Primer group, detection kit and detection method for detecting pathogenic microorganisms of rodent experimental animals

Abstract

The invention relates to the technical field of pathogenic microorganism detection, and particularly discloses a primer group, a detection kit and a detection method for detecting pathogenic microorganisms of rodent experimental animals. The primer group disclosed by the invention is used for simultaneously detecting pasteurella pneumophila, klebsiella pneumoniae and staphylococcus aureus; the primer group comprises a primer pair aiming at pasteurella pneumophila, and the sequences of the primer pair are SEQ ID NO: 1 and SEQ ID NO: 2; the sequences of the primer pair aiming at the klebsiella pneumoniae are SEQ ID NO: 7 and SEQ ID NO: 8; the sequences of the primer pair aiming at the staphylococcus aureus are SEQ ID NO: 9 and SEQ ID NO: 10. The primer group and the detection method disclosed by the invention can realize rapid detection of three bacteria on a molecular level, have the advantages of simplicity and convenience in operation, high detection efficiency and accurate result, and are suitable for rapid screening and monitoring of health conditions of experimental animals.

Description

Technical Field

[0001] The present application relates to the technical field of pathogenic microorganism detection, and in particular to a primer set, a detection kit, and a detection method for detecting pathogenic microorganisms in rodent laboratory animals. Background Art

[0002] Laboratory animals play a vital role in biomedical research, and their health directly impacts the reliability of experimental results. Traditional detection methods typically rely on biochemical identification, which is cumbersome, time-consuming, and requires high operator skill. Single PCR detection methods require testing each animal individually, increasing both time and financial costs.

[0003] Therefore, a method that can quickly and economically detect the three bacteria is urgently needed. Summary of the Invention

[0004] In order to solve the above technical problems, the present application provides a primer set, a detection kit, and a detection method for detecting pathogenic microorganisms in rodent laboratory animals.

[0005] In the first aspect, the present application provides a primer set for detecting pathogenic microorganisms in rodent experimental animals, which is used for the parallel and simultaneous detection of Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus; the primer set includes The primer pair for Pasteurella pneumophila has the sequences of SEQ ID NO: 1 and SEQ ID NO: 2; The primer pair for Klebsiella pneumoniae has the sequences of SEQ ID NO: 7 and SEQ ID NO: 8; The primer pair for Staphylococcus aureus has the sequences of SEQ ID NO: 9 and SEQ ID NO: 10.

[0006] In a second aspect, the present application provides a multiplex PCR detection kit for pathogenic microorganisms of rodent laboratory animals, comprising the above-mentioned primer set.

[0007] Preferably, the multiplex PCR detection kit comprises Taq Master Mix and H2O.

[0008] Preferably, the PCR reaction system of the multiplex PCR detection kit is 20 m L, including: 2 × Taq MasterMix 10 m L, 8-12 m The primer concentration is 0.8-1.2 m L, DNA template 1.8-2.2 μL, supplemented with ddH2O.

[0009] Preferably, the PCR reaction system of the multiplex PCR detection kit is 20 m L, including: 2 × Taq MasterMix 10 m L, 1 m 1 M concentration of primers m L, 2 μL DNA template, supplemented with ddH2O.

[0010] In a third aspect, the present application provides a multiplex PCR detection method for pathogenic microorganisms in rodent laboratory animals for non-disease diagnosis purposes, using the above-mentioned primer pair combination or the above-mentioned multiplex PCR detection kit for detection, comprising the following steps in sequence: Extract bacterial DNA; amplify by PCR; and obtain electrophoresis pattern.

[0011] Preferably, among the PCR amplification conditions, the reaction conditions for PCR amplification are: pre-denaturation: 94-96°C, 4-6 min; cycle extension: 93-95°C, 25-35 s; annealing: 50-60°C, 25-35 s; cycle extension: 70-74°C, 40-50 s; cycle 33-38 times; and cycle extension: 70-75°C, 8-15 min.

[0012] Preferably, in the PCR amplification conditions, the reaction conditions for PCR amplification are: pre-denaturation: 95°C, 5 min; cycle extension: 94°C, 30 s; annealing: 55°C, 30 s; cycle extension: 72°C, 45 s; cycle 35 times; and outer cycle extension: 72°C, 10 min.

[0013] Preferably, the electrophoresis condition is that the PCR product is electrophoresed in a 1.2-1.7% agarose gel at 120V for 30-50 minutes.

[0014] In a specific embodiment, the electrophoresis condition is that the PCR product is electrophoresed in a 1.5% agarose gel at 120V for 40 minutes.

