A visual detection method for Pasteurella multocida and its application

Abstract

The present invention provides a visual detection method for Pasteurella multocida, which comprises the following steps: using primers 2KMT1‑F and primers 2KMT1‑R to perform RPA amplification reaction on the extracted genomic DNA of the sample to be tested at a temperature of 38°C for 15 minutes to obtain an RPA amplification product, using crRNA, ssDNA and the RPA amplification product to establish an RPA‑CRISPR / Cas12a detection system, reacting for 15 minutes at a temperature of 37°C to obtain an RPA‑CRISPR / Cas12a reaction product; observing the fluorescence state under a blue light gel imager, the positive sample containing Pasteurella multocida fluoresces under blue light, and the negative sample does not fluoresce. The method can be used to prepare a kit for detecting Pasteurella multocida. The present invention has no cross-reaction to a variety of common sheep pathogenic microorganisms such as Mo, Mh, BCoV, E.coli, S.aureus and Strep, has good specificity and high sensitivity, and can detect samples with a small viral load, with a minimum detection limit of 5×10 ‑ 1 copies / μL.

Description

Technical Field

[0001] The invention belongs to the technical field of Pasteurella multocida detection, and in particular relates to a visual detection method of Pasteurella multocida and application thereof. Background Art

[0002] Pasteurella multocida (Pm) belongs to the genus Pasteurella. It is a Gram-negative bacterium with a short rod or club shape with blunt ends. It mainly causes pneumonia and hemorrhagic sepsis in clinical practice. Pm has been detected in many places in my country, including Xinjiang, Ningxia, Sichuan, Qinghai, Gansu and Inner Mongolia, with a detection rate of 10.2% to 40.7%. This data shows that the pathogen has been widely prevalent in some sheep breeding areas in my country and is closely related to respiratory diseases. Therefore, rapid and accurate detection is the primary condition for controlling Pm infection.

[0003] The main methods for detecting Pm are pathogen isolation and culture and PCR. Isolation and culture require a lot of work, and because the morphology of various bacterial colonies is similar, misdiagnosis and missed diagnosis often occur. This method is inefficient and needs to be operated in a laboratory environment, which is not conducive to the rapid diagnosis of grassroots pathogens. Established molecular biological detection methods include PCR, fluorescent quantitative PCR, and nested PCR detection methods, but these methods all rely on expensive thermal cycle systems and are not suitable for grassroots detection.

[0004] Recombinase polymerase amplification (RPA) is a new isothermal nucleic acid amplification technology that only requires simple constant temperature equipment such as a water bath or metal bath, and can complete nucleic acid amplification in 5 to 30 minutes at 37°C to 42°C. It has been proven to be a rapid, specific, sensitive and reliable molecular diagnosis method for pathogenic microorganisms. However, because the amplified product often contains proteins and other impurities, it needs to be purified before gel electrophoresis, making it inconvenient to read the results and posing safety risks.

[0005] Based on the non-specific cleavage activity of Cas12a protein in the CRISPR / Cas12a system, adding ssDNA with a fluorescent label into the system can realize the visualization of the test results. The entire detection process takes a short time and has high sensitivity. It can get rid of the sterile environment and thermal cycle system of the laboratory, which is very suitable for grassroots development and application. However, its own sensitivity is low, and it is often combined with other amplification technologies to complete the detection.

[0006] At present, the RPA-CRISPR / Cas12a detection method established by combining RPA with CRISPR / Cas12a system has been applied in the rapid detection of various pathogens and has achieved good results. In order to improve the diagnostic efficiency of Pm, it provides technical support for the diagnosis and prevention and control of respiratory diseases in sheep at the grassroots level. This study mainly targets the conserved regions of genes, designs specific primers, and establishes an RPA-CRISPR / Cas12a detection method suitable for the field, which can quickly, effectively and visually detect Pm, aiming to provide a new and reliable technical support for the grassroots prevention and control and field diagnosis of Pasteurella multocida.

