Primer group for detecting common pathogenic bacteria of pneumonia of moschus berezovkii and application thereof

Abstract

This invention relates to the field of medical testing technology, and in particular to a primer set for detecting common pneumonia pathogens in musk deer and its application. This invention provides a primer set for detecting common pneumonia pathogens in musk deer (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Pasteurella multocida), and successfully establishes a multiplex PCR method capable of simultaneously amplifying four pathogens using the principle of polymerase chain reaction. This method exhibits high specificity and sensitivity; in a 25 μL PCR reaction system, the detection limit for Escherichia coli is 9.2 × 10⁻⁶. 4 The detection limit for CFU / mL and Klebsiella pneumoniae is 1.3 × 10⁻⁶. 5 The detection limit for CFU / mL and Pseudomonas aeruginosa is 1.5 × 10⁻⁶. 5 The detection limit for CFU / mL and Pasteurella multocida is 3.7 × 10⁻⁶. 4 CFU / mL; the method has been shown to have good reproducibility in clinical trials.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to primer sets for detecting common pneumonia pathogens in forest musk deer and their applications. Background Technology

[0002] The pathogens causing bacterial respiratory diseases in musk deer are complex and diverse, with the main pathogens including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Pasteurella multocida. In musk deer farming, pneumonia frequently causes animal mortality, resulting in significant economic losses for the industry.

[0003] In recent years, molecular biology techniques have been increasingly widely used in animal disease detection, with polymerase chain reaction (PCR) being particularly prominent. This technology specifically amplifies fragments of pathogen genetic material, making them easier to identify in samples. It boasts advantages such as high sensitivity and specificity, and has become one of the important tools for detecting various animal diseases. However, traditional PCR methods can only amplify a single target in a single reaction. To diagnose the specific pathogen causing musk deer pneumonia, each pathogen must be tested individually, a time-consuming and labor-intensive process. Therefore, there is an urgent need to establish a multiplex PCR method capable of simultaneously detecting multiple common musk deer pneumonia pathogens to achieve rapid, accurate, sensitive, and efficient diagnosis. Summary of the Invention

[0004] The purpose of this invention is to provide a primer set for detecting common pneumonia pathogens in musk deer and its application, thereby addressing the problems existing in the prior art. This invention establishes a highly efficient, specific, and sensitive multiplex PCR detection technique to rapidly and accurately identify four pathogens simultaneously: *Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Pasteurella multocida*.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a primer set for detecting common pathogens causing pneumonia in musk deer, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Pasteurella multocida.

[0007] The primer set includes primer pairs for detecting Escherichia coli, primer pairs for detecting Klebsiella pneumoniae, primer pairs for detecting Pseudomonas aeruginosa, and primer pairs for detecting Pasteurella multocida.

[0008] The primer pair for detecting Escherichia coli includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2;

[0009] The primer pair for detecting Klebsiella pneumoniae includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4;

[0010] The primer pair for detecting Pseudomonas aeruginosa includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6;

[0011] The primer pair for detecting Pasteurella multocida includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8.

[0012] This invention provides the application of the above-mentioned primer set in the preparation of products for detecting common pneumonia pathogens in musk deer.

[0013] Optionally, the product includes reagents, reagent kits, and chips.

[0014] Optionally, the method of using the product includes the step of using the supernatant of the test sample as a template and performing PCR amplification using the primer set described above.

[0015] Optionally, the PCR amplification system, in 25 μL, includes 12.5 μL of 2×ES Taq Mastermix, 0.5 μL each of the upstream and downstream primers as described in claim 1, 1 μL of template, and the remainder sterile ddH2O;

[0016] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; and a final extension at 72℃ for 10 min.

[0017] Optionally, the working concentration of both the upstream and downstream primers is 0.1 μM.

[0018] Alternatively, the test sample may be lung tissue.

[0019] This invention provides a product for detecting common pneumonia pathogens in forest musk deer, the product comprising the aforementioned primer set.

[0020] Optionally, the working concentration of both the upstream and downstream primers in the primer set is 0.1 μM.

[0021] Optionally, the product also includes 2×ES Taq Mastermix.

[0022] Optionally, the product includes reagents, reagent kits, and chips.

