Method for separating, screening and identifying staphylococcus in chick embryo sample

Abstract

The invention discloses a method for separating, screening and identifying staphylococcus in a chick embryo sample, which comprises the following steps: S1, sterilizing collected chick embryo tissues, cutting into pieces, mixing with an SCB liquid culture medium, and culturing in a shaking table to obtain a culture solution; s2, scribing the obtained culture solution into a BP (Back Propagation) culture plate, putting the plate into an incubator for standing culture, after monoclone grows out of a culture dish, picking the monoclone, and uniformly blowing and beating the monoclone in PBS (Phosphate Buffer Solution) to obtain a mixed bacteria solution; s3, incubating the obtained mixed bacteria solution in lysostaphin to obtain a mixed solution I; s4, Tris-HCl and protease K are added into the obtained mixed solution I, incubation is conducted firstly, then heating is conducted, and a mixed solution II is obtained; and S5, carrying out PCR amplification by taking the obtained mixed solution II as a template to obtain a product, and identifying. The method is accurate, rapid, economical and efficient.

Description

Technical Field

[0001] The invention belongs to the technical field of separation, screening and identification of microorganisms, and particularly relates to a method for separation, screening and identification of staphylococci in chicken embryo samples. Background Art

[0002] Chicken embryos contain a variety of nutrients necessary for the human body. However, the quality of chicken embryo eggs on the market varies greatly. Poor quality chicken embryo eggs may contain a variety of harmful bacteria, posing a food safety hazard. In addition, bacterial contamination in chicken embryos will not only affect the production and processing of chicken embryo egg products in the later stage, but also affect the growth and development of chicks in the chicken embryos, causing resource losses to the poultry industry.

[0003] Staphylococcus is one of the common contaminating bacteria in chicken embryos. Among them, Staphylococcus aureus of the genus Staphylococcus can cause chicken staphylococcal disease, which can attack chickens of any age, including chicken embryos. Therefore, understanding the situation of Staphylococcus in chicken embryos can lay the foundation for animal husbandry and food production safety.

[0004] Staphylococcus is a Gram-positive bacterium with a thick cell wall structure, which mainly contains peptidoglycan and teichoic acid. 90% of Staphylococcus aureus cells contain protein A on their surface, which combines with the peptidoglycan in the cell wall to form a dense covalent cross-linked structure, increasing the difficulty of extracting the genomic DNA of the bacterium. At present, most methods for isolating Staphylococcus from samples require ① picking bacterial monoclonals, ② culturing bacteria overnight, ③ using chemical methods (large amounts of lysostaphin) or mechanical methods (high-speed homogenization, bead milling) to break Staphylococcus, ④ then using a genome extraction kit to extract the bacterial genome, ⑤ then performing polymerase chain reaction (PCR), ⑥ sequencing and identification of the amplified products, etc. These methods are cumbersome, time-consuming, labor-intensive, and costly, which is not conducive to the rapid and economical identification of samples. Although some methods for saving labor and reducing costs have been published, such as direct boiling method and denaturant lysis method, the success rate of these methods is not high. It is difficult to obtain bacterial genomes that meet the requirements of direct PCR, and it is impossible to obtain sufficient PCR products for subsequent dideoxy sequencing (Sanger sequencing). Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a method for separation, screening and identification of Staphylococcus aureus, which can effectively simplify the steps of separation of Staphylococcus aureus, shorten the separation and identification time, and reduce the separation and identification cost.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: The present invention provides a method for separating, screening and identifying Staphylococcus aureus in a chicken embryo sample, the method comprising the following steps: S1: Collect chicken embryo tissue and prepare culture medium; S2: Cultivate the bacterial solution to obtain a single clone colony, pick a single clone and pipette it evenly in PBS to obtain a mixed bacterial solution; S3: placing the mixed bacterial solution obtained in S2 in lysostaphin and incubating it to obtain mixed solution 1; S4: adding Tris-HCl and proteinase K to the mixed solution 1 obtained in S3, incubating first, and then heating to obtain a mixed solution 2; S5: Using the mixed solution 2 obtained in S4 as a template, PCR amplification is performed to obtain a product, and then the product is identified.

