained plasmid are shown in Figure 21 The pMD-19T-Movi was aligned in the NCBI database, the reference sequence information is shown in Table 25, and the homology results are shown in Name . It is shown that the gene fragment of Mo has been successfully connected to the 19T vector, and the reference sequence downloaded from NCBI has a homology of 99.4% and above.

[0282] Table 25: Reference sequence information published in GenBank

[0283] Accession number Year Isolation site Full length (bp) Dovre KJ433280 Norway 2014 FJ-SM 1466 KU870650 Fuzhou, China 2016 MYCO21 1526 MK789495 Turkey 2019 NR025989 1370 Y-98 Sweden 2019 XJ-3f 1475 DQ000588 Heilongjiang, China 2005 MoGH3-3 1508 EF687778 Gansu, China 2007 06OR03 1464 EU265779 USA 2008 GZ-TZ 1457 JN257120 Guizhou, China 2011 GZ-WN 1149 JN257121 Guizhou, China 2011 Altay 1148 PQ444053 Xinjiang, China 2024 Figure 22 1402

[0284] Step 3.4.5 Sensitivity test results

[0285] It is determined that the Mo positive template plasmid concentration is 120 ng / μL (2.78 x 10 11 copies / μL). The positive plasmid was used as a template and diluted to 10 dilutions, i.e. 120 ng / μL, 12 ng / μL, 1.2 ng / μL, 120 pg / μL, 12 pg / μL, 1.2 pg / μL, 120 fg / μL, 12 fg / μL, 1.2 fg / μL, 0.12 fg / μL. The plasmid with different dilutions was detected by constant temperature nucleic acid rapid amplification technology, and it was found that the positive could still be detected after 10 times dilution, and the copy number was calculated to be 278 copies / μL, and the results are shown in :pMD-19T-Movi concentration (ng / μL) , Table 26.

[0286] Table 26: Mo MIRA sensitivity test corresponding Ct value

[0287] Ct Negative control 1.2 x 10 2 ]]> 3.14 1.2 x 10 1 ]]> 1.81 1.2 x 10 0 ]]> 1.48 1.2 x 10 -1 ]]> 1.66 1.2 x 10 -2 ]]> 1.35 1.2 x 10 -3 ]]> 5.28 1.2 x 10 -4 ]]> 4.22 1.2 x 10 -5 ]]> 5.35 1.2 x 10 -6 ]]> 6.21 1.2 x 10 -7 ]]> 9.72 Figure 23 -

[0288] Step 3.4.6 Reproducibility test results

[0289] Three different dilutions (high concentration group 1.2 x 10 1 ng / μL, medium concentration group 1.2 x 10 -1 ng / μL, low concentration group 1.2 x 10 -3 ng / μL) were selected, and the Mo positive standard plasmid was subjected to reproducibility test, and the results are shown in : Different dilutionTable 27, the fluorescence curve of the same dilution is well reproducible.

[0290] Table 27: Ct values corresponding to Mo MIRA reproducibility test

[0291] Ct High concentration 1 High concentration 2 3.69 High concentration 3 2.94 Medium concentration 1 3.44 Medium concentration 2 5.14 Medium concentration 3 4.92 Low concentration 1 5.23 Low concentration 2 13.27 Low concentration 3 12.32 Figure 24 15.08

[0292] Step 3.4.7 Specificity test results

[0293] The established method was used for specificity test, the nucleic acid of Mo was amplified and fluorescence signal was detected, while the rest of the pathogens were not amplified and no fluorescence signal was detected, such as : Sample source This test proved that the method can detect Mo, but cannot detect M. bovis, MS, OrfV, FPV, PRV and CP, and the method has good specificity.

[0294] Step 3.4.8 Clinical sample test results

[0295] Using the established Mo MIRA detection method, 82 clinical samples from three farms in Ninglang, Shidian and Lancang were detected, and a total of 49 samples showed positive, with a positive rate of 59.8%, as shown in Table 28; the number of positive samples detected by PCR in the three areas was 38, with a positive rate of 46.3%, as shown in Table 29.

[0296] Table 28: MIRA clinical sample test results

[0297] Detection number Positive number Positive rate Ninglang Shidian 20 9 45.0％ Lancang 30 20 66.7％ Luquan 12 5 41.7％ Total 20 15 75％ Sample source 82 49 59.8％

[0298] Table 29: PCR clinical sample test results

[0299] Detection number Positive number Positive rate Ninglang Renzhe 20 8 40.0％ Lancang 30 14 46.7％ Luquan 12 2 16.7％ Total 20 14 70.0％ ​ 82 38 46.3％

[0300] Finally: the above only describes the preferred embodiments of the present application and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A rapid on-site detection method for Mycoplasma pneumoniae in sheep based on MIRA technology, characterized in that, Includes the following steps: Extract DNA from the sample to be tested; Using the DNA as a template, a MIRA amplification reaction was performed using specific primer pairs and probes; The upstream primer sequence of the primer pair is shown in SEQ ID NO:1, and the downstream primer sequence is shown in SEQ ID NO:2; The probe sequence is shown in SEQ ID NO:3, with a FAM fluorescent group labeled at the 5' end, a BHQ1 quenching group labeled at the 3' end, and a tetrahydrofuran site inserted at a position 30-35nt from the 5' end. The reaction was carried out at a constant temperature of 41℃ for 20-25 minutes, and the fluorescence signal was monitored in real time. The result is determined based on the fluorescence signal growth curve: an "S"-shaped curve indicates a positive result, otherwise it indicates a negative result.

2. The method according to claim 1, characterized in that, The MIRA amplification reaction system comprises: Buffer A; Upstream primer; Downstream primer; probe; DNA template mixed with ddH2O; B buffer.

3. The method according to claim 1 or 2, characterized in that, The reaction requires premixing for 3 minutes.

4. A primer-probe combination for detecting Mycoplasma pneumoniae MIRA in sheep, characterized in that: The upstream primer sequence is SEQ ID NO:1: 5'-CGTTAAATGATCCGCCTGAGTAGTATGCTCGCAAG-3'; The downstream primer sequence is SEQ ID NO:2: 5'-GCCATTGTAGCACGTTGTGTTGCCCCACTCGTAAGA-3'; The probe sequence is SEQ ID NO:3: 5'-GTCGTCAGCTCGTGTCGTGAGATGTTAGGT[FAM]AA[THF][BHQ1]CCTGCAACGAGCGCA-3'.

5. A kit for detecting Mycoplasma pneumoniae MIRA in sheep, characterized in that... It includes the primer-probe combination as described in claim 4.

6. The reagent kit according to claim 5, characterized in that, The kit is a lyophilized preparation containing recombinase, single-stranded DNA binding protein, DNA polymerase, and dNTPs.

7. The kit according to claim 5 or 6, characterized in that, The kit is used in conjunction with a portable fluorescence detection device, and the detection is completed within 25 minutes at a constant temperature of 41°C.

8. The use of the method according to any one of claims 1-3 or the kit according to any one of claims 5-7 in the preparation of diagnostic products for Mycoplasma pneumoniae in sheep.

9. The application of the method according to any one of claims 1-3 or the kit according to any one of claims 5-7 in the rapid on-site detection of contagious pleuropneumonia in sheep.

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