Primer group, detection kit and detection method for detecting proteus mirabilis of rodent experimental animal

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 proteus mirabilis of rodent experimental animals. The primer group provided by the invention is used for detecting proteus mirabilis of rodent experimental animals; the sequences of the primer group are as shown in SEQ ID NO: 1 and SEQ ID NO: 2. According to the primer group and the detection method disclosed by the invention, the proteus mirabilis can be rapidly detected on the molecular level, and the primer group and the detection method have the advantages of simplicity and convenience in operation, high detection efficiency and accurate result, and are suitable for rapidly screening and monitoring the health condition of rodent experimental animals.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pathogenic microorganism detection, and particularly relates to a primer set for detecting Proteus mirabilis of rodent experimental animals, a detection kit and a detection method. BACKGROUND

[0002] Experimental animals are important carriers for biomedical research, and their health status has a key influence on the reliability of experimental results. Among common diseases of experimental animals, Proteus mirabilis infection is a problem that needs to be focused on. At present, the detection of the pathogen mainly focuses on cats, cows, rabbits and other animals, and the detection method for rodent experimental animals (such as mice) is relatively insufficient. The traditional detection method relying on biochemical identification has limitations such as complicated operation, long time consumption and high requirement for technical personnel. Therefore, there is an urgent need for a detection method for Proteus mirabilis of rodent experimental animals. SUMMARY

[0003] In order to solve the above technical problems, the present application provides a primer set, a detection kit and a detection method for detecting Proteus mirabilis of rodent experimental animals.

[0004] In a first aspect, the present application provides a primer set for detecting Proteus mirabilis of rodent experimental animals, which is used for amplifying Proteus mirabilis; the nucleotide sequence of the primer set is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0005] In the test process, it is found that Proteus mirabilis and Salmonella typhimurium have similar colony morphologies on DHL medium and cannot be distinguished, and the biochemical identification method has the possibility of misjudgment. Therefore, the present application discloses a primer sequence and a detection method for Proteus mirabilis of rodent experimental animals. The method can realize rapid detection of whether rodent experimental animals are infected with Proteus mirabilis at the molecular level, 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 rodent experimental animals.

[0006] In a second aspect, the present application provides a plasmid for detecting Proteus mirabilis of rodent experimental animals, which is obtained by cloning the sequencing results of the amplification fragment of the primer set.

[0007] In a third aspect, the present application provides a PCR detection kit for detecting Proteus mirabilis of rodent experimental animals, which comprises the primer set or the plasmid.

[0008] Preferably, the PCR detection kit comprises Taq Master Mix and H2O.

[0009] Preferably, the PCR reaction system of the PCR detection kit is 20μ L, including: 2 x Taq Master Mix 10 μ L, 8-12 μ M concentration of each primer 0.8-1.2 μ L, DNA template 1.8-2.2 μ L, ddH2O supplemented.

[0010] Preferably, the PCR reaction system of the PCR detection kit is 20 μ L, including: 2 x Taq Master Mix 10 μ L, 1 μ M concentration of each primer 1 μ L, DNA template 2 μ L, ddH2O supplemented.

[0011] In a fourth aspect, the present application provides a PCR detection method for detecting Proteus mirabilis in rodent experimental animals for non-disease diagnosis purposes, which uses the above primer pair combination or uses the above PCR detection kit for detection, comprising the following steps in sequence: extracting bacterial DNA; PCR amplification; electrophoresis.

[0012] Preferably, in the PCR amplification condition, the reaction condition of PCR amplification is: pre-denaturation: 94-96℃, 4-6min; cycle extension: 93-95℃, 25-35s; annealing: 50-60℃, 25-35s; cycle extension: 70-74℃, 40-50s; cycle 33-38 times; cycle outside extension: 70-75℃, 8-15min.

[0013] Preferably, in the PCR amplification condition, the reaction condition of PCR amplification is: pre-denaturation: 95℃, 5min; cycle extension: 94℃, 30s; annealing: 60℃, 30s; cycle extension: 72℃, 45s; cycle 35 times; cycle outside extension: 72℃, 10min.

