Specific molecular markers, primer sets and applications for screening Staphylococcus xylosus

Abstract

The present invention discloses specific molecular markers, primer sets and applications for screening Staphylococcus xylosus, and relates to the field of microbial detection technology. The nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1 or SEQ ID NO: 2; the sequence of the primer set for amplifying the molecular marker is shown in SEQ ID NO: 3 to SEQ ID NO: 6; and also provides the application of the molecular marker and primer set in screening, detecting or identifying Staphylococcus xylosus for non-disease diagnosis purposes. The molecular markers of the present invention have a greater coverage rate of target bacteria and non-target bacteria in specificity evaluation, thereby enhancing accuracy. The detection method of the present invention can accurately quantify Staphylococcus xylosus, greatly improving the efficiency of Staphylococcus xylosus identification, and has the advantages of strong specificity, simple operation, easy result determination, short detection time and low cost, providing technical support for the detection and industrial application of Staphylococcus xylosus.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial testing, and in particular to a specific molecular marker, a primer set and an application thereof for screening Staphylococcus xylosus. Background Art

[0002] Staphylococcus xylosus is ( Staphylococcus xylosus , S. xylosus ) is a coagulase-negative, Gram-positive staphylococcus with a diameter of 0.5 μm to 1.5 μm. It is often found singly, in pairs, in short chains, or in irregular grape-like arrangements. Staphylococcus xylosus is widely distributed in nature and has been found in air, soil, food, and on the body surfaces and mucous membranes of humans and animals.

[0003] Currently, detection methods for Staphylococcus xylosus mainly include traditional culture, immunoassays, molecular biology, and mass spectrometry. Traditional culture methods require inoculating a sample onto a specific culture medium and culturing it under appropriate conditions. Identification is achieved by observing colony morphology and color, as well as performing a series of biochemical reactions. This method is relatively simple and inexpensive, but has a long detection cycle, typically requiring 48 hours or longer. It can easily miss low-concentration bacterial cultures and is susceptible to interference from other microorganisms in complex samples, resulting in poor accuracy. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and latex agglutination tests, utilize the principle of specific antigen-antibody binding. These methods are relatively fast and offer reasonable specificity and sensitivity. However, the production of specific antibodies is costly, and inconsistent antibody quality can affect results. Complex samples are also prone to nonspecific reactions, leading to false positives. Mass spectrometry, which relies on bacterial protein fingerprinting, is fast and highly accurate. However, the instrumentation is expensive, maintenance is high, and it relies on a large database of standard mass spectra. Accurate identification of new variants or rare strains can be difficult due to a lack of reference spectra. In recent years, with the development of molecular biology, molecular detection methods based on PCR have gradually become one of the most promising technologies in microbial detection due to their rapidity, accuracy and simplicity.

[0004] Specific molecular detection targets are the key to ensuring the specificity and sensitivity of molecular detection methods. At present, there are relatively few molecular detection targets and primers for one-step identification of Staphylococcus xylosus by PCR detection methods at home and abroad. gehMTargets used for the detection of Staphylococcus xylosus lack the specificity and sensitivity to meet the practical needs of rapid detection. Thanks to the development of high-throughput sequencing and bioinformatics analysis, omics technologies based on pan-genomic analysis are widely used in studies of bacterial genetic variation, gene function enrichment, and evolutionary analysis. Therefore, pan-genomic analysis can efficiently identify targets for the molecular identification of Staphylococcus xylosus and further develop simple, rapid, economical, efficient, and highly sensitive PCR identification and qPCR quantitative detection technologies to meet the needs of diverse fields for the efficient identification and quantitative detection of Staphylococcus xylosus. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the prior art by providing a specific molecular marker, primer set, and application thereof for screening for Staphylococcus xylosus. The specific molecular marker and primer set of the present invention can accurately identify Staphylococcus xylosus in a sample with good specificity and high accuracy.

