Primer pair for detecting combined lactobacillus salivarius, kit, detection method and application

Abstract

The invention discloses a primer pair for detecting combined lactobacillus salivarius, a kit, a detection method and application, and belongs to the technical field of microbiological detection. In order to solve the problems of low detection efficiency and high cost of the traditional detection method of the combined lactobacillus salivarius in the prior art, the invention provides the primer pair, the kit and the detection method for detecting the combined lactobacillus salivarius, the primer pair and the kit can specifically judge whether the combined lactobacillus salivarius is the combined lactobacillus salivarius, and the detection result is accurate and reliable; the detection method is simple and easy to implement, the fastest detection time of the combined lactobacillus salivarius is only 24 hours, the detection efficiency is improved, and meanwhile, the detection cost is reduced. The primer pair, the kit and the detection method provided by the invention have great application value for judging whether the fermented dairy product or the fermented beverage product is added with the saliva combined lactobacillus or not.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, and in particular relates to a primer pair, kit, detection method and application for detecting Lactobacillus saliva. Background Technology

[0002] Lactobacillus salivans ( Ligilactobacillussalivarius ) is produced by Lactobacillus salivarius ( Lactobacillussalivarius It was renamed; this name change was made after the 2020 recombination of lactic acid bacteria classification, and the original genus was... Lactobacillus spp Sixteen strains with strong host adaptability were reclassified into a new genus, *Lactobacillus assemblica* (…). Ligilactobacillus spp. Lactobacillus salivarius is an important probiotic, and its main biological characteristics are as follows: 1) Morphological characteristics: Gram-positive bacteria, with varying sizes, often appearing as slender rods, but also exhibiting pleomorphism under specific environmental conditions; under an electron microscope, the size is approximately 0.5-1.0 μm × 2.0-10.0 μm, the bacteria are often arranged in chains or palisade, and can also appear singly or in pairs. Some strains have darker staining at both poles, appearing as granules or strips, without spores or capsules, and most are non-motile. 2) Growth characteristics: ① Temperature: The growth temperature range of Lactobacillus salivarius is 30-43℃, with the optimal growth temperature around 37℃; ② pH value: The optimal pH value range is 5.0-5.5; ③ Oxygen: It is suitable for growth under facultative or absolute anaerobic conditions, with better growth in anaerobic environments, but it can also grow in microaerobic environments; 3) Metabolic characteristics: Lactobacillus salivarius can metabolize fructooligosaccharides to produce lactic acid and acetic acid; it produces lactic acid and is acid-resistant, tolerating acid up to pH 2.5; it is resistant to bile salts, tolerating bile salts up to 0.4% for 4 hours; when cultured on MRS (abbreviation of Man, Rogosa and Sharpe) agar medium, its colonies are usually coarse, 1-3 mm in diameter, and white to light gray.

[0003] Furthermore, *Lactobacillus salivarius*, as a probiotic, participates in a series of physiological processes including immunity, nutrition, digestion, and protection, playing an important role in protecting human health. It has been used as a starter or co-starter in fermented milk and fermented beverages. However, whether it is actually added and whether it can survive in fermented dairy products and beverages requires rapid identification by an accredited testing institution with CNAS qualifications. Traditional detection methods for *Lactobacillus salivarius* are time-consuming and laborious, typically requiring 3-5 days for confirmation; moreover, according to the national standard GB4789.34-2016, traditional detection methods require a sample volume of at least 25 g or 25 mL, therefore the sensitivity is usually >4 CFU / g or >4 CFU / mL.

[0004] As can be seen from the above, the traditional detection methods for Lactobacillus by combining saliva in the existing technology have the problems of low detection efficiency and high detection cost. Summary of the Invention

[0005] This invention addresses the problems of low detection efficiency and high cost in traditional detection methods for Lactobacillus salivarius in the prior art by providing a primer pair, kit, detection method, and application for detecting Lactobacillus salivarius.

