Specific molecular marker for screening staphylococcus xylosus, primer group and application

Abstract

The invention discloses a specific molecular marker for screening staphylococcus xylosus, a primer group and application, and relates to the technical field of microbiological detection. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The sequences of the primer group for amplifying the molecular marker are as shown in SEQ ID NO: 3-SEQ ID NO: 6; the invention also provides application of the molecular marker and the primer group in screening or detecting or identifying staphylococcus xylosus for a non-disease diagnosis purpose. The molecular marker disclosed by the invention is higher in coverage rate of target bacteria and non-target bacteria in specificity evaluation, and the accuracy is enhanced. The detection method can accurately quantify the staphylococcus xylosus, greatly improves the identification efficiency of the staphylococcus xylosus, has the advantages of high specificity, simplicity in operation, easiness in result judgment, short detection time and low cost, and provides technical support for detection and industrial application of the staphylococcus xylosus.

Description

Technical Field

[0001] The invention relates to the technical field of microbial testing, and in particular to specific molecular markers, primer sets and applications 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-1.5 μm, often arranged singly, in pairs, in short chains or in irregular grape-like arrangements. Staphylococcus xylosus is widely distributed in nature and has been found in the air, soil, food, and on the body and mucous membranes of humans and animals.

[0003] At present, the detection methods for Staphylococcus xylosus mainly include traditional culture method, immunoassay method, molecular biology detection method and mass spectrometry detection method. The traditional culture method requires the sample to be inoculated on a specific culture medium and cultured under suitable conditions, and the identification is carried out by observing the morphology and color of the colonies and performing a series of biochemical reactions. This method is relatively simple to operate and relatively low in cost, but the detection cycle is long, generally requiring 48 hours or even longer, and it is easy to miss low-concentration bacterial liquids. It is also susceptible to interference from other microorganisms in complex samples, and the accuracy is poor. Immunoassay methods use the principle of antigen-antibody specific binding, such as enzyme-linked immunosorbent assay (ELISA) and latex agglutination test. The detection speed is relatively fast, with certain specificity and sensitivity. However, the cost of preparing specific antibodies is high, and the unstable antibody quality will affect the results. In addition, complex samples are prone to non-specific reactions, resulting in false positives. Mass spectrometry detection is based on bacterial protein fingerprint identification, which is fast and accurate. However, the instrument is expensive, the maintenance cost is high, and it needs to rely on a large standard mass spectrometry database. For new variants or rare strains, it may be difficult to accurately identify them due to the lack of reference maps. In recent years, with the development of molecular biology, molecular detection methods based on PCR have gradually become one of the most promising new 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. oeLhThe specificity and sensitivity of the target for the detection of Staphylococcus xylosus are difficult to meet the needs of actual rapid detection. Thanks to the development of high-throughput sequencing and bioinformatics analysis technology, omics technology based on pan-genomic analysis is widely used in studies such as bacterial genetic variation, gene function enrichment and evolutionary analysis. Therefore, based on pan-genomic analysis technology, molecular identification targets of Staphylococcus xylosus can be efficiently mined, and simple, rapid, economical, efficient and highly sensitive PCR identification and qPCR quantitative detection technologies can be further developed to meet the needs of different fields for efficient identification and quantitative detection of Staphylococcus xylosus. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide a specific molecular marker, a primer set and an application for screening Staphylococcus xylosus. The specific new 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 solution of the present invention is as follows: The 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.

[0007] The present invention obtains xylosus Staphylococcus through pan-genome analysis, and contains specific sequence fragments as shown in SEQ ID NO: 1 to SEQ ID NO: 2, which can be used as specific molecular markers for identifying xylosus Staphylococcus. The molecular marker has a coverage rate of 100% for target bacteria genes and a coverage rate of 0% for non-target bacteria.

[0008] A second aspect of the present invention provides a primer set for amplifying the specific molecular marker, wherein the sequence of the primer set is shown in SEQ ID NO: 3 to SEQ ID NO: 6.

[0009] 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; 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.

[0010] 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.

[0011] 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.

[0012] The molecular marker and primer set of the present invention can accurately detect Staphylococcus xylosus in a sample with good specificity and high accuracy.

[0013] 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.

[0014] By using the molecular markers and primer sets of the present invention to prepare products such as detection kits for screening, detecting or identifying Staphylococcus xylosus, rapid and convenient detection can be achieved.

[0015] Preferably, the product comprises a detection kit, and the detection kit comprises the primer set.

[0016] A fifth aspect of the present invention provides a method for identifying Staphylococcus xylosus for non-disease diagnosis purposes, comprising the following steps: S1, using the primer set to perform PCR amplification on the genomic DNA of the sample to be tested to obtain an amplified product; S2. Observe whether the amplified product has a target band. If the target band appears, it is determined that the sample to be tested contains Staphylococcus xylosus.