[0006] The technical solutions of the present invention are as follows:

[0007] A first aspect of the present invention provides a specific molecular marker for screening Staphylococcus xylosus, wherein the nucleotide sequence of the specific molecular marker is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] Through pan-genomic analysis, the present invention identified specific sequence fragments in Staphylococcus xylosus, as shown in SEQ ID NO: 1 to SEQ ID NO: 2. These fragments can serve as specific molecular markers for identifying Staphylococcus xylosus. Furthermore, the molecular markers achieved 100% coverage of target bacterial genes and 0% coverage of non-target bacteria.

[0009] A second aspect of the present invention provides a primer set for amplifying the specific molecular marker, wherein the sequences of the primer set are shown in SEQ ID NO: 3 to SEQ ID NO: 6.

[0010] Specifically, the primer set shown in SEQ ID NO: 3 and SEQ ID NO: 4 is used to amplify the molecular marker shown in SEQ ID NO: 1;

[0011] The primer set shown in SEQ ID NO: 5 and SEQ ID NO: 6 is used to amplify the molecular marker shown in SEQ ID NO: 2.

[0012] The present invention designs a primer set capable of amplifying the above-mentioned specific molecular markers, and the target band can be amplified by the primer set with high sensitivity.

[0013] The third aspect of the present invention provides the use of the specific molecular marker or the primer set in screening, detecting or identifying Staphylococcus xylosus for non-disease diagnosis purposes.

[0014] The molecular marker and primer set of the present invention can accurately detect Staphylococcus xylosus in a sample with good specificity and high accuracy.

[0015] The fourth aspect of the present invention provides the use of the specific molecular marker or the primer set in preparing a product for screening, detecting or identifying Staphylococcus xylosus.

[0016] By using the molecular markers and primer sets of the present invention to prepare products for screening, detecting or identifying Staphylococcus xylosus, such as detection kits, rapid and convenient detection can be achieved.

[0017] Preferably, the product comprises a detection kit, and the detection kit comprises the primer set.

[0018] A fifth aspect of the present invention provides a method for identifying Staphylococcus xylosus for non-disease diagnosis purposes, comprising the following steps:

[0019] S1, using the primer set to perform PCR amplification on the genomic DNA of the sample to be tested to obtain an amplified product;

[0020] S2. Observe whether a target band appears in the amplified product. If a target band appears, it is determined that the sample to be tested contains Staphylococcus xylosus.

[0021] The present invention uses a PCR reaction to amplify specific molecular markers using detection primers. The presence of the Staphylococcus xylosus can be determined by observing whether the amplified product is at the expected position. The present invention improves detection efficiency by setting up multiple PCR systems and simultaneously amplifying the target DNA using different primers.

[0022] Preferably, in step S1,

[0023] The PCR amplification system includes: 2 μL of 10× PCR reaction buffer, 2 μL of 20 mmol / L MgCl2, 1 μL of 2 mmol / L dNTP, 100 ng of template DNA, 0.5 μL each of 10 μmol / L forward and reverse primers, 1 U of Tag enzyme, and sterile double-distilled water to make up the volume to 20 μL;

[0024] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58.5°C for 30 s; denaturation, annealing, and extension for 35 cycles; and finally extension at 72°C for 5 min.

[0025] A sixth aspect of the present invention provides a method for quantitatively detecting Staphylococcus xylosus for non-disease diagnosis purposes, comprising the following steps:

[0026] S1, using the primer set to perform qPCR amplification on the genomic DNA of the sample to be tested;

[0027] S2. The logarithm of the concentration of the pure culture of the standard Staphylococcus xylosus strain is used as the horizontal axis, and the corresponding real-time Ct value of qPCR is used as the vertical axis. The obtained curve is the standard curve for quantifying Staphylococcus xylosus;

[0028] S3. Analyze the amplification results and determine the content of Staphylococcus xylosus in the sample by comparing the fluorescence signal value with the standard curve.

[0029] The present invention uses a qPCR system to amplify specific molecular markers using a detection primer set, and quantitatively detects the concentration of Staphylococcus xylosus in the system by determining the intensity of the fluorescent signal in the system. The present invention improves detection efficiency by setting up multiple qPCR systems and simultaneously amplifying the target DNA using different primers.

