Detection primer composition of mycoplasma hyorhinis MIRRA-CRISPR / Cas12i3 mutant detection system and application of detection primer composition

Abstract

The invention discloses a detection primer composition of a mycoplasma hyorhinis MIRAA-CRISPR / Cas12i3 mutant detection system and application of the detection primer composition, and belongs to the technical field of microbiological detection. The composition comprises crRNA, an upstream primer Mhr P37 PrF1 and a downstream primer Mhr P37 PrR2, wherein the upstream primer Mhr P37 PrF1 and the downstream primer Mhr P37 PrR2 are used for multi-enzyme constant-temperature nucleic acid amplification. A rapid detection method suitable for clinical field is established by using a CRISPR / Cas12i3 mutant system and an MIRA isothermal amplification technology, a detection result can be obtained within 30 min through a fluorescence signal or fluorescence color development, mycoplasma hyorhinis can be specifically detected, and the sensitivity can reach 0.25 copies / [mu] L. According to the method, the detection of the mycoplasma hyorhinis becomes simple and convenient, the requirements on the environment and instruments are low, the dependence on expensive test equipment is avoided, the method can be used for clinical on-site rapid detection, and a new technical support is provided for rapid screening and purification of the mycoplasma hyorhinis.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a detection primer composition for a MIRA-CRISPR / Cas12i3 mutant detection system for porcine mycoplasma nasopharyngitis and its application. Background Technology

[0002] Mycoplasma hyorhinis (Mhr) is a common strain in the respiratory tract of pigs and is considered a minor pathogen causing endemic pneumonia (also known as swine enzootic pneumonia). Mhr infection can induce polyserositis and polyarthritis in suckling piglets, and can also cause pneumonia, otitis media, conjunctivitis, and abortion, significantly reducing the growth performance of infected piglets. Mhr can also synergistically interact with various pathogens and environmental factors, exacerbating swine respiratory disease syndrome, leading to increased mortality and severe economic losses to the pig industry. In recent years, the infection rate of Mhr in pig herds and its harm to pig farms have shown an upward trend, attracting increasing attention from pig farming enterprises. Studies have shown that Mhr infection has a significant correlation with various human tumors and cancers, and may also pose a threat to human health. However, in vivo diagnosis of mycoplasma infection is challenging; clinical diagnosis of Mhr mainly relies on clinical manifestations and pathological changes for initial diagnosis, while definitive diagnosis still requires laboratory testing.

[0003] Currently, real-time fluorescence PCR (qPCR) has become the most commonly used laboratory diagnostic method. Compared with gel-based and nested PCR methods, qPCR has higher specificity and sensitivity, but it is more expensive and requires specialized laboratory equipment and operators. Isothermal amplification technologies such as RPA, RAA, and LAMP have advantages over traditional nucleic acid amplification technologies, including ease of operation, no reliance on large instruments, speed, and high sensitivity. However, their non-specific amplification can lead to false positives. Multienzyme Isothermal Rapid Amplification (MIRA) is a relatively new isothermal amplification technology that emerged in 2019. It relies on the synergistic action of multiple functional proteins at room temperature to achieve rapid nucleic acid amplification, but it also suffers from non-specific amplification leading to false positives. CRISPR / Cas12a has specific double-strand cleavage target functionality. While targeting and cleaving double-stranded DNA, CRISPR / Cas12a also has a non-specific cleavage effect on surrounding single-stranded DNA, thus finding applications in nucleic acid detection. Compared to CRISPR / Cas12a, CRISPR... While possessing nucleic acid detection capabilities, Cas12i3 has a smaller molecular weight and more compact structure, making it a potential candidate for developing high-fidelity gene editing systems. The optimal temperature for CRISPR-Cas12i3 is similar to that of MIRA, allowing for one-step detection in the MIRA-CRISPR / Cas12i3 detection system. Furthermore, due to its dual specificity in recognizing the target gene, the MIRA-CRISPR / Cas12i3 detection system exhibits numerous advantages, including high specificity, high sensitivity, and ease of operation, and has been successfully applied to the detection of some microorganisms.

