Primer group for detecting pathogenic microorganisms of rodent experimental animals and application of primer group
By designing primer sets and detection methods for multiplex PCR, the problems of cumbersome and time-consuming operation in the detection of Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus in existing technologies have been solved, enabling rapid, simple, and accurate detection of pathogens in rodent laboratory animals.
Patent Information
- Application Number
- CN202511611257.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-05
- Publication Date
- 2025-12-09
AI Technical Summary
Existing detection methods for pathogens such as Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus are cumbersome, time-consuming, and costly, making it difficult to achieve rapid and economical simultaneous detection.
A primer set for multiplex PCR detection was designed, including specific primer pairs for three bacteria, along with a matching PCR detection kit and detection method, to simultaneously detect the three pathogens in the same system through multiplex PCR reactions.
It enables rapid, simple, and accurate detection of three pathogens in rodent laboratory animals, significantly shortening the detection time, improving detection efficiency and accuracy, and avoiding cumbersome biochemical identification steps.
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Abstract
Description
Technical Field
[0001] This application relates to the technical field of pathogenic microorganisms in laboratory animals, specifically to a primer set for detecting pathogenic microorganisms in rodent laboratory animals and its application. Background Technology
[0002] Currently, squamous dermatitis in nude mice is a systemic hyperkeratotic dermatitis, with typical clinical symptoms including white, scaly scales covering the entire skin. SCID mice exhibit symptoms such as hair loss and bald patches. *Corynebacterium bovis* is generally considered the pathogen causing this condition. Nude mice infected with *Corynebacterium bovis* can act as a source of infection, spreading the infection to other nude mice through direct contact and contaminating their cages. However, clinically diagnosed cases suggest that infections with *Staphylococcus xylose* and *Staphylococcus squirrelii* can also cause the same clinical symptoms; these three bacteria are the most common clinically.
[0003] *Corynebacterium bovis* (C. bovis) is a Gram-positive, opportunistically pathogenic bacillus widely distributed worldwide. It infects various animals, causing hyperkeratosis (squamous skin disease) in nude mice and is also a common cause of mastitis in dairy cows. Hairless mice and immunodeficient mice, once infected, almost always carry the bacteria for life. *Staphylococcus xylose* is an opportunistically pathogenic commensal bacterium residing in the skin of mammals, but it exhibits significant pathogenicity in immunodeficient and gene knockout mice, manifesting as dermatitis and abscesses. This bacterium can be isolated from skin samples from these mice. *Staphylococcus squirrelii* is a coagulase-negative, Gram-positive coccus with high pathogenicity and wide distribution. It can be isolated from humans and animals, but rarely from humans, and animals are more susceptible. Cases of endocarditis, endophthalmitis, scalded skin syndrome, urinary tract infection, pelvic inflammatory disease, catheter-related bacteremia, and septic shock have been reported. Staphylococcus aureus can be isolated from infected skin.
[0004] Traditional detection methods typically rely on biochemical identification, which is cumbersome, time-consuming, and requires highly skilled operators. Single PCR detection methods require individual testing, increasing both time and cost. Currently, there is an urgent need for a rapid and economical method to detect three bacteria. Summary of the Invention
[0005] To address the aforementioned technical problems, this application provides a primer set for detecting pathogenic microorganisms in rodent laboratory animals and its application.
[0006] In a first aspect, this application provides a primer set for detecting pathogenic microorganisms in rodent laboratory animals, for the simultaneous detection of Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus; the primer set includes: The primer pair targeting Corynebacterium bovis has the sequences SEQ ID NO: 1 and SEQ ID NO: 2; The primer pair targeting Staphylococcus xylosus has the sequences SEQ ID NO: 3 and SEQ ID NO: 4; The primer pair targeting Staphylococcus aureus has the sequences SEQ ID NO: 5 and SEQ ID NO: 6.
[0007] Secondly, this application provides a multiplex PCR detection kit for pathogenic microorganisms in rodent laboratory animals, including the aforementioned primer set.