[0030] Preferably, the qPCR amplification system is: 10 μL of 2× TB Green Premix reaction solution, 1 μL of template DNA, 0.5 μL each of 10 μmol / L forward and reverse primers, and sterile double-distilled water to make up the volume to 20 μL;

[0031] The qPCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s; annealing at 59°C for 40 s; extension at 72°C for 1 min; and 40 cycles of denaturation and annealing.

[0032] The present invention has at least one of the following beneficial effects:

[0033] The present invention provides specific molecular markers for identifying Staphylococcus xylosus, and provides primer sets related to detecting specific molecular markers, as well as corresponding PCR detection methods. The detection molecular markers of the present invention have a greater coverage of target bacteria and non-target bacteria in specificity evaluation, thereby enhancing accuracy. The detection method of the present invention is capable of accurately quantifying Staphylococcus xylosus, greatly improving the efficiency of Staphylococcus xylosus identification and enhancing practicality. The detection method of the present invention also has the advantages of strong specificity, simple operation, easy result determination, short detection time, and low cost, providing technical support for the detection and industrial application of Staphylococcus xylosus. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Schematic diagram of the electrophoresis results of the specificity evaluation of the PCR detection method for Staphylococcus xylosus in Example 3, wherein A in the figure is a schematic diagram of the electrophoresis results of the specificity evaluation using primer set 1, and B in the figure is a schematic diagram of the electrophoresis results of the specificity evaluation using primer set 2.

[0035] Figure 2 Schematic diagram of the amplification curve and melting curve of the qPCR quantitative detection method for Staphylococcus xylosus in Example 5, wherein A in the figure is the amplification curve and B in the figure is the melting curve.

[0036] Figure 3 Schematic diagram of the sensitivity evaluation results of the qPCR quantitative detection method for artificially spiked sausage Staphylococcus xylosus in Example 6, wherein A and B in the figure are the real-time fluorescence amplification curve and standard curve for quantitative detection using primer set 1, respectively, and C and D in the figure are the real-time fluorescence amplification curve and standard curve for quantitative detection using primer set 2, respectively.

[0037] Figure 4 Schematic diagram of the evaluation results of the anti-interference ability of the qPCR quantitative detection method for Staphylococcus xylosus in Example 7, wherein A in the figure is a schematic diagram of the results of quantitative detection using primer set 1, and B in the figure is a schematic diagram of the results of quantitative detection using primer set 2. DETAILED DESCRIPTION

[0038] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] Example 1 Screening and obtaining specific molecular markers of Staphylococcus xylosus

[0040] Pan-genome analysis was performed based on the genome data of 12 Staphylococcus xylosus strains and 750 non-Staphylococcus xylosus strains from the NCBI website; two specific gene fragments of Staphylococcus xylosus were screened and obtained. The nucleotide sequences of the gene fragments are shown in SEQ ID NO: 1 to SEQ ID NO: 2.

[0041] The xylosus-specific molecular markers obtained by screening were all from Staphylococcus xylosus CP060271.1, and the gene fragment sequences of the molecular markers corresponding to the gene loci are shown in Table 1 below.

[0042] Table 1 Genomic loci of specific molecular markers of Staphylococcus xylosus

[0043]

[0044] Example 2 PCR method for identifying Staphylococcus xylosus

[0045] 1) Primer design

[0046] Specific PCR amplification primers (including forward primers and reverse primers) were designed based on the sequences SEQ ID NO: 1 to SEQ ID NO: 2 in Example 1. The sequences of primer set 1 and primer set 2 are shown in Table 2 below.

[0047] Table 2 Specific PCR detection primer sets

[0048]

[0049] 2) Method for identifying Staphylococcus xylosus, the steps are as follows:

[0050] S1. DNA template preparation: The strains to be tested were cultured in LB liquid medium, and their genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as the template to be tested;

[0051] S2. PCR amplification: PCR amplification was performed on the DNA of the sample to be tested using primer set 1 and primer set 2 described in Example 2, respectively. The specific method is as follows:

[0052] ①PCR detection system:

[0053]

[0054] ②PCR amplification procedure:

[0055]

[0056] S3: Take the PCR amplification product and perform gel electrophoresis;

[0057] S4: Observe whether a single amplified band exists at the position corresponding to the product size of each primer set. If a single amplified band exists, it indicates that the target Staphylococcus xylosus is present in the sample. If no corresponding single amplified band appears, the target Staphylococcus xylosus is not present in the sample.