[0023] The present invention discloses the following technical effects:

[0024] This invention provides a primer set for detecting common pneumonia pathogens in musk deer (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Pasteurella multocida). Based on the principle of polymerase chain reaction (PCR), a multiplex PCR method was successfully established that can simultaneously amplify four pathogens (product sizes of 787 bp, 558 bp, 361 bp, and 244 bp, respectively). This method exhibits high specificity and sensitivity; in a 25 μL PCR reaction system, the detection limit for Escherichia coli is 9.2 × 10⁻⁶. 4 The detection limit for CFU / mL and Klebsiella pneumoniae is 1.3 × 10⁻⁶. 5 The detection limit for CFU / mL and Pseudomonas aeruginosa is 1.5 × 10⁻⁶. 5 The detection limit for CFU / mL and Pasteurella multocida is 3.7 × 10⁻⁶. 4 CFU / mL; the method has been shown to have good reproducibility in clinical trials. Attached Figure Description

[0025] 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.

[0026] Figure 1 Electrophoresis images of singleton PCR reaction systems at different annealing temperatures; where A represents the PCR results of *Escherichia coli*, B represents the PCR results of *Klebsiella pneumoniae*, C represents the PCR results of *Pseudomonas aeruginosa*, and D represents the PCR results of *Pasteurella multocida*; M is the marker, 1-9 represent 55℃, 56℃, 57℃, 58.2℃, 60.6℃, 61.8℃, 63℃, 64℃, and 65℃ respectively, and NC is the negative control;

[0027] Figure 2Electrophoresis images of different annealing temperatures in a multiplex PCR reaction system; where M is the marker, 1-9 are 55℃, 56℃, 57℃, 58.2℃, 60.6℃, 61.8℃, 63℃, 64℃ and 65℃ respectively, and NC is the negative control;

[0028] Figure 3 Electrophoresis images of primers at different concentrations in a singleton PCR reaction system; where A represents the PCR results of *Escherichia coli*, B represents the PCR results of *Klebsiella pneumoniae*, C represents the PCR results of *Pseudomonas aeruginosa*, and D represents the PCR results of *Pasteurella multocida*; M is the marker, and 1-9 represent 1.25 μM, 0.625 μM, 0.3125 μM, 156.25 nM, 78.125 nM, 39.06 nM, 19.53 nM, 9.77 nM, and 4.88 nM respectively; NC is the negative control.

[0029] Figure 4 Electrophoresis images of primers at different concentrations in a multiplex PCR reaction system; where M is the marker, 1-9 are 1.25 μM, 0.625 μM, 0.3125 μM, 156.25 nM, 78.125 nM, 39.06 nM, 19.53 nM, 9.77 nM and 4.88 nM respectively, and NC is the negative control;

[0030] Figure 5 This is an electrophoresis image for sensitivity testing of a multiplex PCR reaction system; where M is the marker, 1-9 are template bacterial solutions mixed in equal proportions after tenfold serial dilution, and NC is the negative control;

[0031] Figure 6 Electrophoresis diagrams for stability testing of multiplex PCR reaction systems; where A represents the PCR results of Escherichia coli culture, B represents the PCR results of Klebsiella pneumoniae culture, C represents the PCR results of Pseudomonas aeruginosa culture, and D represents the PCR results of Pasteurella multocida culture; M is the marker, 1-4 represent the PCR results of the four bacteria mixture, the first-generation culture PCR results, the second-generation culture PCR results, and the third-generation culture PCR results, respectively, and NC represents the negative control;

[0032] Figure 7Electrophoresis images show the specific detection results of the multiplex PCR reaction system; where M is the marker, 1-14 are the PCR results of four bacteria mixed together, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pasteurella multocida, Streptococcus pneumoniae, Proteus mirabilis, Enterococcus faecalis, Enterococcus thamnianus, Acinetobacter lophiae, Acinetobacter baumannii, Bacillus polymyxa, Bacillus amyloliquefaciens, and Enterobacter cloacae, respectively; NC is the negative control.