[0007] Preferably, the present invention provides a method for separating, screening and identifying Staphylococci in chicken embryo samples, the method comprising the following steps: S1: sterilize the collected chicken embryo tissue, cut it into pieces, mix it with SCB liquid culture medium, and culture it in a shaker to obtain culture medium; S2: Streak the obtained culture solution onto a BP culture plate, place it in an incubator and culture it statically. After a single clone grows out of the culture dish, pick the single clone and pipette it evenly in PBS to obtain a mixed bacterial solution; S3: incubating the mixed bacterial solution obtained in S2 with lysostaphin to obtain a mixed solution 1; S4: adding Tris-HCl and proteinase K to the mixed solution 1 obtained in S3, incubating first, and then heating to obtain a mixed solution 2; S5: Using the mixed solution 2 obtained in S4 as a template, PCR amplification is performed to obtain a product, and then the product is identified.

[0008] HCl Further, the chicken embryo tissue in step S1 is mixed with the SCB liquid culture medium in a volume ratio of 1:5-1:15; the shaking incubator culture conditions are: 37° C., 160-220 rpm, and culture for 24-36 h.

[0009] Furthermore, the static culture conditions in step S2 are: 37° C., 24 h.

[0010] Furthermore, in step S3, the volume ratio of the mixed bacterial solution to lysostaphin is 1:19; the concentration of lysostaphin is 50-100 μg / mL; and the incubation conditions are: 37° C., 30 min.

[0011] Furthermore, in step S4, the volume ratio of Tris-HCl to proteinase K is 3:1; the concentration of Tris-HCl is 0.1 M, pH=7.5, and the concentration of proteinase K is 100 μg / mL; the incubation conditions are: 37° C., 15 min; and the heating conditions are: 100° C., 10 min.

[0012] Furthermore, in step S5, the PCR amplification system is: template 1 μL, upstream primer 1 μL, downstream primer 1 μL, 2×TaqMaster Mix 15 μL, ddH2O 12 μL. Mixture 2 is directly used as a template.

[0013] Furthermore, in step S5, the upstream primer in the PCR system is 16s RNA primer 27F, and the downstream primer is 16s RNA primer 1492R, and the corresponding nucleotide sequences are shown in SEQ ID: 1 and SEQ ID: 2, respectively; then the identification method is: directly performing Sanger sequencing on the PCR amplification product to identify the strain species.

[0014] Furthermore, in step S5, the upstream primer in the PCR system is the Staphylococcus aureus specific gene nuc-F, the downstream primer is nuc-R, and the corresponding primer nucleotide sequences are: SEQ ID: 3 and SEQ ID: 4; the identification method is: directly subjecting the PCR amplification product to nucleic acid electrophoresis to identify whether the gene exists in the strain.

[0015] Furthermore, the PCR amplification conditions in step S5 are: (1) 95°C for 3 min, (2) 95°C for 15 sec, (3) 50-55°C for 15 sec, (4) 72°C for 60 sec / kb, (5) steps (2) to (4) are cycled 30 times, and (6) extension at 72°C for 5 min.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The method of the present invention performs pretreatment when obtaining a single colony from a sample, so that the crude product can be used for PCR identification of Staphylococcus aureus. It is not necessary to use a genome kit to extract a complete Staphylococcus genome. The crude product can be directly used as a template to amplify sufficient products with a success rate of 100%. On the one hand, the cost of purchasing a kit is saved, and on the other hand, the amount of lysostaphin required for the use of the matching kit is reduced (the method only requires 0.45-0.9 μg of lysostaphin, while the genome kit generally requires more than 5 μg of lysostaphin). Since the unit price of the fund extraction kit and lysostaphin is relatively high, the method of the present invention greatly reduces the cost of separating and identifying Staphylococcus aureus.