[0008] Preferably, the multiplex PCR detection kit includes Taq Master Mix and H2O.
[0009] Preferably, the PCR reaction system of the multiplex PCR detection kit is 20 μL, comprising: 10 μL of 2×Taq MasterMix, 0.8-1.2 μL each of primers at a concentration of 8-12 μM, 1.8-2.2 μL of DNA template, and ddH2O to make up the difference.
[0010] Preferably, the PCR reaction system of the multiplex PCR detection kit is 20 μL, comprising: 10 μL of 2×Taq MasterMix, 1 μL of each primer at a concentration of 10 μM, 2 μL of DNA template, and ddH2O to make up the difference.
[0011] Thirdly, this application provides a multiplex PCR detection method for pathogenic microorganisms in rodent laboratory animals for non-disease diagnostic purposes, using the above-mentioned primer set or the above-mentioned multiplex PCR detection kit, including the following steps in sequence: Bacterial DNA was extracted; PCR amplification was performed; and electrophoresis was used to obtain an electrophoretic image.
[0012] Preferably, the PCR amplification conditions are as follows: pre-denaturation: 94-96℃, 4-6 min; 33-38 cycles: 93-95℃ 25-35 s, 50-60℃ 25-35 s, 70-74℃ 40-50 s; extension cycle: 70-75℃, 8-12 min.
[0013] Preferably, the PCR amplification conditions are as follows: pre-denaturation: 95℃, 5 min; 35 cycles: 94℃ 30 s, 55℃ 30 s, 72℃ 45 s; cycle extension: 72℃, 10 min.
[0014] Preferably, the electrophoresis conditions are as follows: PCR products are electrophoresed in a 1.2-1.7% agarose gel at 120V for 30-50 minutes.
[0015] In one specific implementation, the electrophoresis conditions are: PCR products in a 1.5% agarose gel, electrophoresis at 120V for 40 minutes.
[0016] The purpose of this application is to provide a multiplex PCR detection method that can simultaneously detect three pathogens (Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus) in rodent laboratory animals. This method enables the simultaneous detection of three pathogenic microorganisms in the same system, achieving rapid detection of three common bacteria that cause hyperkeratosis in immunodeficient animals at the molecular level. It has the advantages of simple operation, high detection efficiency, and accurate results, and is suitable for rapid screening and monitoring of the health status of laboratory animals.
[0017] The primer information and detection method for experimental animal Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus described in this application involve primer design, combined with multiplex PCR requirements to clearly distinguish bands and design bacterial-specific regions; then bacterial nucleic acid extraction is performed; and finally, stability, specificity, sensitivity tests and practical application tests are conducted.
[0018] In summary, the technical solution of this application has the following effects: The primer set, detection kit, and detection method provided in this application have the following advantages: high efficiency and speed: rapid detection of three common pathogens in experimental animals simultaneously, significantly shortening the detection time; simple operation: avoiding cumbersome culture steps such as biochemical identification; accurate results: detection at the gene level, improving the accuracy and reliability of the detection. Attached Figure Description
[0019] Figure 1 Electrophoresis diagrams comparing the amplification effects of different primer sets for *Corynebacterium bovis*, *Staphylococcus xylose*, and *Staphylococcus squirrelii* in Example 1; Note: (M)D2000 DNA Ladder; (1) *Corynebacterium bovis*: primer pair sequences are SEQ ID NO: 1 and SEQ ID NO: 2; (2) *Corynebacterium bovis*: primer pair sequences are SEQ ID NO: 7 and SEQ ID NO: 8; (3) *Staphylococcus xylose*: primer pair sequences are SEQ ID NO: 3 and SEQ ID NO: 4; (4) *Staphylococcus xylose*: primer pair sequences are SEQ ID NO: 9 and SEQ ID NO: 10; (5) *Staphylococcus squirrelii*: primer pair sequences are SEQ ID NO: 5 and SEQ ID NO: 6; (6) *Staphylococcus squirrelii*: primer pair sequences are SEQ ID NO: 11 and SEQ ID NO: 12.