[0033] Figure 8 Electrophoresis images of partial samples tested in a multiplex PCR reaction system; where M is the marker, 1-23 are the PCR results of partial samples tested, and NC is the negative control. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1

[0040] 1. Experimental Materials

[0041] 1.1 Strains and Primers

[0042] Escherichia coli (ATCC25922, E. coli), Klebsiella pneumoniae (ATCC700603, K. pneumoniae), Pseudomonas aeruginosa (ATCC27853, P. aeruginosa), and Pasteurella multocida (ATCC12453, P. multocida) were all purchased from the National Veterinary Microbiology (Virus) Culture Collection Center. Streptococcus pneumoniae, Proteus mirabilis, Enterococcus faecalis, Enterococcus thamnsis, Acinetobacter lophiae, Acinetobacter baumannii, Bacillus polymyxa, Bacillus amyloliquefaciens, and Enterobacter cloacae, totaling nine bacterial colony samples, were identified and preserved by the Animal Infectious Disease Prevention and Control Technology Laboratory of the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences. The applicant promises to release these samples for 20 years from the date of application.

[0043] Lung tissue samples: From September 2023 to March 2024, 22 lung tissue samples were collected from musk deer in Nyingchi City, Tibet Autonomous Region, Haibei Tibetan Autonomous Prefecture, Qinghai Province, and Zizhou County, Shaanxi Province. Bacterial colonies were isolated. Subsequent 16S rRNA detection identified the four bacteria with the highest infectivity rates as *Escherichia coli* (14 samples), *Klebsiella pneumoniae* (14 samples), *Pseudomonas aeruginosa* (8 samples), and *Pasteurella multocida* (9 samples). Therefore, this study focused on the four bacteria with the highest infectivity rates.

[0044] Based on the gene sequence information included in GenBank and referring to relevant literature, including the gene gryB encoding the DNA gyrase B subunit of Escherichia coli, the gene PhoE encoding the outer membrane phosphoporin of Klebsiella pneumoniae, the quorum sensing gene Las I of Pseudomonas aeruginosa, and the highly conserved gene Kmt 1 of Pasteurella multocida, primer sequences were designed. The primer information is shown in Table 1. The primers were synthesized by Sangon Biotech (Changchun) Co., Ltd.

[0045] Table 1 Primer Information

[0046]

[0047] 1.2 Main Reagents

[0048] Brain heart and brain extract broth (BHI) was purchased from Qingdao Haibo Biotechnology Co., Ltd.; agarose was purchased from Beijing Solarbio Science & Technology Co., Ltd.; 2×ES Taq Mastermix (containing Es Taq DNA Polymerase, 3 mM MgCl2 and 400 μM dNTPs) was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; agarose gel powder was purchased from Beijing Lanjieke Technology Co., Ltd.; 50×TAE was purchased from Sangon Biotech (Shanghai) Co., Ltd.; ddH2O was provided by the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences; Brain heart and brain extract agar medium (BHIA agar medium) was prepared by dissolving 15 g BHI and 10 g agar in 500 mL ddH2O, autoclaving, and then pouring the solution onto plates; Brain heart and brain extract liquid medium (BHIB liquid medium) was prepared by dissolving 15 g BHI in 500 mL ddH2O, autoclaving, and then pouring the solution onto plates.

[0049] 1.3 Main Instruments and Equipment

[0050] Pipettes of various volumes were purchased from Eppendorf (Shanghai) International Trading Co., Ltd.; gradient PCR instrument was purchased from Jena Analytical Instruments (Beijing) Co., Ltd.; gel imaging system and electrophoresis kit were purchased from Bio-Rad Life Sciences (Shanghai) Co., Ltd.

[0051] 2. Experimental Methods

[0052] 2.1 Calculation of bacterial concentration

[0053] In a clean bench, 100 μL of standard bacterial suspensions of *Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Pasteurella multocida* were evenly spread onto BHIA agar medium and incubated at 37°C for 24 hours. After incubation, single colonies of each of the four bacteria were inoculated onto BHIB liquid medium and cultured at 37°C with shaking at 180 rpm for 24 hours, followed by incubation at 4°C for 4-6 hours. The resulting bacterial suspensions were serially diluted 10-fold, and the concentration of the template bacterial suspension was determined by colony counting. The effective viable count of *E. coli* strains in the *E. coli* template bacterial suspension was 2.3 × 10⁻⁶. 9 The effective viable count of Klebsiella pneumoniae strain in the Klebsiella pneumoniae template culture was 3.2 × 10⁻¹⁰ CFU / mL. 9 The effective viable count of *Pseudomonas aeruginosa* strain in the template culture was 3.8 × 10⁻⁶ CFU / mL. 9 The effective viable count of Pasteurella multocida strain in the Pasteurella multocida template solution was 9.2 × 10⁻⁶ CFU / mL. 8 CFU / mL.