[0017] The present invention optimizes and improves the method for separating and identifying staphylococci in chicken embryo samples. After picking monoclonal colonies, there is no need to culture bacteria overnight, which simplifies the separation process, reduces the cumbersome intermediate steps, and shortens the time by at least 1 day. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Screen colonies for Staphylococci grown on BP plates; Figure 2 The electrophoresis diagrams of 16sRNA and nuc gene nucleic acid of PCR amplification of Staphylococcus aureus standard strain Newman, chicken embryo-derived Staphylococcus aureus, and chicken embryo-derived Staphylococcus epidermidis under the methods of Example 1, Comparative Example 1, and Comparative Example 2 are shown as follows: wherein lane M is a Marker; lanes 1-18 respectively represent: 1 is a band obtained by amplifying the nuc gene of Staphylococcus aureus standard strain Newman in Example 1; 2 is a band obtained by amplifying the nuc gene of Staphylococcus aureus derived from chicken embryos in Example 1; 3 is Staphylococcus epidermidis derived from chicken embryos in Example 1, and the nuc gene cannot be amplified; 4 is a band obtained by amplifying the 16s RNA gene of Staphylococcus aureus standard strain Newman in Example 1; 5 is a band obtained by amplifying the 16s RNA gene of Staphylococcus aureus derived from chicken embryos in Example 1; 6 is a band obtained by amplifying the 16s RNA gene of Staphylococcus epidermidis derived from chicken embryos in Example 1 RNA gene obtained bands; 7-9 are comparative example 1 method, Staphylococcus aureus standard strain Newman, chicken embryo-derived Staphylococcus aureus and chicken embryo-derived Staphylococcus epidermidis cannot amplify the nuc gene; 10-12 comparative example 1 method, Staphylococcus aureus standard strain Newman, chicken embryo-derived Staphylococcus aureus and chicken embryo-derived Staphylococcus epidermidis cannot amplify the 16s RNA gene; 13-15 are comparative example 2 method, Staphylococcus aureus standard strain Newman, chicken embryo-derived Staphylococcus aureus and chicken embryo-derived Staphylococcus epidermidis cannot amplify the nuc gene; 16-18 are comparative example 2 method, Staphylococcus aureus standard strain Newman, chicken embryo-derived Staphylococcus aureus and chicken embryo-derived Staphylococcus epidermidis cannot amplify the 16s RNA gene.

[0019] Figure 3 The electrophoresis diagram of the PCR-amplified nuc gene nucleic acid of the strain isolated from the chicken embryo in Example 2 is shown in FIG. DETAILED DESCRIPTION

[0020] The present invention is further described below by means of specific examples, but the present invention is not limited to the scope of the embodiments described.

[0021] It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise clearly stated in the text, the singular forms "a", "an" and "this" include plural forms. Unless otherwise clearly stated in the text, professional terms or abbreviations follow the conventional practices in the field, for example, "Tris-HCl concentration is 0.1 M" where M is mol / L; "15 sec" where sec is the time unit: seconds; "60 sec / kb" where kb is kilobase pairs, meaning that the amplification time for every 1000bp is 60 seconds.

[0022] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and the numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0023] The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions in the art, or according to the product specifications. The raw materials and reagents not specifically specified in the examples can be purchased through commercial channels, and the experimental instruments used are all conventional laboratory instruments.

[0024] Chicken embryo-derived Staphylococcus aureus and chicken embryo-derived Staphylococcus epidermidis were both homemade, isolated from chicken embryo samples, and the strains were identified by Sanger sequencing.

[0025] Example 1 Chicken embryo-derived Staphylococcus aureus and chicken embryo-derived Staphylococcus epidermidis were selected to isolate and identify the strains according to the following steps, and a standard strain of Staphylococcus aureus Newman was set as a positive control group.

[0026] Step 1 (S1): Sample processing and bacterial culture S101. Take a breeding egg with no damage on the surface, wipe the surface of the eggshell with a cotton ball dipped in an appropriate amount of disinfectant alcohol; use sterilized tweezers to break the eggshell and take out the chicken embryo.

[0027] S102. Cut the chicken embryo into pieces with sterilized surgical scissors, mix with SCB liquid culture medium in a ratio of 1:10, put into a sterilized shaking bag, put into a shaker, and culture at 37°C, 180 rpm for 30 h to obtain culture medium.

[0028] In some cases, the steps of cutting up the chicken embryo sample in S101 and S102 can be omitted. For example, when a Staphylococcus aureus standard strain is used as a positive control group, these two steps can obviously be omitted.

