Molecular markers, primer probe set and detection method for detecting lactobacillus plantarum s58

Abstract

The application discloses a molecular marker, a primer group and a kit for identifying lactobacillus plantarum S58, and discloses a method for identifying lactobacillus plantarum S58. The application obtains a specific fragment of lactobacillus plantarum S58 through a large number of screening, and uses the specific fragment for constructing a primer group and a probe, and designs a quantitative primer probe group of lactobacillus plantarum S58, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No. 1, the nucleotide sequence of a downstream primer is shown as SEQ ID No. 2, and the nucleotide sequence of a probe is shown as SEQ ID No. 3. The primer probe group has good specificity and high sensitivity, and can quickly and accurately detect whether lactobacillus plantarum S58 is contained in a sample and the concentration of lactobacillus plantarum S58.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a molecular marker, primer-probe set, and detection method for detecting Lactobacillus plantarum S58. Background Technology

[0002] Probiotics are a class of live bacteria composed of one or more microorganisms. When ingested in certain doses, they can promote human health by improving the balance of the gut microbiota. With the deepening research on probiotics in recent years, they have been widely used in medicine, the food industry, and animal husbandry.

[0003] Chinese Patent ZL201911131324.8 (Invention Title: Lactobacillus plantarum S58 and its Application in the Preparation of Products for Alleviating Obesity) discloses Lactobacillus plantarum S58 with accession number CCTCCNO: M2019595. This Lactobacillus plantarum S58, isolated from kimchi, has been proven to alleviate or prevent damage to the digestive tract caused by spicy foods, as well as obesity or metabolic diseases caused by obesity. It has promising applications in food, pharmaceuticals, and functional health products. Therefore, the detection of Lactobacillus plantarum S58 is a necessary step in the future development of related food, pharmaceutical, and functional health products.

[0004] Furthermore, given the current consensus in the industry regarding the evaluation of probiotic efficacy at the strain level, when conducting research on probiotics in the gut, it is necessary to perform qualitative and quantitative analysis at the strain level. Qualitative analysis determines whether the bacteria have entered the gut alive, and quantitative analysis determines whether a certain dosage has been reached. Currently, research reports on the qualitative and quantitative evaluation of probiotics in the gut at the strain level are still scarce, and sufficient reasonable and effective evaluation methods have not yet been established. Traditional isolation and culture methods are inefficient and cannot identify strains from their morphology; therefore, it is particularly important to develop more qualitative and quantitative analysis methods for probiotics at the strain level. Summary of the Invention

[0005] This invention targets *Lactobacillus plantarum* S58, performing Core / pan analysis on the target strain and reference strain to identify unique genes of the target strain. Specificity is confirmed by comparing the gene nucleic acid sequence with the genome of the reference strain; genes longer than 150 bp are screened; a fixed-length (150 bp) sequence fragment is traversed on the target strain genome and compared with the reference strain genome; specific sequence fragments of the target strain are screened; it is determined whether the specific sequence fragment is located in a coding gene region; the found specific sequences are checked using NCBI, ultimately identifying the S58-specific genomic fragment, and a detection method for *Lactobacillus plantarum* S58 is established based on this.

[0006] Based on this, the present invention provides a molecular marker for identifying Lactobacillus plantarum S58, the molecular marker being located on the CP068767 genome sequence, and the nucleotide sequence of the molecular marker being shown in SEQ ID No. 4.

[0007] This invention also provides a primer set for amplifying the above-mentioned molecular markers, including upstream and downstream primers, the nucleotide sequences of which are as follows:

[0008] The upstream primer sequence is shown in SEQ ID No. 1: 5'-ACGAGTCGGTTAAGAATC-3'.

[0009] The downstream primer sequence is shown in SEQ ID No. 2: 5'-GGTTGGAACAATGATAGTG-3'.

[0010] Preferably, the primer set further includes a probe.

[0011] The nucleotide sequence of the probe is shown in SEQ ID No. 3: 5'-TCGGACTGATCGCCACCATT-3'.

[0012] The probe is preferably labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end; more preferably, the fluorescent reporter group is FAM and the fluorescent quencher group is BHQ1.

[0013] The present invention also provides a kit for identifying Lactobacillus plantarum S58, comprising the primer set described in any of the above claims, and further comprising reaction reagents for PCR amplification or real-time fluorescent PCR amplification.