[0058] Example 3 Specificity Evaluation of Staphylococcus xylosus PCR Detection Method

[0059] Ten strains of Staphylococcus xylosus and 39 strains of non-Staphylococcus xylosus were tested by PCR according to the method of Example 2. S1: DNA template preparation was performed by extracting genomic DNA from each bacterium; S2: PCR amplification used primers set 1 and 2 from Example 2. A blank control was set up, and the template for the blank control was an aqueous solution without genomic DNA from each bacterium.

[0060] The bacterial strains used and the test results are shown in Table 3 below. In the test result column, "+" indicates positive, "-" indicates negative, and the isolated strains indicate the strains isolated by the laboratory. Figure 1 As shown, Figure 1 A in the figure is the electrophoresis result of the PCR product of primer set 1. Figure 1 B in the figure is the electrophoresis result of the PCR product of primer set 2; Figure 1 Numbers 1 to 10 represent 10 strains of Staphylococcus xylosus; numbers 11 to 49 represent 39 strains of non-target Staphylococcus xylosus; M stands for 2000Maker, C stands for negative quality control, and P stands for positive quality control.

[0061] Table 3 Results of the molecular marker specificity evaluation test for Staphylococcus xylosus of the present invention

[0062]

[0063]

[0064] Depend on Figure 1 As can be seen from Table 3, the detection results of primer set 1 and primer set 2 in Example 2 showed only specific amplification bands for Staphylococcus xylosus, and no specific bands were found for other non-target Staphylococci xylosus. This shows that the molecular marker in Example 1 has high specificity, and the method for identifying Staphylococcus xylosus using the primer set obtained by the molecular marker has high specificity.

[0065] Example 4 Comparison of the molecular markers for detecting Staphylococcus xylosus of the present invention with existing molecular markers

[0066] The present invention uses the genome data of 12 strains of Staphylococcus xylosus and 750 strains of Staphylococcus xylosus in the NCBI database and successfully obtains two new molecular markers specific for Staphylococcus xylosus through pan-genomic data analysis. At the same time, it searches for existing molecular markers specific for Staphylococcus xylosus. gehM Based on the results of the local BLAST gene database comparison, it was found that the two molecular markers of the present invention had a 100% coverage rate for the target bacteria gene and a 0% coverage rate for non-target bacteria. The existing technology (Iacumin, L., Cocolin, L., Cantoni, C., & Comi, G. (2007). Preliminary analysis of the lipase gene ( gehM) expression of Staphylococcus xylosus in vitro and duringfermentation of naturally fermented sausages (in situ). Journal of foodprotection, 70(11), 2665-2669.) reported in gehM As a molecular marker for detection, its target bacterial gene coverage was 100%, while the non-target bacterial coverage was 13.2%. Therefore, this example demonstrates that the present Staphylococcus xylosus marker has higher accuracy and better specificity, accurately identifying Staphylococcus xylosus in actual samples, and has significant advantages over existing reporting markers. The specific results are shown in Table 4 below.

[0067] Table 4 Comparison results of the present invention's Staphylococcus xylosus marker and existing markers

[0068]

[0069] Example 5 qPCR quantitative detection method for identifying Staphylococcus xylosus

[0070] 1) Primer selection:

[0071] qPCR amplification of the sample DNA was performed using primer set 1 and primer set 2 described in Example 2, respectively;

[0072] 2) The method for quantitative detection of Staphylococcus xylosus is as follows:

[0073] S1. DNA template preparation: The Staphylococcus xylosus strain was cultured in LB liquid medium, and the bacterial genomic DNA was extracted from each strain using a commercial bacterial genomic DNA extraction kit as a test template; a blank control was set up, and the blank control template was an aqueous solution without the genomic DNA of each bacteria.

