Primers for specifically detecting lactobacillus rhamnosus and application thereof

Abstract

The application belongs to the technical field of DNA molecule detection, and relates to a primer probe for specifically detecting and differentially detecting Lactobacillus rhamnosus and application thereof. A primer group for detecting Lactobacillus rhamnosus is characterized in that the primer group comprises four groups of primer pairs, and sequences of the primer pairs are respectively shown in a sequence table of SEQ ID NO. 1 to SEQ ID NO. 8. The technical scheme provided by the application can realize rapid and specific detection of Lactobacillus rhamnosus from complex samples, greatly shortens detection time, and improves detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of DNA molecular detection technology, and relates to a primer for the specific detection and identification of Lactobacillus rhamnosus and its application. Background Technology

[0002] Lactobacillus rhamnosus ( Lactobacillus rhamnosus Lactobacillus rhamnosus is found primarily in the intestines of humans and animals. Taxonomically, it belongs to the genus *Lactobacillus*, subspecies *Rhamnosus*, and is an anaerobic, acid-resistant, non-spore-forming Gram-positive probiotic. After colonizing and multiplying in the human intestinal environment, *Lactobacillus rhamnosus* attaches to the host's intestinal epithelial cells, forming a biological barrier on the intestinal mucosa. This enhances the host's intestinal mucosal barrier function and thus improves the host's digestive system function. Furthermore, *Lactobacillus rhamnosus* is a non-toxic, side-effect-free probiotic. Its main functional characteristics include regulating intestinal flora, preventing and treating diarrhea, facilitating toxin elimination, and enhancing immunity. It has high application value and meets the basic health needs of modern humans. The excellent functional characteristics of *Lactobacillus rhamnosus* allow it to be added to fermented dairy products, demonstrating significant application value and promising development prospects in food science.

[0003] Currently, the detection cycle for Lactobacillus rhamnosus is relatively long, and the accuracy of the detection is not high enough. Existing detection methods lack efficient, sensitive, specific, convenient, and inexpensive methods. Summary of the Invention

[0004] This invention addresses the problems of long experimental cycles and inaccurate detection in traditional Lactobacillus rhamnosus detection methods by proposing a novel primer for the specific detection and identification of Lactobacillus rhamnosus and its application.

[0005] To achieve the above objectives, the present invention is implemented using the following technical solution: ERIC-PCR involves PCR amplification to obtain ERIC fingerprint fragments of the target strain for comparative analysis. ERIC sequences are highly conserved. Primers are designed based on ERIC fragments to construct a rapid and convenient molecular typing platform, enabling rapid detection of target strains in complex genomic environments. ERIC-PCR is currently a widely used molecular biology method for studying the diversity and complexity of gastrointestinal flora structure. The four primer pairs designed in this invention can be used in scientific research, animal husbandry, and other fields to rapidly and efficiently detect the presence of *Lactobacillus rhamnosus*, providing a convenient and rapid method for its detection.

[0006] Whole-genome DNA was extracted from *Lactobacillus rhamnosus*, and four fragments were recovered. The ERIC target fragments were ligated into a T-vector for sequencing. Four pairs of primers were designed and validated, and their specificity, universality, and detection limit were verified. The specific steps are as follows: 1. Extract the whole genome DNA of Lactobacillus rhamnosus.

[0007] 2. The optimal ERIC-PCR amplification conditions for Lactobacillus rhamnosus genomic DNA were found based on a four-factor, three-level orthogonal experiment, and ERIC polymorphism amplification of Lactobacillus rhamnosus genomic DNA was carried out.

[0008] 3. The ERIC gene fragment of Lactobacillus rhamnosus was recovered and purified.

[0009] 4. The gel-recovered fragments were ligated into a T-vector, followed by sequencing and gene alignment analysis.

[0010] 5. Design four pairs of specific primers and verify the designed primers using conventional PCR.

[0011] 6. Perform PCR reaction on the mixed samples using primers, and verify the primer specificity, universality, detection limit and market application potential by conventional PCR.