[0029] Step 2 (S2): Cultivate bacteria and obtain monoclonal colonies S201. Take 10 μL of the culture medium, streak it onto the BP culture plate, and place it in an incubator at 37°C for 24 h ( Figure 1 ).

[0030] S202. After a single clone grows in the culture dish, pick up the single clone and put it into 20 μL PBS, pipette and mix well to obtain a mixed bacterial solution.

[0031] Step 3 (S3): Preparation of mixed solution 1 S301. Take 1 μL of the mixed bacterial solution in step 4 and place it in 19 μL of 100 μg / mL lysostaphin, incubate at 37°C for 30 min, and obtain mixed solution 1.

[0032] Step 4 (S4): Preparation of mixed solution 2 S401. Add 30 μL 0.1M Tris-HCl (pH=7.5) and 10 μL 100μg / mL proteinase K to the mixed solution in step 5, incubate at 37°C for 15 min, and heat at 100°C for 10 min to obtain mixed solution II.

[0033] Step 5 (S5): Polymerase chain reaction (PCR) amplification and product identification S501. Establish a suitable PCR reaction system and PCR amplification program.

[0034] a. The PCR reaction system is as follows: Take 1 μL of the mixed solution in step 6 as a template and prepare 30 μL of PCR reaction system, which is as follows: Template 1 μL, upstream primer 1 μL, downstream primer 1 μL, 2 × Taq Master Mix (Vazyme) 15 μL, ddH2O 12 μL.

[0035] PCR amplification can be performed using two reaction systems, one for 16s ribosomal RNA (16s rRNA), with the upstream primer 27F and the downstream primer 1492R. Using this reaction system, the amplified product can be further directly sequenced by dideoxy sequencing (Sanger sequencing) to identify the strain species.

[0036] Another one is for the Staphylococcus-specific gene thermostable nuclease gene (nuc), the upstream primer is nuc-F, and the downstream primer is nuc-R. With this reaction system, since nuc is a gene specific to Staphylococcus aureus, the reaction product can be directly detected by nucleic acid electrophoresis to identify whether Staphylococcus aureus exists.

[0037] The above primer sequences are shown in Table 1 below: Table 1 Primer sequences b. The PCR reaction procedure is as follows: The PCR reaction program is set according to the size of the amplified template and the commercial PCR enzyme used. The PCR enzyme used in this example is Novagen 2 × Taq Master Mix (Dye Plus), and the PCR reaction program is: pre-denaturation at 95°C for 3 min, followed by 95°C for 15 sec, 50-55°C for 15 sec, 72°C for 60 sec / kb, for a total of 30 cycles, and finally extension at 72°C for 5 min.

[0038] S502. The product amplified by PCR is subjected to electrophoresis detection. The specific process of gel electrophoresis is: the PCR product is spotted into a 1% agarose gel, in 1×TBE electrophoresis solution (90m M Tris-H3BO3, 2m M EDTA), at a voltage of 150V for 25 minutes. If the PCR product obtained by using SEQ ID: 1 and SEQ ID: 2 as primers needs to be further sequenced by Sanger sequencing to determine the strain type; if the PCR product obtained by using SEQ ID: 3 and SEQ ID: 4 as primers can be directly detected by nucleic acid electrophoresis to identify whether Staphylococcus aureus is present.

[0039] Comparative Example 1 This comparative example differs from Example 1 only in the absence of step S3.

[0040] Comparative Example 2 This comparative example differs from Example 1 only in the absence of step S4.

[0041] Experimental tests and results Figure 2The electrophoresis detection results of Example 1, Comparative Example 1, and Comparative Example 2 are shown. Lanes 1-6 are the results of electrophoresis in Example 1: Lane 1 is a band obtained by amplifying the nuc gene of the standard strain Newman of Staphylococcus aureus under the method of Example 1; Lane 2 is a band obtained by amplifying the nuc gene of Staphylococcus aureus derived from chicken embryos under the method of Example 1; Lane 3 is a band obtained by amplifying the nuc gene of Staphylococcus epidermidis derived from chicken embryos under the method of Example 1; Lane 4 is a band obtained by amplifying the 16s RNA gene of the standard strain Newman of Staphylococcus aureus under the method of Example 1; Lane 5 is a band obtained by amplifying the 16s RNA gene of Staphylococcus aureus derived from chicken embryos under the method of Example 1; Lane 6 is a band obtained by amplifying the 16s RNA gene of Staphylococcus epidermidis derived from chicken embryos under the method of Example 1; The results show that nuc can be used as a specific gene of Staphylococcus aureus, can accurately identify Staphylococcus aureus, can avoid the interference of Staphylococcus epidermidis, and has good specificity.