[0054] 2.2 Establishment of PCR method

[0055] 2.2.1 Single PCR Annealing Temperature Gradient Test

[0056] Singleton PCR reaction system: 12.5 μL of 2×ES Taq Mastermix, 0.5 μL each of the specific primers (10 μM stock concentration) for one of the four strains, 1 μL of template bacterial solution (add the corresponding template bacterial solution obtained in step 2.1), and sterile ddH2O to make up the reaction volume to 25 μL.

[0057] Singleton PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at different annealing temperatures for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 10 min.

[0058] The annealing temperature of the singleton PCR reaction program was set in a gradient manner, with annealing temperatures of 55℃, 56℃, 57℃, 58.2℃, 60.6℃, 61.8℃, 63℃, 64℃ and 65℃, while other reaction conditions remained unchanged, to explore the suitable annealing temperature range.

[0059] 2.2.2 Multiplex PCR Annealing Temperature Gradient Test

[0060] Multiplex PCR reaction system: 12.5 μL of 2×ES Taq Mastermix, 0.5 μL of each of the 8 primer solutions (10 μM stock concentration) (total 4 μL), 1 μL of each of the 4 template bacterial cultures (total 4 μL), and sterile ddH2O to make up the reaction volume to 25 μL.

[0061] Multiplex PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at different annealing temperatures for 30 s, extension at 72℃ for 1 min, 30 cycles; final extension at 72℃ for 10 min.

[0062] The annealing temperature of the multiplex PCR reaction program was set in a gradient manner, with annealing temperatures of 55℃, 56℃, 57℃, 58.2℃, 60.6℃, 61.8℃, 63℃, 64℃ and 65℃, while other reaction conditions remained unchanged, to explore the suitable annealing temperature range.

[0063] 2.3 Optimization of PCR conditions

[0064] 2.3.1 Single PCR primer concentration gradient test

[0065] Take 10 μL each of a pair of specific upstream and downstream primers (stock concentration of 10 μM) for a certain bacterium and mix them. Add 60 μL of ddH2O. By diluting the primer concentration, control the primer stock concentration to 1.25 μM, 0.625 μM, 0.3125 μM, 156.25 nM, 78.125 nM, 39.06 nM, 19.53 nM, 9.77 nM and 4.88 nM. Repeat this operation to obtain 4 groups of mixed primers with different concentrations. Use 60℃, determined in steps 2.2.1 and 2.2.2, as the suitable annealing temperature for testing. Keep other conditions unchanged to explore the concentration range of each primer reaction.

[0066] 2.3.2 Multiplex PCR primer concentration test

[0067] 10 μL of each of the four pairs of specific upstream and downstream primers (stock concentration of 10 μM) targeting four bacteria were mixed. The primer stock concentrations were controlled by diluting the primers to 1.25 μM, 0.625 μM, 0.3125 μM, 156.25 nM, 78.125 nM, 39.06 nM, 19.53 nM, 9.77 nM, and 4.88 nM. The annealing temperature of 60℃, determined in steps 2.2.1 and 2.2.2, was used for testing, with other conditions remaining unchanged, to explore the concentration range of each primer reaction.

[0068] 2.4 Performance analysis of multiplex PCR reaction

[0069] 2.4.1 Sensitivity detection of multiplex PCR reaction

[0070] Take 100 μL of each of the four template bacterial solutions from step 2.1, vortex, and then perform 10-fold serial dilution to obtain diluted template bacterial solutions. Use the template bacterial solutions of each concentration gradient as templates and perform the reaction under optimized multiplex PCR amplification conditions.

[0071] Multiplex PCR reaction system: 12.5 μL of 2×ES Taq Mastermix, 0.5 μL of each of the 8 primer solutions (0.625 μM stock concentration) (total 4 μL), 4 μL of template bacterial solution (obtained by mixing template bacterial solutions of various dilutions in equal proportions), and sterile ddH2O to make up the reaction volume to 25 μL.

[0072] Multiplex PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 10 min.

[0073] 2.4.2 Stability detection of multiplex PCR

[0074] Four standard bacterial strains were passaged three times consecutively, and singleton PCR was performed on the passaged strains. Nucleic acid electrophoresis was then performed, and the samples were sent to Sangon Biotech (Changchun) Co., Ltd. for sequencing. The sequencing results were compared with the reference sequence using DNAMAN to determine homology.