[0012] As described in step 1, the whole genome DNA of Lactobacillus rhamnosus HP-B1083 was extracted. HP-B1083 cells were picked and inoculated onto MRS plates and cultured anaerobically at 37 ℃ for 2 days. A small amount of bacterial cells was scraped into an EP tube, and 450 μL of 1×TE and 50 mg·mL⁻¹ were added. -1 20 mL of lysozyme, 950 rpm·min -1 Incubate at 37℃ for 12-14 h; add 50 μL of 20% SDS and 5 μL of a 20 mg / mL solution. -1 Proteinase K, 950 rpm·min -1 Shake well for 1 min, incubate in a 55 ℃ water bath for 60 min, removing and gently inverting every 15 min to mix; add 550 μL of a solution of phenol, chloroform and isoamyl alcohol in a ratio of 25:24:1, mix by blowing and aspiration, incubate at 12500 rpm·min -1 Centrifuge for 10 min, transfer the supernatant to another test tube, add 550 μL of a solution of phenol, chloroform and isoamyl alcohol in a ratio of 25:24:1, mix by pipetting, and centrifuge at 12500 rpm·min. -1 Centrifuge for 10 min; take the supernatant and add 800 μL of anhydrous ethanol and 80 μL of 3 mol·L⁻¹. -1 Sodium acetate, let stand at room temperature for 30 min; 12500 rpm·min -1Centrifuge for 10 min, discard the supernatant, add 200 μL of 70% anhydrous ethanol, gently shake to wash salt, centrifuge at 12500 rpm·min. -1 Centrifuge for 5 min, discard ethanol; bake at 37 ℃ for 25 min, add 50 μL 1×TE to dissolve DNA, and store at -20 ℃ for later use.

[0013] As described in step 2, the Lactobacillus rhamnosus ERIC-PCR reaction system is as follows: 1 μL DNA template, 1 μL each of the above primers, 12.5 μL 2×Taq Master Mix, 9.5 μL ddH2O, and a total reaction volume of 25 μL. The optimal reaction program is: 92-98℃ pre-denaturation for 8-12 min, 92-98℃ denaturation for 1 min, 44-46℃ annealing for 35-45 s, 65-78℃ extension for 210 s, for 30-34 cycles, followed by a final extension at 16℃. The DNA template concentration is 100-300 ng / mL. -1 Primer concentration 30-50 μmol·L -1 Mg 2+ Concentration range: 1.75-5.0 mmol·L -1 .

[0014] As described in step 3, agarose gel electrophoresis was performed using 1×TBE as the raw material, stained with ethidium bromide, and electrophoresed on a 0.8% agarose gel at 120V for 25 min.

[0015] As described in step 5, specific primers were designed based on the four ERIC gene fragments. The sequencing results were then assembled using DNAMAN software. The assembled results were imported into Primer Premier 5 and Oligo 7, resulting in the following four pairs of specific primers: 1083-1-dF: 5'-CGTCCTTAGTATCACCCACCAGC-3', 1083-1-dR: 5'-GATGTGAATCAGATTACCGCATTGCCG-3'; 1083-2-aF: 5'-GACAATGGTCAACTGAATATGAGGC-3', 1083-2-aR: 5'-CACCACCATGACGAGCATTG-3'; 1083-3-ERIC-F:5'-GCCTGCTATTGTCGTGGCGATTATAAC-3', 1083-3-ERIC-R: 5'-GTTCACTGTTGCATCGTTGCCAGC-3'; 1083-4-F: 5'-CACTCAATCCATCAGTCAGAATGTGG-3', 1083-4-R: 5'-CAGCTATCAAGCCGTTCATCCATC-3'.

[0016] Analysis by SnapGene shows that the theoretical product lengths of the four primer pairs are 1366bp, 1173bp, 963bp, and 408bp.

[0017] As described in step 5, the designed primers were validated using agarose gel electrophoresis. Primers 1083-1-dF / 1083-1-dR; 1083-2-aF / 1083-2-aR; 1083-3-ERIC-F / 1083-3-ERIC-R; and 1083-4-F / 1083-4-R were used for Lactobacillus rhamnosus PCR. The reaction system consisted of 1 μL DNA template, 1 μL of each of the above primers, 12.5 μL of 2×Taq Master Mix, and 9.5 μL of ddH2O, for a total reaction volume of 25 μL. The optimal reaction program was: 92-98℃ pre-denaturation for 8-12 min, 92-98℃ denaturation for 1 min, 54-56℃ annealing for 35-45 s, and 65-78℃ extension for 120 s, for 30-34 cycles, followed by a final extension at 16℃. It can successfully obtain target gene bands of approximately 2300bp, 1800bp, 1200bp, and 600bp from the genome.