[0004] For the detection of Mhr, a MIRA-CRISPR / Cas detection method has not yet been successfully established. On the one hand, the Mhr genome has a unique structure with numerous repetitive sequences and high GC content regions. While MIRA technology offers the advantage of rapid amplification, primer design is extremely difficult when dealing with this complex genome structure. Repetitive sequences easily lead to primer mismatches, causing non-specific amplification, while high GC content regions hinder the strand substitution process in the MIRA reaction, reducing amplification efficiency. Simultaneously, the CRISPR / Cas system relies on the specific recognition of target nucleic acid sequences. The Mhr genome lacks sufficient specific recognition sites or contains certain modified structures, making it difficult for Cas proteins to accurately bind to and cleave target nucleic acids, thus affecting the sensitivity and accuracy of detection. On the other hand, the reaction conditions of the MIRA-CRISPR / Cas detection system are not perfectly suited to the biological characteristics of Mhr. The MIRA reaction requires precise temperature, ion concentration, and other conditions, while Mhr is extremely sensitive to the environment during in vitro culture, and its nucleic acids are prone to degradation or structural changes after extraction. When these unstable nucleic acid samples enter the MIRA-CRISPR / Cas detection system, they interfere with the entire reaction process. In addition, Mhr secretes some special metabolites during its growth process. These metabolites may remain in the nucleic acid sample and inhibit the enzyme activity of MIRA or the cleavage activity of Cas, causing the detection system to fail to function properly. Summary of the Invention

[0005] The purpose of this invention is to provide a detection primer composition for a MIRA-CRISPR / Cas12i3 mutant detection system for porcine mycoplasma and its application, thereby addressing the problems existing in the prior art. This invention utilizes the CRISPR / Cas12i3 mutant system and MIRA isothermal amplification technology to establish a rapid and clinically applicable detection method. Results can be observed through fluorescence signals or fluorescence color development, and it can specifically detect porcine mycoplasma with a sensitivity of 0.25 copies / μL.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a detection primer composition for a porcine mycoplasma MIRA-CRISPR / Cas12i3 mutant detection system, the composition comprising crRNA and an upstream primer Mhr P37 PrF1 and a downstream primer Mhr P37 PrR2 for MIRA nucleic acid amplification;

[0008] The sequence of the crRNA is shown in SEQ ID NO.17;

[0009] The sequence of the upstream primer Mhr P37 PrF1 is shown in SEQ ID NO.1; the sequence of the downstream primer Mhr P37 PrR2 is shown in SEQ ID NO.4.

[0010] The present invention also provides the use of the composition in the preparation of a detection product for the porcine mycoplasma MIRA-CRISPR / Cas12i3 mutant.

[0011] The present invention also provides a detection product for the MIRA-CRISPR / Cas12i3 mutant of porcine mycoplasma nasalis, the product comprising the aforementioned detection primer composition.

[0012] Furthermore, the product is a reagent kit.

[0013] Furthermore, the kit also contains the Cas12i3 mutant protein and a fluorescent reporter molecule.

[0014] Furthermore, the kit also contains MIRA amplification reagents.

[0015] The present invention also provides a method of using the aforementioned testing product, comprising the following steps:

[0016] S1. Extract DNA from the sample to be tested;

[0017] S2. The DNA obtained in step S1 is amplified and detected using the upstream primer Mhr P37 PrF1, the downstream primer Mhr P37 PrR2 and crRNA as described in claim 1.

[0018] S3. After the reaction is complete, fluorescence detection is used to determine whether the sample is infected with Mycoplasma hyopneumoniae.

[0019] Furthermore, the amplification detection was performed at 39°C for 30 min.