[0015] The purpose of this application is to provide a multiplex PCR detection method that can simultaneously detect three pathogens, which can realize the simultaneous detection of three pathogenic microorganisms in the same system, and has the characteristics of simple operation, high detection efficiency, and accurate results.

[0016] The primer information and detection methods for experimental animal Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus described in this application are designed by combining primers with the requirements of multiplex PCR to clearly distinguish bands and design bacterial-specific regions; then bacterial nucleic acids are extracted; and the results have passed stability, specificity, sensitivity tests, and practical application tests.

[0017] The detection kit and detection method provided in this application have the following advantages: high efficiency and rapidity: rapid detection of three common pathogens in experimental animals at the same time, significantly shortening the detection time; simple operation: avoiding tedious culture steps such as biochemical identification; accurate results: detection at the genetic level, improving the accuracy and reliability of the detection.

[0018] In summary, the technical solution of this application has the following effects: This application discloses a multiplex PCR primer sequence and detection method for Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus. This method enables rapid molecular-level detection of these three common bacteria in rodents. It offers advantages such as ease of use, high detection efficiency, and accurate results, making it suitable for rapid screening and monitoring of the health status of experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The electrophoretic diagrams are comparative images of the amplification effects of different primers for Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus in Example 1; Note: (M) D2000 DNA Ladder; (1) Pasteurella pneumophila: primer pair sequence is SEQ ID NO: 1 and SEQ ID NO: 2; (2) Pasteurella pneumophila: primer pair sequence is SEQ ID NO: 3 and SEQ ID NO: 4; (3) Klebsiella pneumoniae: primer pair sequence is SEQ ID NO: 5 and SEQ ID NO: 6; (4) Klebsiella pneumoniae: primer pair sequence is SEQ ID NO: 7 and SEQ ID NO: 8; (5) Staphylococcus aureus: primer pair sequence is SEQ ID NO: 9 and SEQ ID NO: 10; (6) Staphylococcus aureus: primer pair sequence is SEQ ID NO: 11 and SEQ ID NO: 12.

[0020] Figure 2 The electrophoresis diagram of the stability test in Example 1; Note: (M) D2000 DNA Ladder; (1-3) positive control; (4-6) Klebsiella pneumoniae; (7-9) Staphylococcus aureus; (10-12) Pasteurella pneumophila; (13) negative control.

[0021] Figure 3 This is the electrophoresis diagram of the specific detection in Example 1; Note: (M) D2000 DNA Ladder; (1) positive control; (2-4) Pseudomonas aeruginosa; (5-7) Salmonella typhimurium; (8-10) Helicobacter; (11-13) beta-hemolytic Streptococcus; (14) negative control.

[0022] Figure 4The electrophoresis diagram of the sensitivity test in Example 1; Note: (M) D2000 DNA Ladder; (1-6) concentrations are 0.1 pg / m L, 1pg / m L, 10pg / m L, 100pg / m L, 1ng / m L, 10ng / m L.

[0023] Figure 5 The test results of the laboratory-stored samples in Example 1 are shown in the figure; Note: (M) D2000 DNA Ladder; (1) positive control; (2, 6, 10, 11, 13) Klebsiella pneumoniae positive; (3, 7, 14) Klebsiella pneumoniae and Staphylococcus aureus positive; (4, 8) Pasteurella pneumophila positive; (5, 15, 17, 18, 19) negative; (9) Klebsiella pneumoniae and Pasteurella pneumophila positive; (12, 16) Staphylococcus aureus positive; (20) negative control. DETAILED DESCRIPTION

[0024] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in this application. Example Example 1

[0025] Example 1 provides a primer set, a detection kit, and a detection method for detecting pathogenic microorganisms in rodent laboratory animals.

[0026] (1) Experimental materials (1.1) Strains and samples Target strains: Pasteurella pneumophila ATCC 35149, Klebsiella pneumoniae ATCC 13883, and Staphylococcus aureus ATCC 25923 were purchased from Beina Biotechnology Co., Ltd.

[0027] Control strains: Pseudomonas aeruginosa, Salmonella typhimurium and other common experimental animal-related bacterial nucleic acids were obtained from samples stored in our laboratory.

[0028] Clinical samples: 18 stool nucleic acid samples preserved by this laboratory.