[0006] One of the objectives of this invention is to provide a primer pair for detecting Lactobacillus salivarius, the primer pair comprising dnaA-L with nucleotide sequences shown in SEQ ID NO.1 and dnaA-R with nucleotide sequences shown in SEQ ID NO.2.

[0007] The second objective of this invention is to provide a kit for detecting Lactobacillus saliva-associated, the kit comprising the aforementioned primer pair.

[0008] In a preferred embodiment of the present invention, the kit further includes 1×PCR reaction buffer, Mg2+, dNTP and Taq DNA polymerase.

[0009] The third objective of this invention is to provide the application of the above primer pairs and kit in the detection of Lactobacillus saliva.

[0010] The fourth objective of this invention is to provide a method for detecting *Lactobacillus saliva*, the method comprising the following steps: S1. Using the genomic DNA of the sample to be tested as a template, perform PCR amplification using the above primer pairs to obtain PCR amplification products; S2. Perform gel electrophoresis on the PCR amplification products obtained in S1, and determine the salivary lactobacilli based on the electrophoresis results.

[0011] In a preferred embodiment of the present invention, the PCR amplification system in S1 is: 1×PCR reaction buffer, 10-15 mmol / L Mg 2+ 0.2-0.3 mmol / L dNTPs, 0.1-0.3 μM primer dnaA-L, 0.1-0.3 μM primer dnaA-R, 0.01-0.1 U / μL Taq DNA polymerase, and 10-100 ng / μL genomic DNA of the sample to be tested.

[0012] In a preferred embodiment of the present invention, the PCR amplification system is: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+0.25 mmol / L dNTP, 0.2 μM primer dnaA-L, 0.2 μM primer dnaA-R, 0.04 U / μL Taq DNA polymerase, 11.6 ng / μL genomic DNA of the sample to be tested.

[0013] In a preferred embodiment of the present invention, the PCR amplification program in S1 is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min, cooling to 12°C, and ending.

[0014] In a preferred embodiment of the present invention, the gel electrophoresis detection in S2 is 1.5%-2.0% agarose gel electrophoresis detection.

[0015] In a preferred embodiment of the present invention, the criterion for determining the result based on the electrophoresis detection in S2 is as follows: if a single amplification product exists at the 338 bp position of the PCR amplification product, it indicates that the sample to be tested contains *Lactobacillus salivarius*; if a single amplification product does not exist at the 338 bp position of the PCR amplification product, it indicates that the sample to be tested does not contain *Lactobacillus salivarius*.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a primer pair, kit, and detection method for detecting *Lactobacillus salivarius*. The primer pair includes dnaA-L with nucleotide sequences shown in SEQ ID NO.1 and dnaA-R with nucleotide sequences shown in SEQ ID NO.2. The provided primer pair and kit can specifically determine whether it is *Lactobacillus salivarius*, and the detection results are accurate and reliable. The provided detection method has a sensitivity of 4.41 pg / PCR, which is high, simple and easy to perform, and the detection time for *Lactobacillus salivarius* is as fast as 24 hours, which not only improves the detection efficiency but also reduces the detection cost.

[0017] This invention provides a primer pair, kit, and detection method for detecting Lactobacillus salivarius, offering a simple, rapid, and sensitive detection method for fermented dairy products or fermented beverages. It has significant application value in determining whether fermented dairy products or fermented beverages contain Lactobacillus salivarius. Attached Figure Description