[0020] The present invention discloses the following technical effects:

[0021] This invention, through design and screening, obtained a set of detection primer compositions for the MIRA-CRISPR / Cas12i3 mutant detection system for *Mycoplasma hyopneumoniae*. Utilizing the CRISPR / Cas12i3 mutant system and MIRA isothermal amplification technology, a rapid and clinically applicable detection method was established. Results can be observed through fluorescence signals or fluorescence colorimetry, enabling specific detection of *Mycoplasma hyopneumoniae*. Compared to traditional detection methods, this invention's detection method has a detection limit of 0.25 copies / μL for DNA, exhibiting high specificity. Furthermore, it allows for real-time monitoring and acquisition of sample detection results within 30 minutes, making it more suitable for rapid on-site screening. This invention meets the clinical requirements for efficient and rapid detection, excellent sensitivity, and no instrument dependence, providing technical support for the clinical monitoring and purification of *Mycoplasma hyopneumoniae* in large-scale poultry farms. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The results are Mhr MIRA amplification results, where M is DL1000 DNA Marker; 1 is primer 1 for the P37 gene; 2 is primer 2 for the P37 gene; 3 is primer 3 for the P37 gene; 4 is primer 4 for the P37 gene; 5 is primer 5 for the P37 gene; 6 is primer 6 for the P37 gene; and 7 is primer 7 for the P37 gene.

[0024] Figure 2 The image shows the fluorescence intensity results for screening Mhr crRNA. Gene 1 is Mhr CVCC361 DNA; gene 2 is Mhr isolate DNA; 1 represents system 2+Cr2-1; 2 represents system 6+Cr46-1; and 3 represents system 6+Cr46-2.

[0025] Figure 3 This is a specificity test amplification curve, where 1 represents Mycoplasma hyopneumoniae; 2 represents Mycoplasma hyopneumoniae; 3 represents Mycoplasma floccosum; 4 represents Mycoplasma synoviae; 5 represents Escherichia coli; 6 represents Staphylococcus aureus; 7 represents Salmonella; 8 represents Streptococcus; 9 represents Haemophilus parasuis; and 10 represents the negative control.

[0026] Figure 4The image shows fluorescence intensity for specific tests. 1 represents *Mycoplasma hyopneumoniae*; 2 represents *Mycoplasma hyopneumoniae*; 3 represents *Mycoplasma floccosum*; 4 represents *Mycoplasma synoviae*; 5 represents *Escherichia coli*; 6 represents *Staphylococcus aureus*; 7 represents *Salmonella*; 8 represents *Streptococcus*; 9 represents *Haemophilus parasuis*; and 10 represents the negative control.

[0027] Figure 5 This is a fluorescence intensity graph from a sensitivity test, where 1 represents 10. 4 copies / μL of positive plasmid; 2 is 10 3 copies / μL of positive plasmid; 3 is 10 2 4 is a positive plasmid with 10 copies / μL; 5 is a positive plasmid with 1 copy / μL; 6 is a positive plasmid with 0.5 copies / μL; 7 is a positive plasmid with 0.25 copies / μL; 8 is a negative control.

[0028] Figure 6 This is a fluorescence intensity graph for repeatability testing, where 1 represents 10. 6 copies / μL of positive plasmid; 2 is 10 5 copies / μL of positive plasmid; 3 is 10 4 copies / μL positive plasmid; 4-6 represent negative template DNA respectively; 7 represents negative control; 8 represents positive control. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] In this embodiment of the invention, *Mycoplasma hyopneumoniae* CVCC361 was purchased from the National Veterinary Microbial Culture Collection Center; isolates of *Mycoplasma hyopneumoniae* (Mhp), *Mycoplasma hyopneumoniae* (Mhr), *Mycoplasma floccosum* (MF), *Mycoplasma synoviae* (MS), *Escherichia coli* (E. coli), *Staphylococcus aureus* (S. aureus), *Salmonella*, *Streptococcus* (SS), and *Haemophilus parasuis* (HPS) were all isolated, identified, and preserved by the inventors; 24 nasal swabs and 24 lung samples from pigs were collected from slaughterhouses in Shandong Province; the MIRA CRISPR one-step detection kit (SF-EA01-1) was purchased from Shandong Shunfeng Biotechnology Co., Ltd., and the FastPure Viral DNA / RNA Mini... The nucleic acid extraction kit (RC311) was purchased from Nanjing Novizan Biotechnology Co., Ltd., and the real-time fluorescence PCR detection kit for porcine mycoplasma nasopharynx (GM11017) was purchased from Hunan Guanmu Biotechnology Co., Ltd.