[0042] The electrophoresis results of Comparative Example 1 are as follows: no bands are generated in lanes 7-9, corresponding to the standard strain Newman of Staphylococcus aureus, Staphylococcus aureus derived from chicken embryos, and Staphylococcus epidermidis derived from chicken embryos, and the nuc gene cannot be amplified; no bands are generated in lanes 10-12, corresponding to the standard strain Newman of Staphylococcus aureus, Staphylococcus aureus derived from chicken embryos, and Staphylococcus epidermidis derived from chicken embryos, and the 16s RNA gene cannot be amplified; The electrophoresis results of Comparative Example 2 are as follows: no bands are generated in lanes 13-15, correspondingly, the nuc gene cannot be amplified from the standard strain Newman of Staphylococcus aureus, Staphylococcus aureus derived from chicken embryos, and Staphylococcus epidermidis derived from chicken embryos; no bands are generated in lanes 16-18, correspondingly, the 16s RNA gene cannot be amplified from the standard strain Newman of Staphylococcus aureus, Staphylococcus aureus derived from chicken embryos, and Staphylococcus epidermidis derived from chicken embryos.

[0043] Figure 2 The results showed that the method of Example 1 can effectively separate Staphylococci, and the strain type can be identified by electrophoresis detection of PCR amplification products, with good specificity, rapidity and high efficiency.

[0044] Example 2 The method in Example 1 was used to detect 17 randomly selected chicken embryo samples to be tested, numbered 1, 2, 3 ... 17, wherein SEQ ID: 3 and SEQ ID: 4 were used as primers to amplify the nuc gene by PCR, and the electrophoresis detection results were obtained as follows: Figure 3 The results showed that among the 17 samples tested, 8 strains, including 2-6, 15-17, could detect the nuc gene.

[0045] Thermostable nuclease gene (nuc) is a marker gene of Staphylococcus aureus. When the nuc gene exists, the strain is likely to be Staphylococcus aureus. In order to finally determine the species of the strain, the 16S RNA gene amplification product is further directly subjected to Sanger sequencing, and the final result is determined after sequence comparison.

[0046] The selected 16S RNA gene amplification products are as follows: The standard strain of Staphylococcus aureus Newman and the Staphylococcus aureus from chicken embryo amplified in Example 1 are numbered NM and Control, respectively; Five strains were randomly selected from the eight strains that could amplify the nuc gene in the 17 samples and were numbered 270221, 270222, 270232, 270251, and 270252; The 9 strains that failed to amplify the nuc gene among the 17 samples were numbered 270051, 270081, 270151, 270101, 270102, 270191, 270192, 270211, and 270271; The above PCR products were sent to Sangon Biotechnology Co., Ltd. (Nanjing) for Sanger sequencing, and the results after comparison are shown in Table 2.

[0047] Table 2 Sanger sequencing results From the results in Table 2 above, it can be seen that the PCR products amplified by the method provided by the present invention can be directly subjected to Sanger sequencing, and most of the obtained products are of high quality and good sequencing quality, and can be directly sequenced to identify the strain species. Figure 3 The data in Table 2 prove that the Sanger sequencing result is consistent with the electrophoresis identification result after the PCR amplification of the nuc gene. ② 5 samples are all Staphylococcus aureus, and ③ 9 samples are all non-Staphylococcus aureus, which once again verifies that the method of the present invention can accurately separate and identify Staphylococcus in chicken embryos, and the success rate reaches 100%. It is worth noting that the Staphylococcus in the chicken embryo samples separated by the method of the present invention does not need to be complicated overnight culture mode, does not need to use a specific gene extraction kit, and the crude product can effectively carry out PCR amplification, and the amplified product can also directly carry out Sanger sequencing, which meets the requirements of separation amount and purity. It is thus explained that the method of the present invention can achieve the purpose of accurately, quickly and low-cost separation and identification of strain species in chicken embryos.