[0018] Figure 1The image shows the primer results verified by 2.0% agarose gel electrophoresis of the PCR products in Example 1; lanes 1-7 are Lactobacillus salivarius CICC21374, Lactobacillus salivarius M3184, Lactobacillus salivarius M3047, Lactobacillus salivarius M3050, Lactobacillus salivarius M3060, Lactobacillus salivarius M3071, Lactobacillus salivarius M3081, N is the ddH2O negative control sample, and M is the DL2000 bp DNA Marker; Figure 2 This is the PCR product electrophoresis pattern used in Example 2 to verify primer specificity using 2.0% agarose gel electrophoresis; lanes 1-19 are, in order: Staphylococcus aureus M3223, Listeria monocytogenes M3261, Enterococcus faecalis ATCC8459, Salmonella ATCC14028, Cronobacter sakazakii ATCC29544, Bifidobacterium lactis HN019, Bacillus licheniformis M2132, Lactobacillus plantarum CICC6240, and thermophilic acid... Bacillus powdery mildew M3304, Bacillus subtilis ATCC6633, Bacillus thuringiensis CICC21708, Lactobacillus acidophilus CICC6075, Lactobacillus curvaturei M3001, Lactobacillus fructosemide M0462, Lactobacillus brunelli M2214, Lactobacillus salivarius CICC21374, Lactobacillus salivarius M3184, Lactobacillus salivarius M3047, Lactobacillus salivarius M3050; N is the ddH2O negative control sample, M is the DL100 DNA marker; Figure 3 This is the primer sensitivity verification experiment using 2.0% agarose gel electrophoresis of the PCR products in Example 2; lanes 1-7 are 44.1 ng / PCR, 4.41 ng / PCR, 441 pg / PCR, 44.1 pg / PCRL, 4.41 pg / PCR, 441 fg / PCR, and 44.1 fg / PCR, respectively; N is the ddH2O negative control sample; and M is the DL2000 bp DNA Marker. Figure 4 The image shows the PCR products of the eight actual samples in Example 3 after electrophoresis on a 2.0% agarose gel. Lanes 1-8 are, in order: fermented milk sample 1, fermented milk sample 2, fermented milk sample 3, fermented milk sample 4, fermented milk sample 5, fermented milk sample 6, fermented milk sample 7, fermented milk sample 8, N is the ddH2O negative control sample, and M is the 2000bp DNA Marker. Detailed Implementation

[0019] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0021] The following examples use *Lactobacillus salivarius* CICC21374, *Enterococcus faecalis* ATCC8459, *Salmonella* ATCC14028, *Cronobacter sakazakii* ATCC29544, *Lactobacillus plantarum* CICC6240, *Bacillus subtilis* ATCC6633, *Bacillus thuringiensis* CICC21708, and *Lactobacillus acidophilus* CICC6075. These standard strains were purchased from the China Industrial Microbial Culture Collection Center. The *Lactobacillus salivarius* M3184, M3047, M3050, M3060, M3071, M3081, *Staphylococcus aureus* M3223, *Listeria monocytogenes* M3261, *Bifidobacterium lactis* HN019, *Bacillus licheniformis* M2132, *Bacillus thermophilus* M3304, *Lactobacillus curvularis* M3001, *Lactobacillus fructose* M0462, *Lactobacillus brunelli* M2214, and *Lactobacillus salivarius* M3184 isolated strains were purchased from bioMérieux Detection Technologies (Shanghai) Co., Ltd.

[0022] Example 1: A primer pair for detecting Lactobacillus saliva Using the gene sequence shown in SEQ ID NO.3 as a template, a primer pair for detecting Lactobacillus salivarius was designed and synthesized by Shanghai Sangon Biotech Co., Ltd. The primer pair includes dnaA-L with nucleotide sequences shown in SEQ ID NO.1 and dnaA-R with nucleotide sequences shown in SEQ ID NO.2, as shown in Table 1.