[0035] Example

[0036] I. Experimental Methods

[0037] 1. Culture and genomic DNA extraction of Mycoplasma hyopneumoniae

[0038] Resuscitate the porcine nasal mycoplasma CVCC361 strain and add it to 1 mL of KM2 liquid medium. Incubate at 37°C for several days until the medium turns yellow. Culture for 2-3 generations using this method. Extract nucleic acids from the cultured bacterial solution using the FastPure Viral DNA / RNA Mini Kit or by boiling. Store the extracted nucleic acids at -20°C for subsequent experiments.

[0039] 2. Sequence conservation analysis

[0040] Download the full sequences of 11 Mhr P37 genes from GeneBank and perform sequence alignment analysis using Megalign software.

[0041] 3. MIRA primer design

[0042] Based on the Mhr P37 gene sequence (KF806458.1) reported in GenBank, seven pairs of Mhr P37 primers were designed based on the relatively conserved sequence (Table 1). The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0043] Table 1 Primer sequences of the Mhr P37 gene

[0044]

[0045] 4. Preparation of plasmid reference standards

[0046] A plasmid reference was prepared based on the Mhr P37 gene sequence information. The selected target sequence contained the target fragment amplified by all MIRA primers. The plasmid was synthesized by Shanghai Sangon Biotech Co., Ltd. and cloned into the pUC-57 vector. The copy number of the recombinant plasmid was calculated using the following formula: copy number (copies) / μL = 6.02 × 10⁻⁶ 23 (copies / mol) × plasmid concentration (ng / μL) / (plasmid length × 660).

[0047] 5. crRNA design

[0048] In the CRISPR / Cas12i3 mutant system, crRNA participates in and guides the Cas12i3 mutant protein to recognize and cleave the target sequence from the T-rich PAM sequence. Using the amplified fragment of MIRA as a template, five Mhr crRNAs were designed (Table 2). The obtained crRNA sequences were imported into the NCBI database for sequence alignment to determine their specificity.

[0049] Table 2 Primer sequences for Mhr crRNA

[0050]

[0051] 6. MIRA-CRISPR Cas12i3 mutant detection system

[0052] The target fragment was amplified and detected using the MIRA CRISPR one-step detection kit. First, the enzyme digestion mixture of T7 Buffer, crRNA (10 μM), Cas 12i3 mutant, and Reporter was prepared according to Table 3. 5 μL of digestion solution was required for each detection reaction. The number of reactions was determined based on the total number of samples to be tested. After vortexing, the mixture was temporarily stored on ice. Then, a 30 μL detection system was prepared on ice: each reaction tube contained 0.5 portions of MIRA lyophilized reagent, 14.7 μL of A Buffer, 1 μL each of forward and reverse primers (10 μM), 5 μL of digestion solution, 2.05 μL of ddH2O, 5 μL of DNA template, and 1.25 μL of B Buffer. To prevent diffusion and contamination of the amplification products, a drop of paraffin oil could be added to the reaction tube to cover the liquid surface. The reaction tubes were placed at 39°C for 30 min for amplification, and then amplified and detected using a fluorescence PCR instrument. Finally, the fluorescence was observed visually on an optical gel spectrometer.

[0053] Table 3 Preparation of Enzyme Digestion Reaction Solution

[0054]

[0055] 7. Screening of MIRA primers and crRNA

[0056] After isothermal amplification of the Mhr CVCC361 genomic DNA using the designed MIRA primers, the amplification products were examined by agarose gel electrophoresis to select the most suitable MIRA primers for subsequent experiments. The designed crRNA was used to detect the MIRA amplification products. Different primers amplify different product sequences, requiring the use of corresponding crRNAs for detection. The templates used were the nucleic acids of Mhr CVCC361 and the isolated strain. The detection system was prepared according to the above method.