[0048] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above-mentioned technical features. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A method for separating, screening and identifying Staphylococci in chicken embryo samples, characterized in that: The method comprises the following steps: S1: Collect chicken embryo tissue and prepare culture medium; S2: Cultivate bacterial solution, streak on BP plate to obtain monoclonal colonies; pick monoclonal colonies and pipette evenly in PBS to obtain mixed bacterial solution; S3: placing the mixed bacterial solution obtained in S2 in lysostaphin and incubating it to obtain mixed solution 1; S4: adding Tris-HCl and proteinase K to the mixed solution 1 obtained in S3, incubating first, and then heating to obtain a mixed solution 2; S5: Using the mixed solution 2 obtained in S4 as a template, PCR amplification is performed to obtain a product, and then the product is identified.

2. The method for separation, screening and identification of Staphylococcus aureus according to claim 1, characterized in that: The method comprises the following steps: S1: sterilize the collected chicken embryo tissue, cut it into pieces, mix it with SCB liquid culture medium, and culture it in a shaker to obtain culture medium; S2: Streak the obtained culture solution onto a BP culture plate, place it in an incubator and culture it statically. After a single clone grows out of the culture dish, pick the single clone and pipette it evenly in PBS to obtain a mixed bacterial solution; S3: incubating the mixed bacterial solution obtained in S2 with lysostaphin to obtain a mixed solution 1; S4: adding Tris-HCl and proteinase K to the mixed solution 1 obtained in S3, incubating first, and then heating to obtain a mixed solution 2; S5: Using the mixed solution 2 obtained in S4 as a template, PCR amplification is performed to obtain a product, and then the product is identified.

3. The method according to claim 2, characterized in that In step S1, the chicken embryo tissue and the SCB liquid culture medium are mixed at a volume ratio of 1:5-1:15; the shaking incubator culture conditions are: 37° C., 160-220 rpm, and culture for 24-36 h.

4. The method according to claim 2, characterized in that: The static culture conditions in step S2 are: 37° C., 24 h.

5. The method according to any one of claims 1 to 2, characterized in that: In step S3, the volume ratio of the mixed bacterial solution to lysostaphin is 1:19; the concentration of lysostaphin is 50-100 μg / mL; and the incubation conditions are: 37° C., 30 min.

6. The method according to any one of claims 1-2, characterized in that: In step S4, the volume ratio of Tris-HCl to proteinase K is 3:1-5:1; the concentration of Tris-HCl is 0.1 M, pH=7.5, and the concentration of proteinase K is 100 μg / mL; the incubation conditions are: 37°C, 15 min; and the heating conditions are: 100°C, 10 min.

7. The method according to any one of claims 1-2, characterized in that: The PCR amplification system in step S5 is: 1 μL of template, 1 μL of upstream primer, 1 μL of downstream primer, 15 μL of 2×Taq Master Mix, and 12 μL of ddH2O.

8. The method according to claim 7, characterized in that The upstream primer is 16s RNA primer 27F, the downstream primer is 16s RNA primer 1492R, and the corresponding nucleotide sequences are shown in SEQ ID: 1 and SEQ ID: 2 respectively; the identification method is: directly performing Sanger sequencing on the PCR amplification product to identify the strain species.

9. The method according to claim 7, characterized in that: The upstream primer is the Staphylococcus aureus specific gene nuc-F, the downstream primer is nuc-R, and the corresponding primer nucleotide sequences are: SEQ ID: 3 and SEQ ID: 4; the identification method is: directly subjecting the PCR amplification product to nucleic acid electrophoresis to identify whether the gene exists in the strain.

10. The method according to any one of claims 1-2, characterized in that: The PCR amplification conditions in step S5 are: (1) 95°C for 3 min, (2) 95°C for 15 sec, (3) 50-55°C for 15 sec, (4) 72°C for 60 sec / kb, (5) steps (2) to (4) are cycled 30 times, and (6) extension at 72°C for 5 min.

Images