[0014] Preferably, the electrophoresis condition is that the PCR product is electrophoresed in 1.2-1.7% agarose gel at 120V for 30-50min.

[0015] In a specific embodiment, the electrophoresis condition is that the PCR product is electrophoresed in 1.5% agarose gel at 120V for 40min.

[0016] The primer information and detection method of Proteus mirabilis in experimental animals described in the present application first designs primers; then extracts bacterial nucleic acids; combines the specificity of PCR requirements to distinguish and analyze the specific bands in the electrophoretogram; and finally completes the specificity and sensitivity test.

[0017] The detection kit and the detection method have the following advantages: high efficiency and rapidness: the nucleic acid extracted from the fecal sample of the experimental animal is detected, which significantly shortens the detection time; simple operation: the cumbersome culture steps such as biochemical identification are avoided; accurate result: the detection is carried out at the gene level, which improves the accuracy and reliability of the detection.

[0018] In summary, the technical scheme of the present application has the following effects: The present application discloses a primer set, a detection kit and a detection method for detecting Proteus mirabilis of rodent experimental animals. The method can realize rapid detection of Proteus mirabilis of rodent experimental animals at the molecular level, has the advantages of simple operation, high detection efficiency and accurate result, and is suitable for rapid screening and monitoring of the health status of rodent experimental animals. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is an electrophoresis map of the 16S rDNA amplification and sequencing detection result in Example 1; note: (M) D2000 DNA Ladder; (1-12) bacterial 16S rDNA amplification band; (NC) negative control.

[0020] Figure 2 It is an electrophoresis map of the primer screening result in Example 1; note: (M) D2000 DNA Ladder; (1) primer set corresponding to SEQ ID NO: 1 and SEQ ID NO: 2; (2) primer set corresponding to SEQ ID NO: 3 and SEQ ID NO: 4; (3) primer set corresponding to SEQ ID NO: 5 and SEQ ID NO: 6.

[0021] Figure 3 It is an electrophoresis map of the detection result of the sensitivity test in Example 1; note: (M) D2000 DNA Ladder; (1-6) the concentrations are 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , 5×10 0 cfu / ml, respectively.

[0022] Figure 4 It is an electrophoresis map of the detection result of the specificity test in Example 1; note: (M) D2000 DNA Ladder; (1-7) are Pasteurella pneumotropica, Klebsiella pneumoniae, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella oxytoca and Proteus mirabilis, respectively.

[0023] Figure 5 It is a schematic diagram of the synthesis of the positive plasmid in Example 1. 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.

[0025] The cholethiocyanate agar medium, nucleic acid dye, PCR premix, and DNA ladder used in this application were purchased from Solarbio. An electrophoresis instrument was purchased from Beijing Liuyi Biotechnology Co., Ltd.; an Eppendorf 5420 desktop high-speed centrifuge; an electric constant-temperature incubator was purchased from Beijing Kewei Yongxing Instrument Co., Ltd.; a gene amplifier was purchased from Hangzhou Biori Technology Co., Ltd.; a clean bench was purchased from Qingdao Haier Special Electric Co., Ltd.; and an Eppendorf micropipette was purchased. Example Example 1

[0026] Example 1 provides a primer set, a detection kit, and a detection method for detecting Proteus mirabilis in experimental rodents.

[0027] (1) Strains and samples Positive mice infected with Proteus mirabilis collected in our laboratory were dissected and the contents of the cecum were collected for DNA extraction. The nucleic acid was extracted by boiling. About 1 g of fecal sample was taken, 2 mL of normal saline was added, and stirred to disperse the feces into the solution. The solution was then heated for 10 min. 4 Dilute 100 μL of the diluted solution and inoculate it onto cholecalciferol agar. Incubate in a 37°C incubator for 24 h.