[0020] Figure 2For stability testing of Example 2, note: (M)D2000 DNA Ladder; (1-3) Triple PCR; (4-6) Staphylococcus xylose; (7-9) Staphylococcus aureus; (10-12) Corynebacterium bovis; (13) Negative control.
[0021] Figure 3 Electrophoresis diagrams for specific detection in Example 2; Note: (M) D2000 DNA Ladder; (1) Positive control; (2-4) Klebsiella pneumoniae; (5-7) Staphylococcus aureus; (8-10) Helicobacter spp.; (11-13) Pasteurella pneumophila; (14) Negative control.
[0022] Figure 4 The image shows the electrophoresis results of the sensitivity detection in Example 3; Note: (M)D2000 DNA Ladder; (1-6) concentrations are 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL, respectively.
[0023] Figure 5 The image shows the detection results of the laboratory-preserved samples in Example 4; Note: (M)D2000 DNA Ladder; (1) Positive control; (8, 14, 16) Staphylococcus xylose positive; (2, 11, 15) Corynebacterium bovis positive; (9) Staphylococcus aureus positive; (4, 5) Corynebacterium bovis and Staphylococcus xylose positive; (6, 19) Staphylococcus aureus and Corynebacterium bovis positive; (10, 13) Both bacteria are positive; (3, 7, 12, 17, 18) All three bacteria are negative; (20) Negative control. Detailed Implementation
[0024] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.
[0025] Example
[0026] Example 1
[0027] Example 1 provides a primer set, detection kit, and detection method for detecting pathogenic microorganisms in rodent laboratory animals.
[0028] (1) Experimental materials (1.1) Strains and Samples Target strains: Corynebacterium bovis (BNCC368427), Staphylococcus xylose (BNCC342448), and Staphylococcus aureus (BNCC342448) were purchased from Beina Biotechnology Co., Ltd. Control strains: The nucleic acids of common laboratory animal-related bacteria such as Klebsiella pneumoniae and Staphylococcus aureus were obtained from samples preserved in our laboratory.
[0029] Clinical samples: 18 nucleic acid samples preserved in this laboratory.
[0030] (1.2) Primer design Primers were designed based on the specific regions of Corynebacterium bovis, Staphylococcus xylosus, and Staphylococcus aureus. Primer genes, primer sequences, product lengths, and literature sources are shown in Table 1. The primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.
[0031] Table 1. Relevant information on primer sequences for Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus.
[0032] (2) Main reagents and instruments Columbia blood agar base, sterile defibrinated Mianyang blood, nutrient agar, nucleic acid dyes, PCR premix, and DNA Ladder were purchased from Solarbio.
[0033] Electrophoresis apparatus from Beijing Liuyi Biotechnology Co., Ltd.; Eppendorf 5420 benchtop high-speed centrifuge; electric thermostatic incubator from Beijing Kewei Yongxing Instrument Co., Ltd.; gene amplification instrument from Hangzhou Borui Technology Co., Ltd.; clean bench from Qingdao Haier Special Electric Appliances Co., Ltd.; Eppendorf micropipette.
[0034] (3) Test kit The PCR reaction system of the detection kit is 20 μL, including: 10 μL of 2×Taq Master Mix, 1 μL of each of the 10 μM primers, 2 μL of DNA template, and ddH2O to make up the volume.
[0035] (4) Detection method (4.1) Extraction of bacterial DNA After reviving and culturing three standard strains of Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus, DNA was extracted using the 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.
[0036] (4.2) PCR amplification The PCR reaction system is 20 μL, including: 10 μL of 2×Taq Master Mix, 1 μL of each primer (10 μM), 2 μL of DNA template, and ddH2O to make up the volume.
[0037] The reaction conditions were as follows: pre-denaturation: 95℃, 5 min; 35 cycles (94℃, 30 s; 55℃, 30 s; 72℃, 45 s); cycle extension: 72℃, 10 min.