[0075] Singleton PCR reaction system: 12.5 μL of 2×ES Taq Mastermix, 0.5 μL each of the upstream and downstream primers of a specific primer for a strain (10 μM stock concentration), 1 μL of template bacterial solution of the corresponding strain (the template bacterial solution obtained in step "2.1 Calculation of bacterial solution concentration"), and sterilized ddH2O to make up the reaction volume to 25 μL.

[0076] Singleton PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 10 min.

[0077] 2.4.3 Specificity detection of multiplex PCR reaction

[0078] Thirteen strains isolated from diseased musk deer specimens were used as templates for multiplex PCR amplification. These strains included Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pasteurella multocida, Streptococcus pneumoniae, Proteus mirabilis, Enterococcus faecalis, Enterococcus thamnsis, Acinetobacter lophiae, Acinetobacter baumannii, Bacillus polymyxa, Bacillus amyloliquefaciens, and Enterobacter cloacae.

[0079] Multiplex PCR reaction system: 12.5 μL of 2×ES Taq Mastermix, 0.5 μL of each of the 8 primer solutions (0.625 μM stock concentration) (total 4 μL), 4 μL of template bacterial solution (obtained by mixing the 4 template bacterial solutions in equal proportions), and sterile ddH2O to make up the reaction volume to 25 μL.

[0080] Multiplex PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 10 min.

[0081] 2.5 Application of Multiplex PCR Method

[0082] Each lung tissue sample was cut into a small piece (approximately 1 cm). 3 Add 1 mL of BHIB liquid culture medium, grind the tissue, and take the supernatant for multiplex PCR detection.

[0083] Multiplex PCR reaction system: 12.5 μL of 2×ES Taq Mastermix, 0.5 μL of each of the 8 primer solutions (0.625 μM stock concentration) (total 4 μL), 1 μL of supernatant, and sterile ddH2O to make up the reaction volume to 25 μL.

[0084] Multiplex PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 10 min.

[0085] 3. Experimental Results

[0086] 3.1 Optimal Annealing Temperature

[0087] In the singlet PCR reaction system, the target bands were observed at annealing temperatures between 55℃, 56℃, 57℃, 58.2℃, 60.6℃, 61.8℃, 63℃, 64℃, and 65℃. Figure 1 ).

[0088] In the multiplex PCR reaction system, the target bands were observed at annealing temperatures between 55℃, 56℃, 57℃, 58.2℃, 60.6℃, 61.8℃, 63℃, 64℃, and 65℃. Figure 2 ).

[0089] The annealing temperature range for conventional PCR is typically 55–72°C. In multiplex PCR, appropriately increasing the annealing temperature helps enhance primer specificity, but excessively high temperatures can hinder primer-template binding. Henegariu et al. pointed out that specific amplification of a single target gene can be achieved within the range of 56°C–60°C, but in multiplex PCR, lowering the annealing temperature by 4°C–6°C effectively promotes the amplification of all target fragments. Although the annealing temperature values ​​provided in the literature are valuable for reference, the final temperature selection still needs to be optimized experimentally to ensure the best amplification effect. This example explored the annealing temperature of singlex and multiplex PCR reactions and found that the annealing temperature has no effect on either singlex or multiplex PCR reactions. Based on Henegariu's suggestion, this example ultimately selected an annealing temperature of 60°C, at which temperature uniform band brightness can be guaranteed.

[0090] 3.2 Optimal primer concentration

[0091] In a singlet PCR reaction system, primers of different concentrations were used for PCR reactions, and the results are as follows: Figure 3 As shown in the figure. The results show that clear bands were visible when the concentrations of each primer added were 1.25 μM, 0.625 μM and 0.3125 μM, with the optimal concentration being 0.625 μM.

[0092] In a multiplex PCR reaction system, different concentrations of primer combinations were used for PCR reactions, and the results are as follows: Figure 4 As shown in the figure. The results show that clear bands are visible when the minimum concentration of each primer is 0.625 μM (the working concentration of each primer in a 25 μL reaction system is 0.1 μM).