[0017] The present invention uses a detection primer to amplify a specific molecular marker through a PCR reaction, and by observing whether the amplified product is at the expected position, it can be determined whether the xylose Staphylococcus exists. The present invention sets up multiple PCR systems and uses different primers to amplify the target DNA at the same time, thereby improving the detection efficiency.

[0018] Preferably, in step S1, The PCR amplification system includes: 10× PCR reaction buffer 2 μL, 20 mmol / L MgCl 2 , 2 μL, 2mmol / L dNTP 1 μL, template DNA 100 ng, 10 μmol / L forward and reverse primers 0.5 μL each, Tag enzyme 1 U, 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; denaturation, annealing, and extension for 35 cycles; and finally extension at 72°C for 5 min.

[0019] A sixth aspect of the present invention provides a method for quantitatively detecting Staphylococcus xylosus for non-disease diagnosis purposes, comprising the following steps: S1, using the primer set to perform qPCR amplification on the genomic DNA of the sample to be tested; S2. The logarithm of the concentration of the pure culture of the standard Staphylococcus xylosus strain is used as the abscissa, and the corresponding real-time Ct value of qPCR is used as the ordinate. The fitted curve is the standard curve for quantifying Staphylococcus xylosus; S3. Analyze the amplification result, and determine the content of Staphylococcus xylosus in the sample by comparing the fluorescence signal value with the standard curve.

[0020] 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 judging the intensity of the fluorescent signal in the system. The present invention improves the detection efficiency by setting up multiple qPCR systems and using different primers to amplify the target DNA at the same time.

[0021] Preferably, the qPCR amplification system is: 10 μL of 2× TB Green Premix reaction solution, 1 μL of template DNA, 0.5 μL 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; extension at 72°C for 1 min; denaturation and annealing were performed for a total of 40 cycles.

[0022] The present invention has at least one of the following beneficial effects: 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. In the specificity evaluation, the new molecular detection molecular markers of the present invention have a greater coverage of target bacteria and non-target bacteria, which enhances accuracy. The detection method of the present invention has the ability to accurately quantify Staphylococcus xylosus, greatly improves the efficiency of Staphylococcus xylosus identification, and enhances 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

[0023] Figure 1 Schematic diagram of electrophoresis results for specificity evaluation of the PCR detection method for Staphylococcus xylosus in Example 3, wherein A in the figure is a schematic diagram of electrophoresis results for specificity evaluation using primer set 1, and B in the figure is a schematic diagram of electrophoresis results for specificity evaluation using primer set 2.

[0024] Figure 2The figure is a schematic diagram of the amplification curve and the dissolution curve of the qPCR quantitative detection method of Staphylococcus xylosus in Example 5, wherein A in the figure is the amplification curve and B in the figure is the dissolution curve.

[0025] Figure 3 It is a schematic diagram of the sensitivity evaluation results of the qPCR quantitative detection method for artificially spiked sausage xylosus Staphylococcus in Example 6, wherein A in the figure 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 in the figure and D in the figure are the real-time fluorescence amplification curve and standard curve for quantitative detection using primer set 2, respectively.

[0026] 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

[0027] 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 in conjunction with 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 used to limit the present invention.

[0028] Example 1 Screening and obtaining specific molecular markers of Staphylococcus xylosus According to the genome data of 12 strains of Staphylococcus xylosus and 750 strains of non-Staphylococcus xylosus in the NCBI website, a pan-genome analysis was performed; two specific gene fragments of Staphylococcus xylosus were screened and obtained, and the nucleotide sequences of the gene fragments are shown in SEQ ID NO: 1 to SEQ ID NO: 2.

[0029] The xylosus-specific molecular markers obtained by screening are all from Staphylococcus xylosus CP060271.1, and the gene fragment sequences of the molecular markers correspond to the gene loci as shown in Table 1 below.

[0030] Table 1 Genomic loci of specific molecular markers of Staphylococcus xylosus Example 2 PCR method for identifying Staphylococcus xylosus 1) Primer design According to the sequences SEQ ID NO: 1 to SEQ ID NO: 2 in Example 1, specific PCR amplification primers (including forward primers and reverse primers) were designed. The sequences of primer set 1 and primer set 2 are shown in Table 2 below.