[0014] Preferably, it also includes DNA extraction reagents, positive controls, and negative controls, and the reaction reagents for PCR amplification or real-time fluorescent PCR amplification include DNA polymerase and DNA polymerase buffer.

[0015] The present invention also provides a method for identifying Lactobacillus plantarum S58, comprising the following steps:

[0016] (1) Extract genomic DNA from the sample to be tested;

[0017] (2) Use the primer set or the kit described above to perform PCR amplification on the extracted genomic DNA;

[0018] (3) Detect whether the PCR amplification product contains the target amplification fragment. The sample containing the target amplification fragment was identified as Lactobacillus plantarum S58, and the size of the target amplification fragment was 97 bp.

[0019] The preferred PCR amplification reaction system is as follows: total volume 25 μl, including: 12.5 μL 2×Taq PCR Master Mix, 1 μL 10 μM upstream primer, 1 μL 10 μM downstream primer, 9.5 μL ddH2O, and 1 μL DNA; PCR reaction conditions are: 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 4 min.

[0020] Agarose gel electrophoresis is preferred for detecting whether the PCR amplification product contains the target band.

[0021] This invention also provides a real-time fluorescent PCR method for identifying Lactobacillus plantarum S58, comprising the following steps:

[0022] (1) Extract genomic DNA from the sample to be tested;

[0023] (2) Using the primer set or the kit described above, the extracted genomic DNA was amplified by real-time fluorescent PCR. The amplification results were used to determine whether the target was Lactobacillus plantarum S58. If Ct≤36, it was determined to be Lactobacillus plantarum S58.

[0024] This invention also provides a method for the detection of Lactobacillus plantarum S58 by real-time PCR, comprising the following steps:

[0025] (1) Preparation of standard: The conserved gene fragment of Lactobacillus plantarum S58, as shown in SEQ ID No.4, is inserted into the cloning vector to obtain a recombinant plasmid containing the conserved gene fragment, which is used as a standard.

[0026] (2) Plotting the standard curve;

[0027] (3) Use the primer set or kit described above to perform real-time fluorescent PCR amplification on the test sample and the standard using the same reaction system;

[0028] (4) Quantitative detection of Lactobacillus plantarum S58 was performed using the standard curve and the Ct value of the sample to be tested.

[0029] Preferably, the cloning vector is a pGM-T vector.

[0030] Preferably, the real-time fluorescence PCR method or the quantitative fluorescence PCR detection method is used. The PCR reaction system is 25 μL, including: 12.5 μL of 2×FastFire qPCRPreMix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 0.5 μL of 10 μM probe, 9 μL of dd H2O, and 1 μL of template DNA; the reaction conditions are: 98℃ for 1 min; 98℃ for 5 s, 65.3℃ for 15 s, for 40 cycles.

[0031] Finally, this invention also provides the application of the above-mentioned molecular markers, or the above-mentioned primer sets, or the above-mentioned kits in the identification of Lactobacillus plantarum S58.

[0032] The beneficial effects of this invention are:

[0033] This invention involves whole-genome sequencing of *Lactobacillus plantarum* S58, a strain exhibiting intestinal tolerance and probiotic functions. The sequencing results were compared with the whole-genome sequences of 164 *Lactobacillus plantarum* strains with complete genome sequences in the NCBI database to identify unique genes specific to *Lactobacillus plantarum* S58. Based on these gene sequences, a specific primer-probe set was designed. This primer-probe set was applied to the detection of *Lactobacillus plantarum* S58, and through optimization of the quantitative real-time PCR reaction system and conditions, a minimum detection limit of 2.1 × 10⁻⁶ was achieved. 2 A rapid detection method for Lactobacillus plantarum S58 with good sensitivity, specificity, accuracy, repeatability, stability, and efficiency (copies / μL). Attached Figure Description

[0034] Figure 1 The graph shows the gene alignment results of Lactobacillus plantarum S58. The horizontal axis represents the number of gene alignments (i.e., how many other Lactobacillus plantarum strains have matched each gene in Lactobacillus plantarum S58), and the vertical axis represents the number of genes. When the horizontal axis is 0, the vertical axis value corresponding to it is the number of genes unique to Lactobacillus plantarum S58.

[0035] Figure 2 This is the standard curve for Lactobacillus plantarum S58.