[0057] 8. Specificity test

[0058] The FastPure Viral DNA / RNA Mini Kit was used to extract nucleic acids from isolates of Mhr, Mhp, Mycoplasma suis (MF), Mycoplasma synoviae (MS), Escherichia coli, Staphylococcus aureus, Salmonella, Streptococcus (SS), and Haemophilus parasuis (HPS). The established Mhr detection method was then used to detect these nucleic acids. The specificity of the detection method was determined by fluorescence signal analysis and visual observation using a gel electrophoresis apparatus.

[0059] 9. Sensitivity Test

[0060] The synthesized Mhr-positive plasmid was converted to plasmid copy number using the plasmid concentration formula, starting from 10. 4 Serial dilutions were performed at 0.25 copies / μL, with ddH2O as a negative control. Positive plasmids at the above dilution gradients were detected using the MIRA-CRISPR / Cas12i3 mutant detection method. The sensitivity of the detection method was analyzed by fluorescence signal and visual observation using a gel electrophoresis apparatus.

[0061] 10. Repeatability test

[0062] The method established in this invention was used to perform three independent replicate tests on three positive samples and three negative samples respectively. A dilution of 10-1 was used. 5 copies / μL, 10 4 copies / μL and 10 3 Plasmids in copies / μL were used as positive samples, and nucleic acids extracted from pathogens that had been verified as negative were used as negative samples. Positive and negative controls were set up, and three MIRA-CRISPR / Cas12i3 mutant detection systems were configured to verify the reproducibility of the method.

[0063] 11. Clinical sample testing

[0064] Twenty-four swabs from pigs and twenty-four lung tissue samples collected from the slaughterhouse were processed, and nucleic acids were extracted for clinical sample testing. The collected swabs were centrifuged at 5000 r / min for 5 min, and 200 μL of the supernatant was collected. The collected lung tissue was washed with PBS, placed on a petri dish, and the surface layer was removed. A small amount of lesion tissue, approximately 5 mg in size (about the size of a sesame seed), was cut from the boundary between lesion and healthy tissue, ground, and mixed with 1 mL of PBS. The mixture was then centrifuged at 5000 r / min for 5 min, and 200 μL of the supernatant was collected. Nucleic acid was extracted from the supernatant using the FastPure Viral DNA / RNA Mini Kit. The MIRA-CRISPR / Cas12i3 mutant detection method and qPCR method established in this study were used to amplify and detect the genomic DNA of the clinical samples, and the results were compared.

[0065] II. Experimental Results and Analysis

[0066] 1. Results of gene conservation analysis

[0067] BLAST analysis of the Mhr P37 gene showed homology of over 99%, indicating that the Mhr P37 gene can be used as the target sequence for MIRA primer design.

[0068] 2. MIRA primer screening

[0069] Seven primer pairs were used to amplify the genomic DNA of Mhr CVCC361 at isothermal temperature. The amplification products were verified by gel electrophoresis to confirm the amplification effect of each primer pair. Electrophoresis results Figure 1 The results showed that the target fragments of MIRA primers 1, 2, 4, and 6, designed based on the P37 gene by Mhr, were between 100 bp and 250 bp, with high band brightness, consistent with the size of the target fragment, and good amplification specificity, making them suitable as primers for MIRA amplification.

[0070] 3. crRNA screening

[0071] The Cas12i3 mutant's targeting and specific binding to the target sequence largely depends on the guidance of the crRNA. Selecting a suitable crRNA significantly impacts the cleavage efficiency of the Cas12i3 mutant. Using the FAM channel, the reaction was performed at 39℃ for 60 min, with fluorescence signals collected every 1 min. The primer and crRNA combination with the highest fluorescence value (Table 4) was selected as the optimal reaction combination. Preliminary experiments excluded Mhr systems 1 and 4, which had no fluorescence or weak fluorescence, before conducting the formal experiment. The templates were MhrCVCC361 and the isolated strain. The results are as follows: Figure 2 As shown, the Mhr system exhibits the best fluorescence intensity for 2+Cr2-1, which will be used in subsequent experiments.