[0023] Table 1

[0024] Example 2: A method for detecting Lactobacillus in saliva S1. Using the genomic DNA of *Lactobacillus salivarius* (including *Lactobacillus salivarius* CICC21374, *Lactobacillus salivarius* M3184, *Lactobacillus salivarius* M3047, *Lactobacillus salivarius* M3050, *Lactobacillus salivarius* M3060, *Lactobacillus salivarius* M3071, and *Lactobacillus salivarius* M3081) as a template, PCR amplification was performed using the primer pairs obtained in Example 1 to obtain PCR amplification products; The process of obtaining Lactobacillus genomic DNA from salivary samples includes the following steps: collecting bacterial cells and extracting genomic DNA using the CTAB method. The specific steps are as follows: 1. Collect bacterial cells: Inoculate Lactobacillus saliva into 5 mL of MRS liquid medium and enrich at 37 °C for 2 h. Then, take 1 mL of bacterial suspension and put it into a 1.5 mL centrifuge tube. Centrifuge at 10000 r / min for 10 min to collect bacterial cells. Resuspend the bacterial cells in sterile double-distilled water, centrifuge and wash, and add 460 μL of sterile TE buffer (10 mM) to obtain a bacterial suspension. 2. Genomic DNA extraction using the CTAB method: (1) Add 24 μL of lysozyme to the above bacterial suspension and incubate in a water bath or metal bath at 37°C for 1-2 h; then add 53 μL of 10% SDS and incubate at 68°C for 15 min; then add 87 μL of 5 M NaCl and 69 μL of 1% CTAB and incubate at 68°C for 15 min. (2) Add 250 μL of phenol and 250 μL of chloroform-isoamyl alcohol (24:1, v / v), let stand, and centrifuge at 12000 r / min for 10 min; (3) Take 400-500 μL of supernatant, add an equal volume (400-500 μL) of chloroform-isoamyl alcohol (24:1, v / v), centrifuge at 12000 r / min for 5 min, take 300 μL of supernatant, add 600 μL of anhydrous ethanol, and place at -20℃ for 30 min-120 min. (4) Centrifuge at 10000 r / min for 2 min, discard the supernatant, add 70% ethanol to wash, centrifuge at 12000 r / min for 2 min, discard the supernatant and air dry at room temperature; add 50-100 μL of sterile deionized water or sterile TE (10 mM) solution to dissolve the DNA, store at -20℃ for later use as a template for subsequent PCR reaction system. The PCR amplification system consisted of: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+0.25 mmol / L dNTPs, 0.2 μM primer dnaA-L, 0.2 μM primer dnaA-R, 0.04 U / μL Taq DNA polymerase, 11.6 ng / μL genomic DNA; The PCR amplification program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min, cooling to 12℃, and ending. S2. The PCR amplification products obtained in S1 were detected by agarose gel electrophoresis, and the salivary lactobacilli were identified based on the electrophoresis results.

[0025] Agarose gel electrophoresis results are as follows Figure 1 As shown, the PCR amplification products of *Lactobacillus salivarius* CICC21374, *Lactobacillus salivarius* M3184, *Lactobacillus salivarius* M3047, *Lactobacillus salivarius* M3050, *Lactobacillus salivarius* M3060, *Lactobacillus salivarius* M3071, and *Lactobacillus salivarius* M3081 all exhibit a single amplified band at the 338 bp position.

[0026] Effect Experiment: 1. Specificity evaluation (1) Obtaining the genomic DNA template of the strain Staphylococcus aureus M3223, Listeria monocytogenes M3261, Enterococcus faecalis ATCC8459, Salmonella ATCC14028, Cronobacter sakazakii ATCC29544, Bifidobacterium lactis HN019, Bacillus licheniformis M2132, Lactobacillus plantarum CICC6240, Bacillus thermophilus M3304, Bacillus subtilis ATCC6633, and Bacillus thuringiensis CIC were collected separately. C21708, Lactobacillus acidophilus CICC6075, Lactobacillus curvularis M3001, Lactobacillus fructose-producing bacteria M0462, Lactobacillus brunelli M2214, Lactobacillus salivarius CICC21374, Lactobacillus salivarius M184, Lactobacillus salivarius M3047, and Lactobacillus salivarius M3050, as shown in Table 2, were collected according to the steps in Example 2, and genomic DNA was extracted using the CTAB method.

[0027] (2) PCR detection to determine if it is *Lactobacillus salivarius* Take 2 μL of genomic DNA (DNA concentration of 44.1 ng / μL) of each strain obtained in step (1) as a PCR amplification template, and perform PCR amplification using the primer pair obtained in Example 1 to obtain PCR amplification products of different strains respectively. The PCR amplification system consisted of: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ The reaction mixture consisted of 0.25 mmol / L dNTPs, 0.2 μM primer dnaA-L, 0.2 μM primer dnaA-R, 0.04 U / μL Taq DNA polymerase, and 11.6 ng / μL genomic DNA; sterile deionized water was used as a negative control. The PCR amplification program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min, cooling to 12℃, and ending. The PCR amplification products of the different strains were detected by agarose gel electrophoresis to determine whether a single amplified band was present at the 338 bp position. If a single amplified band was present at the 338 bp position, it was marked as "+", indicating that the tested strain was *Lactobacillus salivarius*; otherwise, it was marked as "-". The agarose gel electrophoresis results are shown in Table 2, and the agarose gel electrophoresis images are shown below. Figure 2 As shown.