[0007] RPA technology is simple, quick to react, and has low equipment requirements. The results are judged by whether fluorescence is produced, which is suitable for front-line clinical use. Therefore, the present invention provides a simple, fast and efficient method for rapid detection of Pm by combining RPA with CRISPR / Cas12a. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a visual detection method for Pasteurella multocida and its application in view of the shortcomings of the above-mentioned prior art. The method has no cross-reaction to a variety of common sheep pathogenic microorganisms such as Mo, Mh, BCoV, E. coli, S. aureus and Strep, has good specificity and high sensitivity, and can detect samples with a small viral load, with a minimum detection limit of 5×10 -1 copies / μL.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is: a visual detection method for Pasteurella multocida, which is based on the RPA primer design principle, designs specific RPA primers based on the genes in the highly conserved region of Pm, combines RPA with CRISPR / Cas12a to build an RPA-CRISPR / Cas12a detection method, and interprets the results by observing fluorescence under blue light. The method is:

[0010] S1, extracting genomic DNA of the sample to be tested;

[0011] S2.RPA amplification:

[0012] The genomic DNA of the sample to be tested extracted from S1 was subjected to RPA amplification reaction at 38°C for 15 minutes using primers 2KMT1-F and 2KMT1-R to obtain RPA amplification products;

[0013] The system of the RPA amplification reaction is: A buffer 14.7 μL, 1 μL of 10 μmol / L primer 2KMT1-F, 1 μL of 10 μmol / L primer 2KMT1-R, 2 μL of genomic DNA of the sample to be tested, B buffer 1.3 μL, and 5 μL of enzyme-free water;

[0014] The nucleotide sequence of the primer 2KMT1-F is shown in SEQ ID NO: 1;

[0015] The nucleotide sequence of the primer 2KMT1-R is shown in SEQ ID NO: 2;

[0016] S3, RPA-CRISPR / Cas12a reaction:

[0017] The RPA-CRISPR / Cas12a detection system was established using crRNA, ssDNA, and the RPA amplification product obtained in S2, and the reaction was carried out at 37°C for 15 minutes to obtain the RPA-CRISPR / Cas12a reaction product;

[0018] The RPA-CRISPR / Cas12a detection system is: 10×NEBuffer TM r2.1 2.0μL, 50nM Cas12a 1μL, 200nM crRNA 1μL, ssDNA reporter 1.5μL, RPA amplification product 4μL, enzyme-free water 10.5μL;

[0019] The nucleotide sequence of the crRNA is shown in SEQ ID NO: 3;

[0020] The sequence of the ssDNA reporter molecule is 5-FAM-TTATT-BHQⅠ;

[0021] S4. The fluorescence state of the RPA-CRISPR / Cas12a reaction product was observed under a blue light gel imager. The positive samples containing Pasteurella multocida showed fluorescence under blue light, while the negative samples did not fluoresce.

[0022] Preferably, the minimum detection limit of Pasteurella multocida in S4 is 5×10 -1 copies / μL.

[0023] The present invention also provides application of the above-mentioned visual detection method of Pasteurella multocida in preparing a kit for detecting Pasteurella multocida.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The visual detection method of Pasteurella multocida of the present invention has no cross-reaction to a variety of common sheep respiratory microorganisms such as Mo, Mh, BCoV, E. coli, S. aureus and Strep, has good specificity, high sensitivity and high accuracy, and can detect samples with a small viral load, with a minimum detection limit of 5×10 -1 copies / μL.

[0026] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of RPA primer screening in Example 1 of the present invention are shown in Figure 1, where A is the electrophoresis result diagram and B is the grayscale analysis diagram.

[0028] Figure 2 The figure shows the RPA reaction temperature optimization result in Example 1 of the present invention, wherein A is the electrophoresis result diagram.

[0029] Figure 3 The results of RPA reaction time optimization in Example 1 of the present invention are shown in Figure 1, where A is the electrophoresis result diagram.

[0030] Figure 4 This is the best crRNA sequence screening in Example 1 of the present invention, where A is the fluorescence result and B is the grayscale analysis diagram.

[0031] Figure 5 It is the RPA-CRISPR / Cas12a reaction time optimization result in Example 1 of the present invention, wherein A is a blue light instrument observation picture, and B is a gel imager observation picture.

[0032] Figure 6 It is the concentration optimization of crRNA and Cas12a in Example 1 of the present invention, wherein A is the fluorescence result and B is the grayscale analysis diagram.