[0031] Table 2 Specific PCR detection primer sets 2) The method for identifying Staphylococcus xylosus is as follows: S1. Preparation of DNA template: The strains to be tested were cultured in LB liquid medium, and their bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as the template to be tested; S2. PCR amplification: Use the primer set 1 and primer set 2 described in Example 2 to perform PCR amplification on the sample DNA to be tested, respectively. The specific method is as follows: ①PCR detection system: ②PCR amplification procedure: S3: taking the PCR amplification product for gel electrophoresis; S4: Observe whether there is a single amplification band at the position corresponding to the product size of each primer set. If there is, it means that the sample contains the target Staphylococcus xylosus; if no corresponding single amplification band appears, the sample does not contain the target Staphylococcus xylosus.

[0032] Example 3 Evaluation of the specificity of the PCR detection method for Staphylococcus xylosus A total of 10 strains of Staphylococcus xylosus and 39 strains of non-Staphylococcus xylosus were taken and PCR detection was performed according to the method of Example 2. Wherein, S1: DNA template preparation is to extract the genomic DNA of each bacterium respectively; S2: during PCR amplification, the primers used are primer set 1 and primer set 2 in Example 2 respectively. A blank control is set, and the template of the blank control is an aqueous solution without the genomic DNA of each bacterium.

[0033] The bacterial strains used and the test results are shown in Table 3 below. In the table, "+" 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 is 2000Maker, C is negative quality control, and P is positive quality control.

[0034] Table 3 Results of the molecular marker specificity evaluation test of Staphylococcus xylosus of the present invention Depend on Figure 1 As shown in Table 3, in the detection results of primer set 1 and primer set 2 in Example 2, only Staphylococcus xylosus showed specific amplification bands, and other non-target Staphylococcus xylosus had no specific bands, indicating 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.

[0035] Example 4 Comparison results of the molecular markers for detecting Staphylococcus xylosus of the present invention and existing molecular markers 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 to Staphylococcus xylosus through pan-genomic data analysis. At the same time, the existing molecular detection markers specific to Staphylococcus xylosus are searched. oeLh . Based on the comparison results of the local BLAST gene database, 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 the non-target bacteria. The prior art (Iacumin, L., Cocolin, L., Cantoni, C., & Comi, G. (2007). Preliminary analysis of the lipase gene ( oeLh ) expression of Staphylococcus xylosus in vitro and duringfermentation of naturally fermented sausages (in situ). Journal of foodprotection, 70(11), 2665-2669.) reported in oeLh As a molecular marker for detection, its target bacteria gene coverage is 100%, and the non-target bacteria coverage is 13.2%. Therefore, this example illustrates that the xylosus Staphylococcus marker of the present invention has higher accuracy and better specificity, and can accurately identify xylosus Staphylococcus in actual samples, which has obvious advantages over existing reporting markers. The specific results are shown in Table 4 below.

[0036] Table 4 Comparison results of the Staphylococcus xylosus markers of the present invention and existing markers Example 5 qPCR quantitative detection method for identifying Staphylococcus xylosus 1) Primer selection: Use the primer set 1 and the primer set 2 described in Example 2 to perform qPCR amplification on the sample DNA to be tested; 2) The method for quantitative detection of Staphylococcus xylosus is as follows: S1. Preparation of DNA template: The xylose Staphylococcus strain was cultured in LB liquid medium, and the bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as the template to be tested; a blank control was set up, and the template of the blank control was an aqueous solution without the genomic DNA of each bacteria.

[0037] S2, PCR amplification: using the primer set 1 and the primer set 2 to perform qPCR amplification on the DNA of the sample to be tested; ①qPCR detection system: ②qPCR amplification procedure: S3: The qPCR amplification system was performed on a Bio-Rad CFX96 fluorescence quantitative amplification instrument; S4: Use the software Bio-Rad CFX Manager to analyze whether the amplification results are in line with expectations. Under the premise that there is no signal in the blank control, if a fluorescence amplification curve and a corresponding melting curve are generated, it means that the sample contains Staphylococcus xylosus; if no signal is generated, the sample does not contain Staphylococcus xylosus.

[0038] The fluorescence amplification curve and the corresponding melting curve are shown in Figure 2 As shown in the figure, when the sample contains Staphylococcus xylosus, the qPCR system established based on primer set 1 and primer set 2 can produce an obvious fluorescence amplification curve ( Figure 2 A in the figure) and the corresponding dissolution curve ( Figure 2 The blank control showed no fluorescence curve or melting curve.

[0039] Example 6 Evaluation of the sensitivity of the qPCR quantitative detection method for Staphylococcus xylosus artificially spiked sausages After the sausage samples were sterilized by ultraviolet light, physiological saline was added to prepare sterile samples. After culturing 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 operating procedures of 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, and homogenized at 8000 r / min~10000 r / min for 1 min~2 min to make a 1:10 sample solution. The culture was cultured to a concentration of 10 8 The xylose Staphylococcus isolate with 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 7CFU / 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 the 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 1 CFU / mL xylose Staphylococcus aureus liquid sample DNA template, according to the method of Example 5, take 1 μL of the above-mentioned different dilution spiked samples DNA templates for qPCR reaction based on primer set 1 and primer set 2, and perform three parallel experiments for each template. The results were analyzed according to the method of Example 5.