[0074] S2. PCR amplification: using the primer set 1 and primer set 2 to perform qPCR amplification on the sample DNA to be tested;

[0075] ①qPCR detection system:

[0076]

[0077] ②qPCR amplification procedure:

[0078]

[0079] S3: The qPCR amplification system was performed on a Bio-Rad CFX96 fluorescence quantitative amplification instrument;

[0080] S4: Use Bio-Rad CFX Manager software to analyze the amplification results to see if they meet expectations. If a fluorescence amplification curve and a corresponding melting curve are generated in the absence of a signal in the blank control, the sample contains Staphylococcus xylosus. If no signal is generated, the sample does not contain Staphylococcus xylosus.

[0081] Fluorescence amplification curve and corresponding melting curve are as follows Figure 2 As shown in the figure, when the sample contains Staphylococcus xylosus, the qPCR systems established based on primer set 1 and primer set 2 can each produce an obvious fluorescence amplification curve ( Figure 2 A in the figure) and the corresponding melting curve ( Figure 2 The blank control showed no fluorescence curve or melting curve.

[0082] Example 6 Sensitivity Evaluation of qPCR Quantitative Detection Method for Staphylococcus xylosus Artificially Spiked Sausage

[0083] After the sausage samples were sterilized by ultraviolet light, physiological saline was added to prepare sterile samples. After culture on NA nutrient agar plates, the results showed that no microorganisms were present after treatment, ensuring that the microorganisms in subsequent experiments were all from artificial contamination. According to the national standard "GB4789.10-2016", 25 g of sample was weighed and placed in a sterile homogenizer cup containing 225 mL of physiological saline. Homogenization was performed at 8000 r / min~10000 r / min for 1 min~2 min to prepare a 1:10 sample solution. The sample was cultured to a concentration of 10 8 The xylosus Staphylococcus isolate with a CFU / mL was diluted 10-fold with deionized water and spiked into the sausage sample to obtain a final concentration of 10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 CFU / mL mixture, the DNA template of all mixtures was extracted according to Example 5, that is, the spiked 10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1CFU / mL Staphylococcus xylosus liquid was used as the test sample DNA template. 1 μL of the spiked samples at different dilutions was taken for qPCR reactions using Primer Set 1 and Primer Set 2 according to the method of Example 5. Three parallel experiments were performed for each template. The results were analyzed according to the method of Example 5.

[0084] Draw a standard curve: use the logarithm of the concentration of Staphylococcus xylosus in the spiked sausage sample as the horizontal axis and the corresponding qPCR real-time Ct value as the vertical axis. The fitted curve is the standard curve for the quantification of Staphylococcus xylosus.

[0085] Standard curve and real-time fluorescence amplification curve are as follows Figure 3 As shown, the detection limit of primer set 1 of the present invention is 10 2 CFU / g, the fitting standard curve of Staphylococcus xylosus is y = -3.2275x + 42.259, and the correlation coefficient R 2 The detection limit of primer set 2 of the present invention is 10 3 CFU / g, the fitting standard curve of Staphylococcus xylosus is y = -2.5708x +43.642, and the correlation coefficient R 2 It is 0.9688.

[0086] Example 7 Evaluation of the ability of the qPCR quantitative detection method for Staphylococcus xylosus to resist interference from other bacteria

[0087] The new culture was cultured to a concentration of 10 7 The S. xylosus isolate and S. aureus ATCC 25923 at CFU / mL were mixed in specific proportions to obtain mixed bacterial solutions with final concentration ratios of 1:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:15, and 1:20, respectively. DNA template extraction from the mixture was performed according to Example 5. For test samples with different concentration ratios of S. xylosus to S. aureus, qPCR reactions were performed using primer set 1 and primer set 2, respectively, according to Example 5. Three replicates were performed for each template. The results were analyzed according to Example 5.

[0088] Fluorescence amplification curve Figure 4 As shown, primer set 1 and primer set 2 both produced relatively overlapping real-time fluorescence amplification curves, indicating that the qPCR quantitative detection method has a strong ability to resist interference from foreign bacteria.