[0033] Figure 7 The specific detection results in Example 1 of the present invention, wherein A is the fluorescence result and B is the grayscale analysis diagram.

[0034] Figure 8 The sensitivity test results in Example 1 of the present invention are shown in Figure 1, where A is the fluorescence result and B is the grayscale analysis diagram.

[0035] Fig. 9 This is the clinical application test result in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] Example 1

[0037] The visual detection method of Pasteurella multocida in this embodiment is based on the RPA primer design principle, designs specific RPA primers based on the genes in the highly conserved region of Pm, combines RPA with CRISPR / Cas12a to build an RPA-CRISPR / Cas12a detection method, and interprets the results by observing fluorescence under blue light. The method is:

[0038] S1, extracting genomic DNA of the sample to be tested;

[0039] S2.RPA amplification:

[0040] The genomic DNA of the sample to be tested extracted from S1 was subjected to RPA amplification reaction at 38°C for 15 minutes using primers 2KMT1-F and 2KMT1-R to obtain RPA amplification products;

[0041] The system of the RPA amplification reaction is: A buffer 14.7 μL, 1 μL of 10 μmol / L primer 2KMT1-F, 1 μL of 10 μmol / L primer 2KMT1-R, 2 μL of genomic DNA of the sample to be tested, B buffer 1.3 μL, and 5 μL of enzyme-free water;

[0042] A buffer is the reagent in the DNA constant temperature rapid amplification kit (basic type) of Amp Future Co., Ltd.; B buffer is the reagent in the DNA constant temperature rapid amplification kit (basic type) of Amp Future Co., Ltd.;

[0043] The nucleotide sequence of the primer 2KMT1-F is shown in SEQ ID NO: 1;

[0044] The nucleotide sequence of the primer 2KMT1-R is shown in SEQ ID NO: 2;

[0045] S3, RPA-CRISPR / Cas12a reaction:

[0046] The RPA-CRISPR / Cas12a detection system was established using crRNA, ssDNA, and the RPA amplification product obtained in S2, and the reaction was carried out at 37°C for 15 minutes to obtain the RPA-CRISPR / Cas12a reaction product;

[0047] The RPA-CRISPR / Cas12a detection system is: 10×NEBuffer TM r2.1 2.0μL, 50nM Cas12a 1μL, 200nM crRNA 1μL, 10μM ssDNA reporter 1.5μL, RPA amplification product 4μL, enzyme-free water 10.5μL;

[0048] The nucleotide sequence of the crRNA is shown in SEQ ID NO: 3;

[0049] The sequence of the ssDNA reporter molecule is 5-FAM-TTATT-BHQⅠ;

[0050] S4. The fluorescence state of the RPA-CRISPR / Cas12a reaction products was observed under a blue light gel imager. Positive samples containing Pasteurella multocida showed fluorescence under blue light, while negative samples did not fluoresce. The minimum detection limit of Pasteurella multocida was 5×10 -1 copies / μL.

[0051] The above-mentioned visual detection method of Pasteurella multocida can be used to prepare a kit for detecting Pasteurella multocida.

[0052] The condition optimization and design method of the visual detection method of Pasteurella multocida in this embodiment are as follows:

[0053] 1.1 Construction of standard plasmid

[0054] According to the GenBank database, combined with the published Pm gene (GenBank No. CP133492.1) sequence, the standard plasmid was synthesized by a third-party company, Pm was connected to the pMD18-T vector, and then sequencing verification was completed. The plasmid was extracted from the positive bacterial solution with the correct detection according to the instructions using a plasmid mini-extraction kit, and the concentration was measured by a single drop spectrophotometer.

[0055] The copy number of the standard plasmid dilution is calculated according to formula ①:

[0056] Copy number = [6.02 × 10 23 × plasmid concentration (g / mL)] / plasmid relative molecular mass (g / mol)①.