[0028] Table 2

[0029] As shown in Table 2, except for the *Lactobacillus salivarius* strain, none of the other negative control strains showed a specific amplification band (338 bp). Among them, one standard strain of *Lactobacillus salivarius* and six isolated strains of *Lactobacillus salivarius* represent typical strains of all species in this genus. At the same time, this invention used *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus curvaturei*, and *Lactobacillus fructosebacterium*, which are closely related to *Lactobacillus salivarius*, as negative control strains. In addition, 10 other isolated strains were used as negative controls in this experiment.

[0030] If the primer pair for detecting *Lactobacillus salivarius* (nucleotide sequences dnaA-L as shown in SEQ ID NO. 1 and dnaA-R as shown in SEQ ID NO. 2) provided by this invention is used to perform PCR amplification on the above-mentioned negative control strain, and no specific fragment of 338 bp is obtained, then other strains more distantly related to *Lactobacillus salivarius* will be even more difficult to amplify this fragment. Therefore, this invention, through specificity verification with closely related strains, ensures the specificity of the primer pair (dnaA-L and dnaA-R) for detecting *Lactobacillus salivarius* provided by this invention, and also fully demonstrates that this method can amplify any strain within the *Lactobacillus salivarius* species, but will not amplify any strains other than those at the *Lactobacillus salivarius* species level.

[0031] As can be seen from the above examples, the strain to be tested is enriched in MRS medium for no more than 18 hours, genomic DNA is extracted by CTAB method for about 2 hours, and PCR detection to determine whether it is Lactobacillus salivae strain takes about 2 hours. Therefore, the method for detecting Lactobacillus salivae of the present invention takes about 24 hours in total, which is significantly shorter than the traditional method and improves the detection efficiency.

[0032] 2. Sensitivity Evaluation (1) Obtaining the genomic DNA template of the strain In Example 2, the bacterial suspension of Lactobacillus salivarii CICC21374 was collected and genomic DNA was extracted using the CTAB method.

[0033] (2) PCR detection to determine if it is *Lactobacillus salivarius* The genomic DNA of Lactobacillus CICC21374 obtained in step (1) was dissolved in sterile water to obtain a genomic DNA concentration of 22.05 ng / uL. Then, it was serially diluted 10-fold with sterile water to a total of 6 gradients: 2.205 ng / uL, 220.5 pg / uL, 22.05 pg / uL, 2.205 pg / uL, 220.5 fg / uL, and 22.05 fg / uL. 2 μL of each gradient was taken as a template and added to the PCR reaction system. PCR amplification was performed using the primer pair obtained in Example 1 to obtain PCR amplification products of different strains. The PCR amplification system consisted of: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ The reaction mixture consisted of 0.25 mmol / L dNTPs, 0.2 μM primer dnaA-L, 0.2 μM primer dnaA-R, 0.04 U / μL Taq DNA polymerase, and 11.6 ng / μL genomic DNA; sterile deionized water was used as a negative control. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min, cooling to 12℃, and then stopping.

[0034] The PCR amplification products were detected by agarose gel electrophoresis. The agarose gel electrophoresis images are shown below. Figure 3 As shown, a clear band (338 bp) is visible in lane 5, corresponding to a DNA concentration of 4.41 pg / PCR, while no amplification bands are observed after lane 6. Therefore, the detection method for Lactobacillus saliva-based strains provided by this invention has a sensitivity of 4.41 pg / PCR, exhibiting high sensitivity.