[0089] Example 8 Detection results of Staphylococcus xylosus in actual samples

[0090] Ten sausage and bacon samples were collected from local supermarkets and meat and vegetable markets. Samples were processed under sterile conditions in a clean laboratory bench according to the national standard GB4789.10-2016. A template was prepared using a strain identified as Staphylococcus xylosus to extract genomic DNA, specifically following the procedures in Example 6. Finally, qPCR was performed as in Example 6. To ensure the accuracy of the results, one sausage and bacon sample spiked with known Staphylococcus xylosus was identified and identified according to the national standard GB4789.10-2016.

[0091] The test results are shown in Table 5, where "+" indicates a positive test result and "-" indicates a negative test result.

[0092] Table 5 Detection results of Staphylococcus xylosus in actual samples

[0093]

[0094] As shown in Table 5, compared with the national standard method, the accuracy of the qPCR method of the present invention for detecting Staphylococcus xylosus in the 10 real food samples tested was 100%, and the accuracy of the two positive control samples was also 100%.

[0095] The qPCR method of this invention is capable of rapidly identifying Staphylococcus xylosus in real-world samples, significantly improving its efficiency and practicality. It also offers the advantages of simple operation, easy result determination, short detection time, high specificity, and low cost. Subsequent research will further enable on-site, real-time detection of Staphylococcus xylosus by constructing biosensor platforms such as test strips.

[0096] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A use of a specific molecular marker or primer set for screening, detecting or identifying Staphylococcus xylosus for non-disease diagnosis purposes, characterized in that: The nucleotide sequence of the specific molecular marker is shown in SEQ ID NO: 1 or SEQ ID NO: 2; The sequences of the primer set are shown in SEQ ID NO: 3 to SEQ ID NO:

6.

2. Use of a primer set in preparing a product for screening, detecting or identifying Staphylococcus xylosus, characterized in that: The primer set is used to amplify specific molecular markers; The nucleotide sequence of the specific molecular marker is shown in SEQ ID NO: 1 or SEQ ID NO: 2; The sequences of the primer set are shown in SEQ ID NO: 3 to SEQ ID NO:

6.

3. The use according to claim 2, characterized in that The product includes a detection kit, and the detection kit includes the primer set.

4. A method for identifying Staphylococcus xylosus for non-disease diagnosis purposes, characterized in that: The following steps are involved: S1. Performing PCR amplification on the genomic DNA of the sample to be tested using a primer set to obtain an amplified product; wherein the sequence of the primer set is shown in SEQ ID NO: 3 to SEQ ID NO: 6; S2. Observe whether a target band appears in the amplified product. If a target band appears, it is determined that the sample to be tested contains Staphylococcus xylosus.

5. The method according to claim 4, characterized in that In step S1, The PCR amplification system included: 2 μL of 10× PCR reaction buffer, 2 μL of 20 mmol / L MgCl2, 1 μL of 2 mmol / L dNTPs, 100 ng of template DNA, 0.5 μL each of 10 μmol / L forward and reverse primers, 1 U of Taq enzyme, and sterile double-distilled water to make up the volume to 20 μL; The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58.5°C for 30 s; extension at 72°C for 1 min; 35 cycles of denaturation, annealing, and extension; and final extension at 72°C for 5 min.

6. A method for quantitatively detecting Staphylococcus xylosus for non-disease diagnosis purposes, characterized in that: The following steps are involved: S1. Performing qPCR amplification on the genomic DNA of the sample to be tested using a primer set; wherein the sequence of the primer set is shown in SEQ ID NO: 3 to SEQ ID NO: 6; S2. The logarithm of the concentration of the pure culture of the standard Staphylococcus xylosus strain is used as the horizontal axis, and the corresponding real-time Ct value of qPCR is used as the vertical axis. The obtained curve is the standard curve for quantifying Staphylococcus xylosus; S3. Analyze the amplification results and determine the content of Staphylococcus xylosus in the sample by comparing the fluorescence signal value with the standard curve.

7. The method according to claim 6, characterized in that The qPCR amplification system was as follows: 10 μL of 2× TB Green Premix reaction solution, 1 μL of template DNA, 0.5 μL each of 10 μmol / L forward and reverse primers, and sterile double-distilled water to make up the volume to 20 μL. The qPCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s; annealing at 59°C for 40 s; and 40 cycles of denaturation and annealing.

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