[0036] Figure 3 The graph shows the sensitivity detection results of the real-time PCR method. The concentrations of *Lactobacillus plantarum* S58 from left to right in the amplification curves are: 2.1 × 10⁻⁶. 9 copies / μL, 2.1×10 8 copies / μL, 2.1×10 7 copies / μL, 2.1×10 6 copies / μL, 2.1×10 5 copies / μL, 2.1×10 4 copies / μL, 2.1×10 3 copies / μL, 2.1×10 2 copies / μL.

[0037] Figure 4 This is a graph showing the specific detection results of the real-time PCR method.

[0038] Figure 5 The image shows the results of quantitative detection of Lactobacillus plantarum S58 in feces using the real-time PCR method. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations. Non-essential modifications and adjustments made by others based on the concept of the present invention are still within the scope of protection of the present invention.

[0040] The instrument used in this invention is the Bio-rad CFX96 real-time PCR instrument.

[0041] The genomic DNA extraction kit, qPCR premix, DNA purification and recovery kit, pGM-T cloning kit, DH5α competent cells, and plasmid small extraction kit used in this invention were all purchased from Tiangen Biotech Co., Ltd., and the primers and probes were synthesized by Takara.

[0042] The *Lactobacillus plantarum* S58 detected in this invention has been disclosed in Chinese Patent ZL 201911131324.8 (Publication No. CN110684701B) and has been deposited. The deposit information is as follows:

[0043] China Center for Type Culture Collection; Address: Wuhan University, Wuhan, China; Date of deposit: August 1, 2019; Accession number: CCTCC NO: M 2019595; Classification and nomenclature: Lactobacillus plantarum S58.

[0044] Example 1: A primer and probe set for detecting Lactobacillus plantarum S58 based on qPCR

[0045] Primer and probe design:

[0046] 1. Search for strain-specific genes

[0047] Whole-genome sequencing was performed on *Lactobacillus plantarum* S58. Comparison analysis was conducted with 164 *Lactobacillus plantarum* strains with complete genome sequences in the NCBI database. A total of 484,447 coding genes were identified in the 164 reference strains, and the 2,828 genes from strain S58 were added, resulting in a total of 487,275 coding genes. First, the coding genes were clustered using the CD-hit clustering software. Genes that could not be clustered with other strains were identified as candidate S58-specific genes. Second, genes longer than 150 bp were filtered out based on length requirements. Finally, these genes were compared with the NCBI NT database (Non-Redundant Genome Sequence Database) to confirm genes that did not match any of the non-S58 strains, resulting in two S58-specific gene sequences. Figure 1 The gene sequence lengths are 423bp and 1164bp, respectively.

[0048] 2. Search for strain-specific genomic fragments

[0049] First, using a step size of 50 bp, all sequence fragments of 150 bp in length on the S58 strain genome were traversed and compared with the genome sequences of all reference strains. Sequence fragments without any alignment results on the S58 strain were screened, and overlapping sequence fragments were merged; the merged sequences were used as candidate specific sequence fragments. Next, the candidate sequence fragments were compared with the NCBI NT database (Non-Redundant Genome Sequence Database) to confirm sequence fragments that could not be aligned with any non-S58 strains. After sequence fragment alignment and result screening, a total of 6 candidate specific sequence fragments longer than 150 bp were found on the S58 strain genome. After comparison with the NCBI NT database, one sequence fragment aligned with more genomes of other strains, while the remaining 5 sequence fragments could only align with the S58 strain genome, resulting in 5 specific sequence fragments for the S58 strain. The lengths of the 5 sequence fragments, from shortest to longest, are 248 bp, 679 bp, 1238 bp, 1420 bp, and 1945 bp. Based on the gene location information of strain S58, the analysis of specific genes revealed that two strain-specific genes were located within the five specific sequence fragments found. Table 2 shows that the lengths of the five sequence fragments, from shortest to longest, are 248 bp, 679 bp, 1238 bp, 1420 bp, and 1945 bp. Ordered by the start site of the sequence fragments in the genome, the third specific sequence fragment contains a coding gene (JMO19_10610), the function of which is unknown. The fifth specific sequence fragment contains a coding gene (JMO19_11120), described as a "DUF262 domain-containing protein".

[0050] The identified specific sequences were checked using NCBI alignment, and primers and probes were designed using software such as Primer Premier 5.0. The CP068767 genome sequence was ultimately selected for primer and probe design. The specific sequence fragment starts at nucleotide 2,135,593 and terminates at nucleotide 2,137,537, with a total length of 1945 bp. The specific sequence is shown in SEQ ID NO. 5.