[0040] 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.

[0041] The 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 fitted 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 fitted standard curve of Staphylococcus xylosus is y = -2.5708x +43.642, and the correlation coefficient R 2 It is 0.9688.

[0042] Example 7 Evaluation of the ability of the qPCR quantitative detection method of Staphylococcus xylosus to resist interference from other bacteria The new culture was cultured to a concentration of 10 7CFU / mL of xylosus Staphylococcus isolate and Staphylococcus aureus ATCC25923 were mixed in a certain ratio to obtain mixed bacterial solutions with final concentration ratios of xylosus Staphylococcus isolate and Staphylococcus aureus ATCC 25923 of 1:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:15, and 1:20, respectively. The DNA template of the mixture was extracted according to Example 5, that is, the samples with different concentration ratios of xylosus Staphylococcus and Staphylococcus aureus were tested, and qPCR reactions based on primer set 1 and primer set 2 were performed according to Example 5, and three parallel experiments were performed for each template. The results were analyzed according to Example 5.

[0043] Fluorescence amplification curve Figure 4 As shown, both primer set 1 and primer set 2 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.

[0044] Example 8 Detection results of Staphylococcus xylosus in actual samples 10 sausage and bacon samples were collected from local supermarkets and meat and vegetable markets. Samples were processed under sterile operation in a clean bench in the laboratory, and the sample processing was carried out according to the national standard "GB4789.10-2016" operation steps, and then the strain identified as xylosus Staphylococcus was used to prepare a template to extract genomic DNA, and the DNA template was extracted specifically according to Example 6. Finally, the qPCR method was used to conduct experiments according to Example 6. At the same time, in order to ensure the accuracy of the results, 1 sausage and bacon sample spiked with known xylosus Staphylococcus was selected, and the identification was carried out according to the national standard "GB4789.10-2016" operation steps.

[0045] The test results are shown in Table 5, in which "+" indicates a positive test result, and "-" indicates a negative test result.

[0046] Table 5 Detection results of Staphylococcus xylosus in actual samples As shown in Table 5, compared with the national standard method, in the 10 real food samples tested, the accuracy of Staphylococcus xylosus detection in the actual samples using the qPCR method of the present invention was 100%, and the accuracy of the two positive control samples was also 100%.

[0047] The qPCR method of the present invention is capable of rapid identification of Staphylococcus xylosus in actual samples, greatly improving the efficiency of identification of Staphylococcus xylosus and enhancing its practicality; the detection method of the present invention also has the advantages of simple operation, easy result determination, short detection time, strong specificity and low cost. In subsequent studies, by constructing biosensor platforms such as test strips, on-site instant detection of Staphylococcus xylosus can be further realized.

[0048] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A specific molecular marker for screening Staphylococcus xylosus, characterized in that: The nucleotide sequence of the specific molecular marker is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A primer set for amplifying the specific molecular marker according to claim 1, characterized in that: The sequences of the primer set are shown in SEQ ID NO: 3 to SEQ ID NO:

6.

3. The primer set according to claim 2, characterized in that 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; 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.

4. Use of the specific molecular marker according to claim 1 or the primer set according to any one of claims 2 to 3 in screening, detecting or identifying Staphylococcus xylosus for non-disease diagnosis purposes.

5. Use of the specific molecular marker according to claim 1 or the primer set according to any one of claims 2 to 3 in preparing a product for screening, detecting or identifying Staphylococcus xylosus.

6. The use according to claim 5, characterized in that: The product includes a detection kit, and the detection kit includes the primer set.

7. 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 the primer set according to claim 2 or claim 3 to obtain an amplified product; S2. Observe whether the amplified product has a target band. If the target band appears, it is determined that the sample to be tested contains Staphylococcus xylosus.

8. The method according to claim 7, characterized in that In step S1, 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 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; 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; denaturation, annealing, and extension for 35 cycles in total; and finally extension at 72°C for 5 min.

9. 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 the primer set according to claim 2 or claim 3; S2. The logarithm of the concentration of the pure culture of the standard Staphylococcus xylosus strain is used as the abscissa, and the corresponding real-time Ct value of qPCR is used as the ordinate. The fitted 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.

10. The method according to claim 9, characterized in that The qPCR amplification system is as follows: 10 μL of 2× TB Green Premix reaction solution, 1 μL of template DNA, 0.5 μL 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 denaturation and annealing for 40 cycles in total.

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