[0018] As described in step 6, the specificity of primers 1083-1-dF / 1083-1-dR; 1083-2-aF / 1083-2-aR; 1083-3-ERIC-F / 1083-3-ERIC-R; and 1083-4-F / 1083-4-R was verified. Eight strains of *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus casei*, *Pediococcus lactis*, *Lactobacillus fermentum*, *Lactobacillus acidophilus*, *Lactobacillus helveticus*, and *Streptococcus thermophilus* were selected. PCR reactions were performed using the four designed primer pairs. The reaction cycle was: pre-denaturation at 92-98℃ for 8-12 min, denaturation at 92-98℃ for 1 min, annealing at 54-56℃ for 35-45 s, extension at 65-78℃ for 120 s, for 30-34 cycles, followed by a final extension at 16℃. 5 μL of each PCR product was then used for conventional PCR verification. The results showed that the primers 1083-1-dF / 1083-1-dR; 1083-2-aF / 1083-2-aR; 1083-3-ERIC-F / 1083-3-ERIC-R; and 1083-4-F / 1083-4-R disclosed in this patent could specifically identify Lactobacillus rhamnosus HP-B1083.

[0019] As described in step 6, to verify the primer universality, Lactobacillus rhamnosus HP-B1083 was selected. Lacticaseibacillus rhamnosus ATCC 11443, Lactobacillus rhamnosus DSM 14870, Lactobacillus rhamnosus ATCC 8530, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei DSM 20011, Lactobacillus paracasei ATCC 334; Genomic DNA was extracted from the above six strains, and PCR verification was performed using the designed primers. The reaction system was as follows: pre-denaturation at 92-98℃ for 8-12 min, denaturation at 92-98℃ for 1 min, annealing at 54-56℃ for 35-45 s, extension at 65-78℃ for 120 s, for 30-34 cycles, followed by a final extension at 16℃. 5 μL of each PCR product was then subjected to agarose gel electrophoresis for verification. The results demonstrate that the four primer pairs involved in this patent can detect all of the above-mentioned types of *Lactobacillus rhamnosus*, exhibiting universality.

[0020] As described in step 6, the detection limit of the primers was tested. Eight bacteria, namely *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus casei*, *Pediococcus lactis*, *Lactobacillus fermentum*, *Lactobacillus acidophilus*, *Lactobacillus helveticus*, and *Streptococcus thermophilus*, were selected and subjected to PCR reactions using four pairs of primers. The genomic DNA extracted from the eight strains was mixed in equal amounts to prepare a *Lactobacillus rhamnosus* HP-B1083 genomic DNA solution with a content of 100% (absolute content 900 ng·mL). -1 ), 70% (absolute content 630 ng·mL) -1 ), 30% (absolute content 270 ng·mL) -1 ), 3% (absolute content 27 ng·mL) -1 0% (absolute content 0 ng·mL) -1 A solution containing 3% (absolute concentration 27 ng / mL) was used. Equal volumes of the product were then subjected to agarose gel electrophoresis for verification. The results showed that when the sample contained 3% (absolute concentration 27 ng / mL)... -1 Lactobacillus rhamnosus can be detected by examining the genomic DNA of Lactobacillus rhamnosus.

[0021] As described in step 6, to test the market application potential of the primers, three ready-to-eat probiotic products on the market that claim to contain Lactobacillus rhamnosus were selected. Genomic DNA of the samples was extracted, and PCR verification was performed using the four pairs of primers designed. Agarose gel electrophoresis was used to identify whether the commercially available products contained Lactobacillus rhamnosus.

[0022] Compared with the prior art, the advantages and positive effects of the present invention are as follows: The technical solution provided by this invention can quickly and specifically detect Lactobacillus rhamnosus from complex samples, greatly shortening the detection time and improving the detection efficiency. Attached Figure Description

[0023] Figure 1 This diagram shows the optimal reaction system for ERIC-PCR.