[0057] 1.2RPA primer design

[0058] According to the conserved gene sequence registered in GenBank (accession number: CP133492.1), two RPA primers were designed as required, and primers suitable for detection were selected from them. The primer sequences are shown in Table 1. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0059] Table 1 Basic RPA primer sequences

[0060]

[0061] 1.3 crRNA and ssDNA design

[0062] The PAM site was found according to the conserved sequence of Pm, and three crRNAs were designed around different PAM sites (as shown in Table 2). The ssDNA reporter sequence was 5-FAM-TTATT-BHQⅠ. Both crRNA and ssDNA reporter molecules were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0063] Table 2 crRNA sequences

[0064]

[0065] The nucleotide sequence of crRNA2 in this embodiment is shown in SEQ ID NO:3, which is an RNA sequence. When input into the WIPOSquence software, the nucleotide sequence must only contain the symbols listed in Part 1 of Annex I of WIPOST.26. When the symbol "t" is used without further explanation, it will be interpreted as asthymine in DNA and uracil in RNA. The t in the SEQ ID NO:3 sequence is actually uracil "U".

[0066] 1.4 Optimization of RPA reaction conditions

[0067] 1.4.1 Primer screening

[0068] RPA amplification was performed using a standard plasmid, the reaction system was 25 μL (as shown in Table 3), Abuffer was a reagent in the DNA Constant Temperature Rapid Amplification Kit (Basic Type) of Amp Future, and Bbuffer was a reagent in the DNA Constant Temperature Rapid Amplification Kit (Basic Type) of Amp Future.

[0069] Table 3 RPA detection system

[0070] Reagents Volume (μL) A buffer 14.7 F(10μmol / L) 1 R(10μmol / L) 1 template 2 B buffer 1.3 Enzyme-free water 5 Volume 25

[0071] The results are as follows Figure 1 As shown: the amplified product is consistent with the expected size. The band size of the amplified product of 1kmt 1 primer pair (1KMT1-F, 1KMT1-R) is 101bp, and the band size of the amplified product of 2kmt 1 primer pair (2KMT1-F, 2KMT1-R) is 191bp ( Figure 1 A). After grayscale analysis, the difference between 2kmt 1 and 1kmt 1 is extremely significant ( Figure 1 B), so the 2kmt1 primer pair was selected, and NC in the figure is a negative control.

[0072] 1.4.2 Optimal reaction temperature

[0073] Amplification was performed at 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C for 30 min. After the reaction, the products were analyzed by electrophoresis and the optimal reaction temperature was selected.

[0074] The results are as follows Figure 2 As shown in the figure, when the reaction temperature of Pm RPA is between 35℃ and 38℃, the band gradually becomes brighter with the increase of temperature, the band is brightest at 38℃, and the amplified band gradually becomes lighter at 39℃ to 40℃. Therefore, the optimal reaction temperature of Pm is 38℃, and NC in the figure is a negative control.

[0075] 1.4.2 Optimal response time

[0076] The copy number is 5×10 6 The plasmid standard of 1000 copies / μL was reacted at a constant temperature for 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively. The products were analyzed by electrophoresis and the best reaction time was selected. Figure 3 As shown in the figure, the bands appeared after 10 minutes of reaction, and the bands became clearer as time went on. Considering the short-term high efficiency of the rapid test method, the optimal reaction time was determined to be 15 minutes, and NC in the figure is a negative control.

[0077] 1.5 Optimization of RPA-CRISPR / Cas12a reaction conditions

[0078] Add Cas12a, crRNA, RPA amplification product, and 10X NEBuffer into the RPA-CRISPR / Cas12a reaction system TM r2.1, ssDNA and enzyme-free water. Different crRNAs and RPA amplification products were used to configure the reaction system, 6 gradients were set for the reaction time, and reaction systems with different concentrations of Cas12a protein and crRNA were configured to optimize the reaction system conditions.

[0079] (1) Selecting the best crRNA

[0080] The three designed crRNAs were reacted separately to select the crRNA that can maximize the cutting efficiency of Cas12a protein. The results showed that the fluorescence of crRNA 2 in the three sets of repeats was stronger than that of crRNA 1 and crRNA 3 under the blue light instrument and gel imaging system ( Figure 4 A). Grayscale analysis results show that crRNA 2 is significantly different from crRNA 1 and crRNA 3 ( Figure 4 B). Therefore, crRNA 2 was selected as the optimal crRNA sequence, NC in the figure is a negative control, and PC is a positive control (Pasteurella multocida DNA).