[0051]

[0052] The upstream primer sequence is shown in SEQ ID No. 1: 5'-ACGAGTCGGTTAAGAATC-3';

[0053] The downstream primer sequence is shown in SEQ ID No. 2: 5'-GGTTGGAACAATGATAGTG-3';

[0054] The probe sequence is shown in SEQ ID No. 3: 5'FAM-TCGGACTGATCGCCACCATT-3'BHQ1.

[0055] Table 1: List of 164 *Lactobacillus plantarum* strains used for comparative analysis on NCBI

[0056]

[0057]

[0058] Note: Strain: strain name; Assembly: strain sequence access ID.

[0059] Table 2: List of Specific Sequence Fragment Results

[0060]

[0061] Explanation: Seq_ID: Genomic sequence ID containing the sequence, Region_Start: Start position of the specific sequence fragment, Region_end: End position of the specific sequence fragment, Region_Length: Length of the specific sequence fragment, ID: Gene ID, Start: Start position of the gene, End: End position of the gene, Strand: Gene orientation, Product: Functional description of the gene.

[0062] The sequence amplified using the above primer and probe set is shown in SEQ ID No. 4:

[0063] 5'-ACGAGTCGGTTAAGAATCCCATCTTAATTGTTTCAACATCGGACTGATCGCCACCATTGATTATTTGATAAGGACCCGCACTATCATTGTTCCAACC-3'.

[0064] Example 2: A method for detecting Lactobacillus plantarum S58 based on real-time quantitative PCR

[0065] (1) Extract Lactobacillus plantarum S58 genomic DNA using the Tiangen Bacterial Genomic DNA Extraction Kit.

[0066] (2) Quantitative real-time PCR amplification was performed using the primer and probe set described in Example 1 and the qPCR premix solution from Tiangen Biotech. The reaction system consisted of 25 μL, including: 12.5 μL of 2×FastFire qPCR PreMix (Probe), 1 μL of 10 μM upstream primer (SEQ ID No. 1), 1 μL of 10 μM downstream primer (SEQ ID No. 2), 0.5 μL of 10 μM probe, 9 μL of ddH2O, and 1 μL of template DNA. The reaction conditions were: 98℃ for 1 min; 98℃ for 5 s, 60℃ for 15 s, for 40 cycles.

[0067] Optimization of reaction conditions for real-time PCR:

[0068] By comparing the results of primer and probe concentrations and annealing temperatures in the reaction system, a reaction system with high sensitivity, low background fluorescence signal, typical S-type amplification fluorescence signal curve, and amplification efficiency close to 1 was selected. The optimized reaction system was: 12.5 μL of 2×FastFire qPCR PreMix (Probe), 0.5 μL of upstream primer, 0.5 μL of downstream primer, 0.5 μL of probe, 10 μL of dd H2O, and 1 μL of template DNA; the optimized reaction conditions were: 98℃ for 1 min; 98℃ for 5 s, 65.3℃ for 15 s, for 40 cycles.

[0069] Establishment of the method for detecting Lactobacillus plantarum S58 according to the present invention:

[0070] 1. Construction of the standard curve for quantitative PCR of Lactobacillus plantarum S58

[0071] (1) Constructing a plasmid containing the S58-specific sequence of Lactobacillus plantarum.

[0072] ① Extract genomic DNA from Lactobacillus plantarum S58;

[0073] ② The specific sequence was amplified by conventional PCR using the primers (SEQ ID No. 1, SEQ ID No. 2) described in Example 1. The reaction system was 25 μL, including: 12.5 μL of 2×Taq PCR Master Mix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 9.5 μL of dd H2O, and 1 μL of DNA. The reaction conditions were: 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 4 min; 4℃ at ∞.

[0074] ③ The amplified products were detected by 2.0% agarose gel electrophoresis, and the gel was excised. The target fragment was recovered using a universal DNA purification and recovery kit (Tiangen), and the target fragment size was 97 bp. The amplified target gene was cloned into the pGM-T vector using the pGM-T cloning kit, transformed into Escherichia coli DH5α strain, positive clones were screened and identified by PCR, and the colonies identified as positive were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The single clones that were correctly sequenced were then expanded and cultured.