[0028] (2) Extraction of bacterial DNA Twelve black single colonies were picked on the cholethiazolyl agar medium and DNA was extracted using the boiling template method. The colonies were boiled in a 100°C water bath for 12 min and then centrifuged at 12,000 rpm for 10 min. The supernatant was used as the template.

[0029] (3) 16S rDNA amplification and sequencing The extracted DNA was used as a template and PCR amplification was performed using bacterial universal primers 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R 5′-ACGGCTACCTTGTTACGACTT-3′.

[0030] PCR amplification: A 20 μL PCR reaction system consisted of 10 μL of 2× Taq Master Mix, 1 μL of each primer (1 μM), 2 μL of DNA template, and ddH₂O to make up the residue. Reaction conditions included initial denaturation at 95°C for 5 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s, followed by extension at 72°C for 10 min. PCR products were electrophoresed on a 1.5% agarose gel at 120 V for 40 min and sequenced.

[0031] 16S rDNA amplification and sequencing: Genomic DNA extracted by boiling was used as a PCR template and PCR amplified using bacterial 16S rDNA universal primers. The resulting PCR product was identified by agarose gel electrophoresis. The electrophoresis diagram of the amplification results is shown in the figure below. Figure 1 As shown (Note: (M) D2000 DNA Ladder; (1-12) bacterial 16S rDNA amplified bands; (NC) negative control). The target band is approximately 1500 bp in size, consistent with the expected size. The sequencing result shows 100% similarity to the sequence of Proteus mirabilis strain A31498 reported in GenBank.

[0032] (4) Primer design and synthesis With reference to the 16S rDNA sequencing results, three sets of primers were designed using Primer 5.0 software. The specific information is shown in Table 1.

[0033] Table 1 Detailed information of the three primer groups in Example 1

[0034] The PCR amplification system was as described above, with initial denaturation at 95°C for 5 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and extension at 72°C for 10 min. PCR products were electrophoresed on a 1.5% agarose gel at 120 V for 40 min.

[0035] (5) Detection kit The PCR reaction system of the detection kit is 20 μ L, including: 2 × Taq Master Mix 10 μ L, 1 μ 1 M concentration of primers μ L, 2 μL DNA template, supplemented with ddH2O.

[0036] (6) Primer screening The PCR effects of the three designed primer sets were verified, and the amplified bands were compared.

[0037] The electropherogram of the primer screening result is shown in Figure 2 ; Note: (M) D2000 DNA Ladder; (1) primer group corresponding to SEQ ID NO: 1 and SEQ ID NO: 2; (2) primer group corresponding to SEQ ID NO: 3 and SEQ ID NO: 4; (3) primer group corresponding to SEQ ID NO: 5 and SEQ ID NO: 6.

[0038] The results show that the amplification effect of the primer group corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 is better; the amplification effect of the primer group corresponding to SEQ ID NO: 3 and SEQ ID NO: 4 is poorer; and the primer group corresponding to SEQ ID NO: 5 and SEQ ID NO: 6 has non-specific amplification. Therefore, the primer group corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 is finally selected for subsequent tests.

[0039] (7) Sensitivity test Proteus mirabilis was used as the detection bacteria, and after overnight culture, it was diluted by 10 times gradient with sterile normal saline, and then the colony counting was performed to obtain the concentration of each gradient: 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , and 5×10 0 cfu / ml, which were detected by the above-mentioned PCR method.

[0040] The sensitivity test detection result: Proteus mirabilis was used as the detection bacteria, and the specific primers were designed for amplification, and the concentration was determined for sensitivity verification. The electropherogram of the sensitivity test detection result is shown in Figure 3 ; Note: (M) D2000 DNA Ladder; (1-6) are 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , and 5×10 0 cfu / ml, respectively; The results show that the sensitivity of the PCR method is 5×10 2 cfu / ml.

[0041] (8) Specificity test According to the above PCR reaction condition and system, the proteus mirabilis used in the present application is detected, and the salmonella, the pasteurella pneumotropica, the klebsiella pneumoniae, the staphylococcus aureus and the pseudomonas aeruginosa are specifically detected.