[0072] Table 4. Mhr MIRA primers and crRNA combinations

[0073]

[0074] 4. Specific detection

[0075] To verify the specificity of the detection method for MIRA / CRISPR-Cas12i3 mutants, an optimized detection system was used to detect genomic DNA of Mhr, Mhp, Mycoplasma floccosum, Mycoplasma synoviae, Escherichia coli, Staphylococcus aureus, Salmonella, Streptococcus, and Haemophilus parasuis. After the reaction, qPCR was used to collect fluorescence data. Except for Mhr, no high fluorescence values ​​were collected for the other pathogens. Figure 3 When the reaction tubes were placed in a blue light spectrometer for visual observation, only the Mhr reaction tube showed obvious green fluorescence, while other pathogens did not show fluorescence. Figure 4 ).

[0076] 5. Sensitivity Testing

[0077] To test the sensitivity of this method, the MIRA-CRISPR / Cas12i3 mutant detection system was used to detect six concentration gradient plasmids of Mhr, with enzyme-free sterile water as a negative control. The results are as follows: Figure 5 As shown, the sensitivity of this method can reach 0.25 copies / μL.

[0078] 6. Repeatability testing

[0079] To verify the repeatability of the detection method of the present invention, Mhr 10 was tested. 5 copies / μL, 10 4 copies / μL and 10 3 Three independent replicate tests were conducted using three gradient concentrations of positive samples (copies / μL) and three negative samples, with negative and positive controls included. Results are as follows: Figure 6 As shown, all positive templates were able to produce fluorescent signals, while no fluorescence was produced in the negative templates, indicating that the MIRA-CRISPR / Cas12i3 mutant detection method established in this study has good reproducibility.

[0080] 7. Clinical sample testing

[0081] The established MIRA / CRISPR-Cas12i3 mutant system was used to detect 48 samples collected from slaughterhouses, and the results were compared with those of a commercial qPCR detection kit. The results are shown in Table 5. In the 48 samples tested, the positive detection rate of the MIRA-CRISPR / Cas12i3 mutant system was higher than that of qPCR, indicating that this method is slightly superior to the commercially available detection kit used in the experiment.

[0082] Table 5 Clinical Sample Detection Results

[0083]

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer composition for detecting porcine mycoplasma MIRA-CRISPR / Cas12i3 mutants, characterized in that, The composition includes crRNA and upstream primer Mhr P37 PrF1 and downstream primer Mhr P37 PrR2 for MIRA nucleic acid amplification; The sequence of the crRNA is shown in SEQ ID NO.17; The sequence of the upstream primer Mhr P37 PrF1 is shown in SEQ ID NO.1; the sequence of the downstream primer Mhr P37 PrR2 is shown in SEQ ID NO.

4.

2. The use of the composition according to claim 1 in the preparation of a detection product for the porcine mycoplasma MIRA-CRISPR / Cas12i3 mutant.

3. A detection product for the porcine mycoplasma MIRA-CRISPR / Cas12i3 mutant, characterized in that, The product comprises the detection primer composition of claim 1.

4. The testing product according to claim 3, characterized in that, The product in question is a reagent kit.

5. The testing product according to claim 4, characterized in that, The kit also contains the Cas12i3 mutant protein and a fluorescent reporter molecule.

6. The testing product according to claim 5, characterized in that, The kit also contains MIRA amplification reagents.

7. A method of using the testing product according to any one of claims 3-6, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. The DNA obtained in step S1 is amplified and detected using the upstream primer Mhr P37 PrF1, the downstream primer Mhr P37 PrR2 and crRNA as described in claim 1. S3. After the reaction is complete, fluorescence detection is used to determine whether the sample is infected with Mycoplasma hyopneumoniae.

8. The method of use according to claim 7, characterized in that, The one-step amplification and detection method was performed at 39°C for 30 minutes.

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