[0038] (4.3) Electrophoresis to obtain electrophoresis diagram The PCR products were electrophoresed in a 1.5% agarose gel at 120V for 40 min to obtain the electrophoresis image.
[0039] (5) Primer screening Two sets of primers were designed for each of Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus to verify the single PCR effect, and the amplified bands were compared.
[0040] Electrophoresis image as shown Figure 1 As shown; Note (M)D2000 DNA Ladder; (1) Corynebacterium bovis: primer pair sequences are SEQ ID NO: 1 and SEQ ID NO: 2; (2) Corynebacterium bovis: primer pair sequences are SEQ ID NO: 7 and SEQ ID NO: 8; (3) Staphylococcus xylose: primer pair sequences are SEQ ID NO: 3 and SEQ ID NO: 4; (4) Staphylococcus xylose: primer pair sequences are SEQ ID NO: 9 and SEQ ID NO: 10; (5) Staphylococcus aureus: primer pair sequences are SEQ ID NO: 5 and SEQ ID NO: 6; (6) Staphylococcus aureus: primer pair sequences are SEQ ID NO: 11 and SEQ ID NO: 12.
[0041] The results showed that both primer pairs for *Corynebacterium bovis* had good amplification effects; primer pairs for *Staphylococcus xylose* with sequences SEQ ID NO: 3 and SEQ ID NO: 4 had good amplification effects; and primer pairs for *Staphylococcus squirrelii* with sequences SEQ ID NO: 5 and SEQ ID NO: 6 had good amplification effects. However, based on the requirements of multiplex PCR experiments, to avoid interference from adjacent amplified bands being of similar size, the primer pairs for *Corynebacterium bovis* with sequences SEQ ID NO: 1 and SEQ ID NO: 2, for *Staphylococcus xylose* with sequences SEQ ID NO: 3 and SEQ ID NO: 4, and for *Staphylococcus squirrelii* with sequences SEQ ID NO: 5 and SEQ ID NO: 6 were ultimately selected for subsequent experiments.
[0042] (6) Stability testing The established multiplex PCR method of "Corynebacterium bovis + Staphylococcus xylose + Staphylococcus squirrelii" was compared with the amplification bands of Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus squirrelii by single PCR method.
[0043] Electrophoresis image as shown Figure 2 As shown; Note: (M)D2000 DNA Ladder; (1-3) Multiplex PCR method; (4-6) Staphylococcus xylose; (7-9) Staphylococcus aureus; (10-12) Corynebacterium bovis; (13) Negative control.
[0044] The results showed that the positive control of the multiplex PCR method simultaneously displayed amplified target bands of Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus, indicating that the multiplex PCR detection method provided in this application has good stability.
[0045] Example 2
[0046] This embodiment tests the specificity of the detection kit and detection method used to detect pathogenic microorganisms in rodent laboratory animals.
[0047] The specificity of the established method was tested by amplifying Klebsiella pneumoniae, Staphylococcus aureus, Helicobacter spp., and Pasteurella multocida using the established multiplex PCR method of "Corynebacterium bovis + Staphylococcus xylose + Staphylococcus aureus"; three nucleic acid samples were prepared for each bacterium.
[0048] Electrophoresis image as shown Figure 3 As shown; Note: (M)D2000 DNA Ladder; (1) Positive control; (2-4) Klebsiella pneumoniae; (5-7) Staphylococcus aureus; (8-10) Helicobacter spp.; (11-13) Pasteurella pneumophila; (14) Negative control.
[0049] The results showed that only the first tube of positive control had the target band amplified, and no other positive products were found, indicating that the multiplex PCR detection method provided in this application has good specificity.
[0050] Example 3
[0051] This embodiment tests the sensitivity of a detection kit and detection method for detecting pathogenic microorganisms in rodent laboratory animals.
[0052] DNA from three pathogens—Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus—was diluted at a ratio of 1:10, resulting in concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL, respectively. The sensitivity of multiplex PCR was then tested.
[0053] Electrophoresis image as shown Figure 4 As shown; Note: (M)D2000 DNA Ladder; (1-6) concentrations are 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL, respectively.