[0042] The specific test detection result is shown in the electrophoretogram as Figure 4 (M) D2000 DNA Ladder; (1-7) are the pasteurella pneumotropica, the klebsiella pneumoniae, the salmonella, the staphylococcus aureus, the pseudomonas aeruginosa, the klebsiella oxytoca and the proteus mirabilis, respectively.

[0043] The results show that in the PCR electrophoretogram, only the sample corresponding to the proteus mirabilis has the target amplification band, and there is no other positive product, indicating that the detection method provided in the present application has good specificity.

[0044] (9) Establishment of positive quality control sample The gene sequence is shown in SEQ ID NO: 7.

[0045] PUC-57 is selected as the carrier plasmid, the chemically synthesized gene fragment SEQ ID NO: 7 is fused and cloned into PUC-57, the positive bacteria liquid is obtained by resistance screening method, and after the correct sequencing verification, the plasmid is extracted, and the glycerol bacteria are reserved for standby. The positive plasmid synthesis schematic diagram is shown in Figure 5 .

[0046] After the successful construction of the plasmid prepared in the present application, it can be used in large quantities, long term and stably, saving the time and labor cost of repeated culture of strains or extraction of DNA. The plasmid is extremely stable under the storage condition of-20℃, and can be stored for a long time without degradation or sequence variation. The purified plasmid only contains the target insertion fragment and the plasmid backbone, avoiding the interference of impurities or non-specific amplification in the PCR process, and because it does not contain complete and active pathogens, it is not easy to produce biological pollution.

[0047] Therefore, the plasmid prepared in the present application as the PCR positive control product has significant advantages in stability, purity, accurate quantification (copy number), standardization, safety, convenience and cost-effectiveness compared with the positive strain or the extracted positive genomic DNA, and is particularly suitable as the positive control product for PCR detection, for performance verification of reaction system, for pollution investigation and primer specificity verification.

[0048] In the current existing research, there is less information about the proteus mirabilis of rodent experimental animals, which is mostly concentrated in human or other animal sources, such as pig source, bovine source, etc. The positive control plasmid established by the present test kit can be used for the detection of proteus mirabilis of rodent experimental animals, and is convenient to store, stable and not easy to produce biological pollution.

[0049] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. It is therefore intended that whatever lies within the scope of the application be covered by the appended claims and that there be no intent, one way or the other, to limit the claims to the precise embodiments described.

Claims

1. A primer set for detecting Proteus mirabilis in rodent experimental animals, characterized in that: The primer set is used to amplify Proteus mirabilis; the nucleotide sequences of the primer set are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. A plasmid for detecting Proteus mirabilis in experimental rodents, characterized in that: The plasmid is cloned by sequencing the amplified fragments of the primer set according to claim 1; the gene sequence of the plasmid is shown in SEQ ID NO:

7.

3. A PCR detection kit for detecting Proteus mirabilis in rodent experimental animals, characterized in that: Comprising the primer set according to claim 1 or the plasmid according to claim 2.

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

5. The PCR detection kit according to claim 3, characterized in that The PCR reaction system of the 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.

6. The PCR detection kit according to claim 3, characterized in that The PCR reaction system of the PCR detection kit is 20 μ L, including: 2 × Taq Master Mix 10 μ L, 1 μ 1 M concentration of primers μ L, DNA template 2 μ L, supplemented with ddH2O.

7. A PCR detection method for detecting Proteus mirabilis in rodent laboratory animals for non-disease diagnosis purposes, characterized in that: Detection using the primer set of claim 1 or the PCR detection kit of any one of claims 3 to 6 comprises the following steps: extracting bacterial DNA; PCR amplification; electrophoresis.

8. The PCR detection method according to claim 7, 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.

9. The PCR detection method according to claim 7, 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: 60°C, 30 s; cycle extension: 72°C, 45 s; cycle 35 times; and outer cycle extension: 72°C, 10 min.

10. The PCR detection method according to claim 7, 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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