[0029] (1.2) Primer design Software is applied based on the specific regions of Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus Primer Premier designed the corresponding primers. The primer genes, primer sequences, and product lengths are shown in Table 1 . The primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

[0030] Table 1 Information on primer sequences for Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus

[0031] (2) Main reagents and instruments Columbia blood agar base, mannitol-sodium chloride medium, cholethiocyanate agar, sterile defibrinated sheep blood, nucleic acid dyes, PCR premix, and DNA ladder were purchased from Solarbio. An electrophoresis instrument from Beijing Liuyi Biotechnology Co., Ltd.; an Eppendorf 5420 desktop high-speed centrifuge; an electric constant-temperature incubator from Beijing Kewei Yongxing Instrument Co., Ltd.; a gene amplifier from Hangzhou Biori Technology Co., Ltd.; and a clean bench from Qingdao Haier Special Electric Co., Ltd.; as well as Eppendorf micropipettes were purchased.

[0032] (3) Detection kit The PCR reaction system of the detection kit is 20 m L, including: 2 × Taq Master Mix 10 m L, 1 m 1 M concentration of primers m L, 2 μL DNA template, supplemented with ddH2O.

[0033] (4) Detection method (4.1) Extraction of bacterial DNA Three standard strains of Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus were revived and cultured, and DNA was extracted using the water-boiling template method. The samples were boiled in a 100°C water bath for 12 minutes and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was used as the template.

[0034] (4.2) PCR amplification The reaction system is: 20 m L, including: 2 × Taq Master Mix 10 m L, 1 m 1 M concentration of primers m L, 2 μL DNA template, supplemented with ddH2O.

[0035] The reaction conditions were as follows: pre-denaturation: 95°C, 5 min; cyclic extension: 94°C, 30 s; annealing: 55°C, 30 s; cyclic extension: 72°C, 45 s; 35 cycles; and cyclic extension: 72°C, 10 min.

[0036] (4.3) Electrophoresis to obtain electrophoretic pattern The PCR products were electrophoresed in 1.5% agarose gel at 120 V for 40 min to obtain the electrophoretic pattern.

[0037] (5) Primer screening The effects of two sets of primers designed for Klebsiella pneumoniae, Staphylococcus aureus and Pasteurella pneumophila were verified by single PCR, and the amplified bands were compared.

[0038] Electrophoresis diagram Figure 1 As shown; Note: (M) D2000 DNA Ladder; (1-2) Pasteurella pneumophila; (3-4) Klebsiella pneumoniae; (5-6) Staphylococcus aureus.

[0039] The results showed that the primer pair for Pasteurella pneumophila, SEQ ID NO: 1 and SEQ ID NO: 2, had the best amplification effect; the two primer pairs for Klebsiella pneumoniae also had the best amplification effect; and the two primer pairs for Staphylococcus aureus also had the best amplification effect. However, based on the requirements of the multiplex PCR test, to avoid interference in the result judgment due to the similar size of adjacent amplified bands, the primer pair for Pasteurella pneumophila, SEQ ID NO: 1 and SEQ ID NO: 2; the primer pair for Klebsiella pneumoniae, SEQ ID NO: 7 and SEQ ID NO: 8; and the primer pair for Staphylococcus aureus, SEQ ID NO: 9 and SEQ ID NO: 10, were ultimately selected for subsequent experiments.

[0040] (6) Stability testing The amplified bands of the established "Klebsiella pneumoniae + Staphylococcus aureus + Pasteurella pneumophila" multiplex PCR method were compared with those of the single PCR method "Klebsiella pneumoniae", "Staphylococcus aureus" and "Pasteurella pneumophila".

[0041] Electrophoresis diagram Figure 2 As shown; Note: (M) D2000 DNA Ladder; (1-3) positive control; (4-6) Klebsiella pneumoniae; (7-9) Staphylococcus aureus; (10-12) Pasteurella pneumophila; (13) negative control.

[0042] The results showed that the positive control simultaneously displayed amplified target bands of Klebsiella pneumoniae, Staphylococcus aureus, and Pasteurella pneumophila, indicating that the multiplex PCR detection method provided by the present application has good stability.

[0043] (7) Specificity detection The specificity of the method was tested by amplifying Pseudomonas aeruginosa, Salmonella typhimurium, Helicobacter spp., and beta-hemolytic Streptococcus using the established multiplex PCR system; three nucleic acid samples were used for each bacterium.

[0044] Electrophoresis diagram Figure 3 As shown; Note: (M) D2000 DNA Ladder; (1) positive control; (2-4) Pseudomonas aeruginosa; (5-7) Salmonella typhimurium; (8-10) Helicobacter; (11-13) beta-hemolytic Streptococcus; (14) negative control.

[0045] The results showed that only the first tube of positive control had the amplified target band, and no other positive products, indicating that the multiplex PCR detection method provided by the application has good specificity.