[0093] 3.3 Sensitivity Testing

[0094] PCR was performed under optimized reaction conditions, and the results are as follows: Figure 5 As shown in the figure. The results showed that the detection limit of Escherichia coli in a 25 μL PCR reaction system was 9.2 × 10⁻⁶. 4 The detection limit for Klebsiella pneumoniae is 1.3 × 10⁻⁶ CFU / mL. 5 The detection limit for Pseudomonas aeruginosa is 1.5 × 10⁻⁶ CFU / mL. 5 The detection limit for Pasteurella multocida (CFU / mL) is 3.7 × 10⁻⁶. 4 CFU / mL.

[0095] 3.4 Stability Testing

[0096] PCR was performed under optimized reaction conditions, and the results are as follows: Figure 6 As shown in the figure. The results showed that after three consecutive passages, bands of the same size were amplified. Sequencing of the three generations of PCR products and comparing the obtained gene sequences with reference gene sequences revealed the following similarities: the PCR products of *Klebsiella pneumoniae* from three generations showed 99.44% similarity to the PhoE reference sequence; the PCR products of *Pseudomonas aeruginosa* from three generations showed 99.39% similarity to the Las I reference sequence; the PCR products of *Pasteurella multocida* from three generations showed 99.06% similarity to the Kmt 1 reference sequence; and the PCR products of *Escherichia coli* from three generations showed 98.54% similarity to the gryB reference sequence. This demonstrates that the multiplex PCR provided by this invention exhibits extremely high stability.

[0097] 3.4 Specificity Detection

[0098] PCR was performed under optimized reaction conditions, and the results are as follows: Figure 7 As shown in the figure. The results show that Klebsiella pneumoniae, Pseudomonas aeruginosa, Pasteurella multocida, and Escherichia coli can amplify the corresponding bands, while other strains cannot amplify the fragments. This demonstrates that the multiplex PCR provided by this invention has extremely high specificity.

[0099] 3.5 Sample Testing

[0100] The established multiplex PCR method was used to detect pneumonia in 47 samples of musk deer. Some of the test results are as follows: Figure 8 As shown in the figure, the results indicated that *Escherichia coli* had the highest detection rate (79.8%), followed by *Klebsiella pneumoniae* (65.4%), *Pseudomonas aeruginosa* (40.1%), and *Pasteurella multocida* (39.9%). Furthermore, the results showed that clinically, mixed infections with two or more bacteria were more common (63.0%), while single-pathogen infections were relatively rare (7.0%). These results were consistent with bacterial isolation and culture, as well as 16S rRNA sequencing results. Therefore, the multiplex PCR detection results provided by this invention are consistent with existing technologies and can effectively detect these four strains.

[0101] 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. The application of primer sets in the preparation of products for detecting common pneumonia pathogens in musk deer, characterized in that, The common pathogen causing pneumonia in the musk deer is Escherichia coli (Escherichia coli). Escherichia coli ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Pasteurella multocida ( Pasteurella multocida ); The primer set consists of primer pairs for detecting Escherichia coli, primer pairs for detecting Klebsiella pneumoniae, primer pairs for detecting Pseudomonas aeruginosa, and primer pairs for detecting Pasteurella multocida. The primer pair for detecting Escherichia coli includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; The primer pair for detecting Klebsiella pneumoniae includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4; The primer pair for detecting Pseudomonas aeruginosa includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6; The primer pair for detecting Pasteurella multocida includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

8.

2. The application according to claim 1, characterized in that, The products include reagents, reagent kits, and chips.

3. The application according to claim 1, characterized in that, The method of using the product includes the steps of using the supernatant of the analyte as a template and performing PCR amplification using the primer set described in claim 1.

4. The application according to claim 3, characterized in that, The PCR amplification system, in 25 μL volumes, includes 2×ES. Taq Mastermix 12.5 μL, upstream and downstream primers as described in claim 1 each 0.5 μL, template 1 μL, and the remainder sterile ddH2O; The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; and a final extension at 72℃ for 10 min.

5. The application according to claim 4, characterized in that, The working concentration of both the upstream and downstream primers is 0.1 μM.

6. A product for detecting common pneumonia pathogens in musk deer, characterized in that, The product comprises the primer set as described in claim 1.

7. The product according to claim 6, characterized in that, The working concentrations of both upstream and downstream primers in the primer set are 0.1 μM.

8. The product according to claim 6, characterized in that, The product also includes 2×ES Taq Mastermix.

9. The product according to claim 6, characterized in that, The products include reagents, reagent kits, and chips.

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