[0054] The results showed that the lowest detectable concentration of Staphylococcus aureus DNA was 1 pg / μL, while the lowest detectable concentration of Staphylococcus xylose and Corynebacterium bovis was 10 pg / μL.
[0055] Example 4
[0056] This embodiment uses the detection kit and detection method provided in Example 1 to detect 18 fecal nucleic acid samples stored in the laboratory.
[0057] Electrophoresis image as shown Figure 5 As shown; Note: (M)D2000 DNA Ladder; (1) Positive control; (8, 14, 16) Staphylococcus xylose positive; (2, 11, 15) Corynebacterium bovis positive; (9) Staphylococcus aureus positive; (4, 5) Corynebacterium bovis and Staphylococcus xylose positive; (6, 19) Staphylococcus aureus and Corynebacterium bovis positive; (10, 13) Both bacteria are positive; (3, 7, 12, 17, 18) All three bacteria are negative; (20) Negative control.
[0058] Multiplex PCR was used to amplify fecal nucleic acid samples preserved in the laboratory. The results were consistent with the expected results, and no non-specific products were amplified. Multiplex PCR was used to detect the nucleic acid of 18 preserved fecal and tracheal secretion samples. The results showed that 3 samples were positive for Staphylococcus xylose, 3 samples were positive for Corynebacterium bovis, 1 sample was positive for Staphylococcus squirrelii, 2 samples were positive for both Corynebacterium bovis and Staphylococcus xylose, 2 samples were positive for both Staphylococcus squirrelii and Corynebacterium bovis, 2 samples were positive for all three bacteria, and 5 samples were negative for all three bacteria.
[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A primer set for detecting pathogenic microorganisms in rodent laboratory animals, characterized in that, For simultaneous and parallel detection of Corynebacterium bovis, Staphylococcus xylose, and Staphylococcus aureus; the primer set includes: The primer pair targeting Corynebacterium bovis has the sequences SEQ ID NO: 1 and SEQ ID NO: 2; The primer pair targeting Staphylococcus xylosus has the sequences SEQ ID NO: 3 and SEQ ID NO: 4; The primer pair targeting Staphylococcus aureus has the sequences SEQ ID NO: 5 and SEQ ID NO:
6.
2. A multiplex PCR detection kit for pathogenic microorganisms in rodent laboratory animals, characterized in that, Includes the primer set as described in 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 μL, including: 10 μL of 2×Taq Master Mix, 0.8-1.2 μL of each primer at a concentration of 8-12 μM, 1.8-2.2 μL of DNA template, and ddH2O to make up the difference.
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 μL, including: 10 μL of 2×Taq Master Mix, 1 μL of each of the 10 μM primers, 2 μL of DNA template, and ddH2O to make up the volume.
6. A multiplex PCR detection method for pathogenic microorganisms in rodent laboratory animals for non-disease diagnostic purposes, characterized in that, The detection is performed using the primer set described in claim 1 or the multiplex PCR detection kit described in any one of claims 2-5, comprising the following steps in sequence: Extracting bacterial DNA; PCR amplification; electrophoresis to obtain electrophoresis image.
7. The multiplex PCR detection method according to claim 6, characterized in that, The PCR amplification conditions are as follows: pre-denaturation: 94-96℃, 4-6 min; 33-38 cycles: 93-95℃ 25-35 s, 50-60℃ 25-35 s, 70-74℃ 40-50 s; extension cycle: 70-75℃, 8-12 min.
8. The multiplex PCR detection method according to claim 6, characterized in that, The PCR amplification conditions are as follows: pre-denaturation: 95℃, 5 min; 35 cycles: 94℃ 30 s, 55℃ 30 s, 72℃ 45 s; cycle extension: 72℃, 10 min.
9. The multiplex PCR detection method according to claim 6, characterized in that, The electrophoresis conditions were as follows: PCR products were electrophoresed in a 1.2-1.7% agarose gel at 120V for 30-50 minutes.