[0024] Figure 2 Four agarose gel electrophoresis images of the recovered and purified genomic DNA target fragments, ranging from approximately 600bp to 2300bp. M: 2K Plus II DNA marker; 1-4: the four fragments recovered from the gel.

[0025] Figure 3 To verify the PCR results using the four designed specific primer pairs with HP-B1083 genomic DNA as a template, agarose gel images were generated. Figure A shows the PCR results using primers 1083-1-dF / 1083-1-dR; Figure B shows the PCR results using primers 1083-2-aF / 1083-2-aR; Figure C shows the PCR results using primers 1083-3-ERIC-F / 1083-3-ERIC-R; and Figure D shows the PCR results using primers 1083-4-F / 1083-4-R.

[0026] Figure 4 Agarose gel electrophoresis images used to verify primer specificity. M: 2K Plus II DNA marker. Phases 1-4, 5-8, 9-12, 13-16, 17-20, 21-24, 25-28, and 29-32 used genomic DNA from *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus casei*, *Pediococcus lactis*, *Lactobacillus fermentum*, *Lactobacillus acidophilus*, *Lactobacillus helveticus*, and *Streptococcus thermophilus* as templates, respectively. Primers used for phases 1, 5, 9, 13, 17, 21, 25, and 29 were 1083-1-dF / 1083-1-dR; primers used for phases 2, 6, 10, 14, 18, 22, 26, and 30 were 1083-2-aF / 1083-2-aR; and primers used for phases 3, 7, 11, 15, 19, 23, 27, and 31 were 1083-3-ERIC-F. The primers used for numbers 4, 8, 12, 16, 20, 24, 28, and 32 are 1083-4-F / 1083-4-R.

[0027] Figure 5 Agarose gel electrophoresis image to test primer universality. M: 2K Plus II DNA marker, lanes 1-4, 5-8, 9-12, 13-16, 17-20, 21-24, 25-28 are respectively filled with Lactobacillus rhamnosus HP-B1083, Lacticaseibacillus rhamnosus ATCC 11443, Lactobacillus rhamnosusDSM 14870, Lactobacillus rhamnosus ATCC 8530, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei DSM 20011, Lactobacillus paracasei ATCC 334 genomic DNA was used as a template. Primers used for primers 1, 5, 9, 13, 17, 21, and 25 were 1083-1-dF / 1083-1-dR; primers used for primers 2, 6, 10, 14, 18, 22, and 26 were 1083-2-aF / 1083-2-aR; primers used for primers 3, 7, 11, 15, 19, 23, and 27 were 1083-3-ERIC-F / 1083-3-ERIC-R; and primers used for primers 4, 8, 12, 16, 20, 24, and 28 were 1083-4-F / 1083-4-R.

[0028] Figure 6 Agarose gel electrophoresis image to verify the detection limit of primers for Lactobacillus rhamnosus concentration. M: 2K Plus II DNA marker; lanes 1-4 contain 100% Lactobacillus rhamnosus HP-B1083 genomic DNA (absolute concentration 900 ng / mL). -1 The verification showed that lanes 5-8 contained 70% genomic DNA of Lactobacillus rhamnosus HP-B1083 (absolute content 630 ng·mL). -1 Lanes 9-12 contain 30% genomic DNA of Lactobacillus rhamnosus HP-B1083 (absolute content 270 ng·mL). -1 Lanes 13-16 contain 3% (absolute content 27 ng / mL) genomic DNA of Lactobacillus rhamnosus HP-B1083. -1 Lanes 17-20 contained 0% genomic DNA of Lactobacillus rhamnosus HP-B1083 (absolute content 0 ng / mL). -1 The primers used for 1, 5, 9, 13, and 17 are 1083-1-dF / 1083-1-dR; the primers used for 2, 6, 10, 14, and 18 are 1083-2-aF / 1083-2-aR; the primers used for 3, 7, 11, 15, and 19 are 1083-3-ERIC-F / 1083-3-ERIC-R; and the primers used for 4, 8, 12, 16, and 20 are 1083-4-F / 1083-4-R.