[0081] (2) Selecting the best response time

[0082] The results showed that weak fluorescence could be observed under a blue light instrument after 5 minutes of reaction, and the fluorescence became stronger after 10 minutes, making it easier to observe with the naked eye ( Figure 5 ). In order to shorten the RPA-CRISPR / Cas12a detection time and make the results more accurate, the optimal reaction time was determined to be 15 minutes. NC in the figure is a negative control. The temperature refers to the Cas12a protein instruction manual, and the reaction temperature is 37°C.

[0083] (3) Optimization of crRNA and Cas12a concentrations

[0084] The results are as follows Figure 6 As shown: With the increase of crRNA and Cas12a concentration, the fluorescence intensity basically shows an increasing trend. When Cas12a / crRNA is 50nM / 500nM, the fluorescence intensity is weaker than 50nM / 200nM and 25nM / 500nM, indicating the necessity of concentration optimization. Based on the principle of low cost of rapid detection method, Cas12a / crRNA is selected as 50nM / 200nM for subsequent detection. With the increase of Cas12a protein, the fluorescence becomes brighter and brighter ( Figure 6 A), line graph after gray value analysis ( Figure 6 B).

[0085] The specific reaction system is as follows (Table 4):

[0086] Table 4 RPA-CRISPR / Cas12a detection system

[0087] Reagents Volume (μL) <![CDATA[10×NEBuffer TM r2.1]]> 2.0 Cas12a 1 crRNA 1 ssDNA (10 μM) 1.5 RPA amplification products 4 Enzyme-free water 10.5 Volume 20

[0088] 1.6Pm RPA-CRISPR / Cas12a specificity test

[0089] cDNA or DNA of Mycoplasma ovipneumoniae (Mo), Mannheimia hemolytica (Mh), bovine coronavirus (BCoV), Escherichia coli (E.coli), Staphylococcus aureus (S.aureus) and Streptococcus (Streptococcus, Strep) were used as templates, and primers 2KMT1-F and 2KMT1-R were used for RPA amplification. 4 μL of RPA reaction product was taken as a template for CRISPR / Cas12a specific detection, and enzyme-free water was set as a negative control. The results were recorded by a blue light instrument and a gel imager, and the fluorescence intensity result graph was analyzed in grayscale using ImageJ software. The results are shown in Figure 7 As shown, only Pm fluoresces under blue light, while the others do not fluoresce ( Figure 7A), and grayscale analysis of the fluorescence intensity results was performed using ImageJ software. The results showed that the Pm fluorescence value was significant and significantly different from the other five groups ( Figure 7 B) This indicates that the method has good specificity, and NC in the figure is a negative control.

[0090] 1.7Pm RPA-CRISPR / Cas12a sensitivity test

[0091] Determine the concentration of Pm plasmid and calculate the copy number according to formula ①. Use the 10-fold dilution method to dilute the Pm recombinant plasmid to 5×10 4 -5×10 -2 The primers 2KMT1-F and 2KMT1-R were used as templates for RPA isothermal amplification. 4 μL of RPA product was taken as target dsDNA, and enzyme-free water was set as negative control. According to the determined optimal reaction system, the detection system was configured using fluorescent reporter molecules. Repeated 3 times, and then placed under a blue light gel imager to observe the fluorescence state and determine the sensitivity. The results are shown in Figure 8 As shown in Figure 2, the lowest concentration that can be detected by the established RPA-CRISPR / Cas12a method is 5×10 -1 copies / μL. The concentration of Pm plasmid ranged from 5×10 4 -5×10 -2 copies / μL fluorescence result diagram ( Figure 8 A), the result after sensitivity gray value analysis ( Figure 8 B), NC in the figure is the negative control.

[0092] 1.8Pm RPA-CRISPR / Cas12a reproducibility test

[0093] To determine the intra-batch reproducibility of the Pm RPA-CRISPR / Cas12a method, the optimized method was used to perform 5×10 6 copies / μL, 5×10 5 copies / μL, 5×10 4 The plasmid standard with 10 copies / μL was used as a template, and each concentration was repeated 3 times for RPA-CRISPR / Cas12a reaction.