[0075] ④ Plasmid extraction and purification were performed on the bacterial culture using a plasmid mini-extraction kit (Tiangen). The plasmid concentration was determined using a Nanodrop micro spectrophotometer, with a mass concentration of 71.8 ng / μL, a DNA length of 3112 bp, and a plasmid copy number of 6.02 × 10⁻⁶. 23 )×(71.8ng / μL×10 -9 ) / (3112×660)=2.1×10 10 copies / μL.

[0076] (2) Construction of standard curve for real-time PCR

[0077] The obtained quantitative standard plasmid was serially diluted 10-fold using ddH2O to a concentration of 2.1 × 10⁻⁶. 9 copies / μL, 2.1×10 8 copies / μL, 2.1×10 7 copies / μL, 2.1×10 6 copies / μL, 2.1×10 5 copies / μL, 2.1×10 4 copies / μL, 2.1×10 3 copies / μL, 2.1×10 2 Copies / μL, three replicates for each dilution. Plot a standard curve (e.g., ...). Figure 1 The results showed that this detection method has a good linear relationship. A standard curve was obtained by plotting the logarithm of DNA copy number (lg[DNA]) on the x-axis and Ct value on the y-axis. Figure 2 The regression equation is y = -3.3475x + 44.611, and the correlation coefficient (R²) of the standard curve is... 2 The amplification efficiency reached 0.9964, with a slope k = -3.3475, therefore the amplification efficiency E = 10. -1 / k -1 = 98.94%. Based on the established standard curve, quantitative real-time PCR at 2.1 × 10⁻¹ 2 -2.1×10 9 A good linear relationship was observed within the dilution range of copies / μL.

[0078] (3) Method sensitivity detection

[0079] The quantitative standard plasmid was serially diluted 10-fold using dd H2O to obtain concentrations of 2.1 × 10⁻⁶. 9 copies / μL, 2.1×10 8 copies / μL, 2.1×10 7 copies / μL, 2.1×10 6 copies / μL, 2.1×10 5 copies / μL, 2.1×10 4 copies / μL, 2.1×10 3 copies / μL, 2.1×10 2 The sensitivity of this method was tested using optimized system and reaction conditions, with three replicates for each concentration. Ct values ​​were recorded, and the amplification results are shown in Table 3. Figure 3 The results show that the detection limit of this method is 2.1 × 10⁻⁶. 2 The final experimental results showed that the standard deviation (SD) of the Ct values ​​ranged from 0.10 to 1.17, and the relative standard deviation (RSD) ranged from 0.31% to 3.69%. The errors were small and negligible, demonstrating that the established real-time quantitative PCR method has good accuracy in quantifying DNA concentration.

[0080] Table 3. Ct values ​​of standard plasmids at different concentrations

[0081]

[0082] (4) Method specificity detection

[0083] Using the primer and probe set (SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3) for detecting *Lactobacillus plantarum* S58 from Example 1, qPCR amplification was performed on 74 other *Lactobacillus* strains and 16 *Lactobacillus plantarum* intraspecific strains preserved in the laboratory. The amplification results are shown in Table 4. Figure 4 The amplification results of 74 other lactobacilli and 16 *Lactobacillus plantarum* strains were all negative. The Ct values ​​of the six parallel reactions corresponding to *Lactobacillus plantarum* S58 were 20.86, 21.44, 21.09, 21.34, 21.27, and 21.43. This indicates that the primer and probe set designed in this invention for detecting *Lactobacillus plantarum* S58 has good specificity for the detection of strain S58.

[0084] Table 4. Results of specificity verification for Lactobacillus plantarum S58

[0085]

[0086]

[0087]

[0088] Example 3: Quantitative detection of Lactobacillus plantarum S58 in fecal samples using the primer and probe set provided by the present invention.

[0089] Feces were collected from rats after gavage administration of *Lactobacillus plantarum* S58. Fecal DNA was extracted and amplified using the primer and probe set provided in this invention, following the reaction conditions in Example 2. The amplification results are as follows: Figure 5 The Ct values ​​for the three parallel reactions were 26.78, 24.91, and 27.88. The average value was used to calculate the concentration of *Lactobacillus plantarum* S58 in feces as 2.1 × 10⁻⁶. 5.4 =5.27×10 5 copies / uL.