[0035] Example 3: Detection of the presence of *Lactobacillus salivarius* in food samples Eight fermented milk samples provided by the Dairy Research Institute of Bright Dairy Co., Ltd. were used as test samples. 25 g of each sample was added to 225 mL of sterile physiological saline for dilution. The Lactobacillus saliva was isolated and identified according to the national standard GB 4789.34-2016 (Table 3). The final identification results were compared with those obtained by PCR.

[0036] Table 3

[0037] Note: - (+): indicates that a small number of strains are positive; + (-): indicates that a small number of strains are negative. Meanwhile, 1 mL of the sample to be tested was used to extract genomic DNA using the CTAB method in Example 2, and the total genomic DNA was diluted to 20 ng / uL as a PCR template. Sterile water was used as a negative control. PCR amplification was performed using the primer pair obtained in Example 1, and PCR amplification products of different strains were obtained respectively. The PCR amplification system consisted of: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ The reaction mixture consisted of 0.25 mmol / L dNTPs, 0.2 μM primer dnaA-L, 0.2 μM primer dnaA-R, 0.04 U / μL Taq DNA polymerase, and 11.6 ng / μL genomic DNA; sterile deionized water was used as a negative control. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min, cooling to 12℃, and then stopping.

[0038] The PCR amplification products were detected by agarose gel electrophoresis. The agarose gel electrophoresis images are shown below. Figure 4 As shown, four samples (sample 1-sample 4) were found to contain a specific fragment (338 bp), and these four samples were also isolated and identified as *Lactobacillus salivarius* by the national standard method. This demonstrates that the detection method for *Lactobacillus salivarius* provided by this invention has very high reliability.

[0039] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A primer pair for detecting *Lactobacillus assemblica* in salivary fluid, characterized in that, The primer pair includes dnaA-L with the nucleotide sequence shown in SEQ ID NO.1 and dnaA-R with the nucleotide sequence shown in SEQ ID NO.

2.

2. A kit for detecting *Lactobacillus saliva*, characterized in that, The kit contains the primer pair as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes 1×PCR reaction buffer, Mg 2+ dNTPs and Taq DNA polymerase.

4. The use of the primer pair of claim 1 and the kit of claim 2 in the detection of Lactobacillus salivae.

5. A method for detecting lactobacilli in saliva, characterized in that, The detection method includes the following steps: S1. Using the genomic DNA of the sample to be tested as a template, perform PCR amplification using the primer pair described in claim 1 to obtain PCR amplification products; S2. Perform gel electrophoresis on the PCR amplification products obtained in S1, and determine the salivary lactobacilli based on the electrophoresis results.

6. The detection method according to claim 5, characterized in that, The PCR amplification system described in S1 is: 1×PCR reaction buffer, 10-15 mmol / L Mg 2+ 0.2-0.3 mmol / L dNTPs, 0.1-0.3 μM primer dnaA-L, 0.1-0.3 μM primer dnaA-R, 0.01-0.1 U / μL Taq DNA polymerase, and 10-100 ng / μL genomic DNA of the sample to be tested.

7. The detection method according to claim 6, characterized in that, The PCR amplification system consisted of: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ 0.25 mmol / L dNTPs, 0.2 μM primer dnaA-L, 0.2 μM primer dnaA-R, 0.04 U / μL Taq DNA polymerase, and 11.6 ng / μL genomic DNA of the sample to be tested.

8. The detection method according to claim 5, characterized in that, The PCR amplification program described in S1 is as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, 30 cycles; extension at 72℃ for 10 min, cooling to 12℃, and ending.

9. The detection method according to claim 5, characterized in that, The gel electrophoresis detection described in S2 is 1.5%-2.0% agarose gel electrophoresis.

10. The detection method according to claim 5, characterized in that, The criteria for judgment based on electrophoresis detection results, as described in S2, are as follows: If a single PCR amplification product is present at the 338 bp position, it indicates that the sample contains Lactobacillus salivarii. If no single amplified product is found at the 338 bp position of the PCR amplification product, it indicates that the sample to be tested does not contain Lactobacillus salivarius.

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