[0094] To determine the inter-batch repeatability of the Pm RPA-CRISPR / Cas12a method, the above conditions were used and the experiment was repeated three times at intervals. The coefficient of variation (CV) of the fluorescence intensity in the intra-batch and inter-batch repeated experiments was calculated by formula ②, and the coefficient of variation was used to reflect the repeatability.

[0095] CV = standard deviation / mean②.

[0096] The results are shown in Table 5. The intra-assay coefficient of variation and the inter-assay coefficient of variation were both less than 5%, and no fluorescence was observed in the negative controls, which demonstrated that the detection method had good repeatability and stability.

[0097] Table 5 Pm repeatability test

[0098]

[0099] 1.9 Testing of clinical samples

[0100] 102 nasal swabs of sheep with respiratory symptoms were collected and tested using the RPA-CRISPR / Cas12a method and the PCR method. The result was that the detection rate of the PCR method was 9.80% (10 / 102) ( Fig. 9 , Table 6), the detection rate of RPA-CRISPR / Cas12a method was 40.20% (41 / 102). This method detects highly conserved sequences selected by Pasteurella multocida, and after constant temperature amplification, there is crRNA to guide the cutting of PAM sites, double protection, so the specificity is high, and the detection sensitivity is high, so the detection rate is high. Comparing the two methods, the detection rate of RPA-CRISPR / Cas12a method is 4.1 times that of PCR method. Among them, the RPA-CRISPR / Cas12a method and the PCR method result are consistent with 100%, which further illustrates that RPA-CRISPR / Cas12a is more sensitive and reliable than the PCR method.

[0101] Table 6 Clinical application test results

[0102] sample PCR RPA-CRISPR / Cas12a Positive / Total Samples 10 / 102 41 / 102 Detection rate 9.80％ 40.20％

[0103] Therefore, the present invention adopts the above-mentioned visual detection method for Pasteurella multocida and its application. The method has no cross-reaction to a variety of common pathogenic microorganisms such as Mo, Mh, BCoV, E. coli, S. aureus and Strep, has good specificity and high sensitivity, and can detect samples with a small bacterial load, with a minimum detection limit of 5×10 -1 copies / μL.

[0104] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A visual detection method for Pasteurella multocida, characterized in that: The method is: S1, extracting genomic DNA of the sample to be tested; S2. RPA amplification: The genomic DNA of the sample to be tested extracted from S1 was subjected to RPA amplification reaction at 38°C for 15 minutes using primers 2KMT1-F and 2KMT1-R to obtain RPA amplification products; The system of the RPA amplification reaction is: A buffer 14.7 μL, 1 μL of 10 μmol / L primer 2KMT1-F, 1 μL of 10 μmol / L primer 2KMT1-R, 2 μL of genomic DNA of the sample to be tested, B buffer 1.3 μL, and 5 μL of enzyme-free water; The nucleotide sequence of the primer 2KMT1-F is shown in SEQ ID NO: 1; The nucleotide sequence of the primer 2KMT1-R is shown in SEQ ID NO: 2; S3, RPA-CRISPR / Cas12a reaction: The RPA-CRISPR / Cas12a detection system was established using crRNA, ssDNA, and the RPA amplification product obtained in S2, and the reaction was carried out at 37°C for 15 minutes to obtain the RPA-CRISPR / Cas12a reaction product; The RPA-CRISPR / Cas12a detection system is: 10×NEBuffer TM r2.1 2.0μL, 50nM Cas12a 1μL, 200nM crRNA 1μL, 10μM ssDNA reporter 1.5μL, RPA amplification product 4μL, enzyme-free water 10.5μL; The nucleotide sequence of the crRNA is shown in SEQ ID NO: 3; The sequence of the ssDNA reporter molecule is 5-FAM-TTATT-BHQⅠ; S4. The fluorescence state of the RPA-CRISPR / Cas12a reaction products was observed under a blue light gel imager. Positive samples containing Pasteurella multocida showed fluorescence under blue light, while negative samples did not fluoresce.

2. A visual detection method for Pasteurella multocida according to claim 1, characterized in that: The minimum detection limit of Pasteurella multocida described in S4 is 5×10 -1 copies / μL.

3. Use of the visual detection method of Pasteurella multocida as claimed in claim 1 in preparing a kit for detecting Pasteurella multocida.

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