[0046] (8) Sensitivity testing The DNA of three pathogens, Klebsiella pneumoniae, Staphylococcus aureus and Pasteurella pneumophila, were diluted at a ratio of 1:10, and the concentrations after dilution were 10 ng / m L, 1ng / m L, 100pg / m L, 10pg / m L, 1pg / m L, 0.1pg / m L, Testing the sensitivity of multiplex PCR.

[0047] Electrophoresis diagram Figure 4 As shown; Note: (M) D2000 DNA Ladder; (1-6) concentrations are 0.1pg / m L, 1pg / m L, 10pg / m L, 100pg / m L, 1ng / m L, 10ng / m L.

[0048] The results showed that the minimum detection concentration of DNA of Pasteurella pneumophila and Staphylococcus aureus was 1pg / m The minimum detection concentration of Klebsiella pneumoniae DNA is 10 pg / L. m L.

[0049] (9) Application The established multiplex PCR method was used to detect 18 stool nucleic acid samples stored in the laboratory.

[0050] Electrophoresis diagram Figure 5As shown; Note: (M) D2000 DNA Ladder; (1) positive control; (2, 6, 10, 11, 13) positive for Klebsiella pneumoniae; (3, 7, 14) positive for Klebsiella pneumoniae and Staphylococcus aureus; (4, 8) positive for Pasteurella pneumophila; (5, 15, 17, 18, 19) negative; (9) positive for Klebsiella pneumoniae and Pasteurella pneumophila; (12, 16) positive for Staphylococcus aureus; (20) negative control.

[0051] The results showed that the established multiplex PCR method could accurately detect the infection of three bacterial species in preserved stool samples.

[0052] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A primer set for detecting pathogenic microorganisms in rodent laboratory animals, characterized in that: Used for the simultaneous detection of Pasteurella pneumophila, Klebsiella pneumoniae, and Staphylococcus aureus; the primer set includes The primer pair for Pasteurella pneumophila has the sequences of SEQ ID NO: 1 and SEQ ID NO: 2; The primer pair for Klebsiella pneumoniae has the sequences of SEQ ID NO: 7 and SEQ ID NO: 8; The primer pair for Staphylococcus aureus has the sequences of SEQ ID NO: 9 and SEQ ID NO:

10.

2. A multiplex PCR detection kit for pathogenic microorganisms in rodent laboratory animals, characterized in that: Comprising the primer set according to claim 1.

3. The multiplex PCR detection kit according to claim 2, characterized in that Includes Taq Master Mix and H2O.

4. The multiplex PCR detection kit according to claim 2, characterized in that The PCR reaction system of the multiplex PCR detection kit is 20 μ L, including: 2 × Taq Master Mix 10 μ L, 8-12 μ The primer concentration is 0.8-1.2 μ L, DNA template 1.8-2.2 μ L, supplemented with ddH2O.

5. The multiplex PCR detection kit according to claim 2, characterized in that The PCR reaction system of the multiplex PCR detection kit is 20 μ L, including: 2 × Taq Master Mix 10 μ L, 1 μ 1 M concentration of primers each μ L, DNA template 2 μ L, supplemented with ddH2O.

6. A multiplex PCR method for detecting pathogenic microorganisms in rodent laboratory animals for non-disease diagnosis purposes, characterized in that: Detection using the primer pair combination of claim 1 or the multiplex PCR detection kit of any one of claims 2 to 5 comprises sequentially performing the following steps: Extract bacterial DNA; PCR amplification; electrophoresis to obtain electropherogram.

7. The multiplex PCR detection method according to claim 6, characterized in that: Among the PCR amplification conditions, the reaction conditions for PCR amplification are: pre-denaturation: 94-96°C, 4-6 min; cycle extension: 93-95°C, 25-35 s; annealing: 50-60°C, 25-35 s; cycle extension: 70-74°C, 40-50 s; cycle 33-38 times; and cycle extension: 70-75°C, 8-15 min.

8. The multiplex PCR detection method according to claim 6, characterized in that: Among the PCR amplification conditions, the reaction conditions for PCR amplification are: pre-denaturation: 95°C, 5 min; cycle extension: 94°C, 30 s; annealing: 55°C, 30 s; cycle extension: 72°C, 45 s; cycle 35 times; and cycle extension: 72°C, 10 min.

9. The multiplex PCR detection method according to claim 6, characterized in that: The electrophoresis condition is as follows: the PCR product is electrophoresed in 1.2-1.7% agarose gel at 120V for 30-50 minutes.

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