Claims

1. A method for identifying Lactobacillus plantarum S58 ( Lactobacillus plantarum S58 The method, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) The extracted genomic DNA was amplified by PCR using a primer set; the primer set included upstream and downstream primers, and the upstream primer sequence was shown in SEQ ID No. 1: 5'-ACGAGTCGGTTAAGAATC-3'. The downstream primer sequence is shown in SEQ ID No. 2: 5'-GGTTGGAACAATGATAGTG-3'; (3) Detect whether the PCR amplification product contains the target amplification fragment. The sample containing the target amplification fragment was identified as Lactobacillus plantarum S58, and the size of the target amplification fragment was 97 bp. The preservation information for Lactobacillus plantarum S58 is as follows: China Center for Type Culture Collection; Address: Wuhan University, Wuhan, China; Date of deposit: August 1, 2019; Accession number: CCTCC NO: M 2019595; Classification and nomenclature: Lactobacillus plantarum S58 ( Lactobacillus plantarum S58 ).

2. The method according to claim 1, characterized in that: The PCR amplification reaction system is as follows: total volume 25 μl, including: 12.5 μL of 2×Taq PCR Master Mix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 9.5 μL of dd H2O, and 1 μL of DNA; The PCR reaction conditions were: 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 4 min. Agarose gel electrophoresis was used to detect whether the PCR amplification products contained the target band.

3. A real-time fluorescent PCR method for identifying Lactobacillus plantarum S58, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) The extracted genomic DNA was amplified by real-time fluorescent PCR using primer sets and probes. The amplification results were used to determine whether the target was Lactobacillus plantarum S58. If Ct≤36, it was determined to be Lactobacillus plantarum S58. The primer set includes upstream and downstream primers. The upstream primer sequence is shown in SEQ ID No. 1: 5'-ACGAGTCGGTTAAGAATC-3', and the downstream primer sequence is shown in SEQ ID No. 2: 5'-GGTTGGAACAATGATAGTG-3'. The nucleotide sequence of the probe is shown in SEQ ID No. 3: 5'-TCGGACTGATCGCCACCATT-3'. The preservation information for Lactobacillus plantarum S58 is as follows: China Center for Type Culture Collection; Address: Wuhan University, Wuhan, China; Date of deposit: August 1, 2019; Accession number: CCTCC NO: M 2019595; Classification and nomenclature: Lactobacillus plantarum S58 ( Lactobacillus plantarum S58 ).

4. The real-time fluorescence PCR method according to claim 3, characterized in that: The probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

5. A method for detecting Lactobacillus plantarum S58 using real-time quantitative PCR, characterized in that, Includes the following steps: (1) Preparation of standard: The sequence shown in SEQ ID No.4 of the conserved gene fragment of Lactobacillus plantarum S58 is inserted into the cloning vector to obtain a recombinant plasmid containing the conserved gene fragment, which is used as a standard; (2) Plotting the standard curve; (3) Real-time fluorescence PCR amplification was performed using the same reaction system for the test sample and the standard, with the primer set and probe being used; the primer set included upstream and downstream primers, the upstream primer sequence being as shown in SEQ ID No. 1: 5'-ACGAGTCGGTTAAGAATC-3', and the downstream primer sequence being as shown in SEQ ID No. 2: 5'-GGTTGGAACAATGATAGTG-3'; the nucleotide sequence of the probe was as shown in SEQ ID No. 3: 5'-TCGGACTGATCGCCACCATT-3'; (4) Quantitative detection of Lactobacillus plantarum S58 was performed using the standard curve and the Ct value of the sample to be tested; The preservation information for Lactobacillus plantarum S58 is as follows: China Center for Type Culture Collection; Address: Wuhan University, Wuhan, China; Date of deposit: August 1, 2019; Accession number: CCTCC NO: M 2019595; Classification and nomenclature: Lactobacillus plantarum S58 ( Lactobacillus plantarum S58) .

6. The method according to claim 5, characterized in that: The cloning vector is the pGM-T vector.

7. The method according to claim 3 or 5, characterized in that: For real-time fluorescence PCR or quantitative fluorescence PCR detection, the PCR reaction system is 25 μL, including: 12.5 μL of 2×FastFire qPCR PreMix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 0.5 μL of 10 μM probe, 9 μL of dd H2O, and 1 μL of template DNA; the reaction conditions are: 98℃ for 1 min; 98℃ for 5 s, 65.3℃ for 15 s, for 40 cycles.

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