[0029] Figure 7To verify the detection of *Lactobacillus rhamnosus* in three commercially available probiotic products using designed primers, agarose gel electrophoresis images were used. Lanes 1-4, 5-8, and 9-12 used genomic DNA from commercially available mixed probiotic products 1, 2, and 3 as templates, respectively. The primers used in lanes 1, 5, and 9 were 1083-1-dF / 1083-1-dR; lanes 2, 6, and 10 used 1083-2-aF / 1083-2-aR; lanes 3, 7, and 11 used 1083-3-ERIC-F / 1083-3-ERIC-R; and lanes 4, 8, and 12 used 1083-4-F / 1083-4-R. Detailed Implementation

[0030] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0031] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification. Example 1

[0032] This embodiment provides the extraction of genomic DNA from Lactobacillus rhamnosus HP-B1083.

[0033] Lactobacillus rhamnosus HP-B1083 was picked and inoculated onto an MRS plate and cultured anaerobically at 37°C for 2 days. A small amount of bacterial cells was scraped into an EP tube, and 450 μL of 1×TE and 50 mg·mL⁻¹ were added. -1 20 mL of lysozyme, 950 rpm·min -1 Incubate at 37℃ for 12-14 h; add 50 μL of 20% SDS and 5 μL of a 20 mg / mL solution. -1 Proteinase K, 950 rpm·min -1 Shake well for 1 min, incubate in a 55℃ water bath for 60 min, removing and gently shaking every 15 min; add 550 μL of a solution of phenol, chloroform and isoamyl alcohol in a ratio of 25:24:1, mix by blowing and aspiration, and incubate at 12500 rpm·min. -1 Centrifuge for 10 min, transfer the supernatant to another test tube, add 550 μL of a solution of phenol, chloroform and isoamyl alcohol in a ratio of 25:24:1, mix by pipetting, and centrifuge at 12500 rpm·min. -1 Centrifuge for 10 min; take the supernatant and add 800 μL of anhydrous ethanol and 80 μL of 3 mol·L⁻¹.-1 Sodium acetate, let stand at room temperature for 30 min; 12500 rpm·min -1 Centrifuge for 10 min, discard the supernatant, add 200 μL of 70% anhydrous ethanol, gently shake to wash salt, centrifuge at 12500 rpm·min. -1 Centrifuge for 5 min, discard ethanol; dry at 37 ℃ for 25 min, add 50 μL 1×TE to dissolve DNA, and store at -20 ℃ for later use. Example 2

[0034] The ERIC-PCR reaction conditions for Lactobacillus rhamnosus HP-B1083 were optimized using a four-factor, three-level orthogonal experiment.

[0035] A four-factor, three-level orthogonal experiment was conducted to find the optimal conditions. By changing the annealing temperature, DNA template concentration, primer concentration, and Mg content of the ERIC-PCR reaction... 2+ Concentration conditions were determined, and experimental results under different conditions were compared to select the optimal reaction conditions. The orthogonal experiment is shown in the four-factor, three-level orthogonal table (Table 1).

[0036] Table 1 Orthogonal Experiment L9(3) 4 Factor Level Table

[0037] Determine the optimal reaction procedure for ERIC-PCR The novel ERIC-PCR degenerate primers were synthesized by Suzhou Genewiz Co., Ltd. Upstream primer: 1083-3-ERIC-F: 5'-ATGTMAGCBCCTGKGGATTCDC-3'; Downstream primer: 1083-3-ERIC-R: 5'-AASTAAGTGACHGGGGTGAYCG-3'.

[0038] Amplification system: 1 μl DNA template, 1 μl each of the above primers, 12.5 μl 2×Taq Master Mix, 9.5 μl ddH2O, total reaction volume 25 μl. DNA template concentration: 100-300 ng / mL. -1 Primer concentration 30-50 μmol·L -1 Mg 2+ Concentration range: 1.75-5.0 mmol·L -1 .

[0039] The optimal reaction program was obtained through the orthogonal experiment in Example 2(1): 92-98℃ pre-denaturation for 8-12 min, 92-98℃ denaturation for 1 min, 44-46℃ annealing for 35-45 s, 65-78℃ extension for 210 s, for 30-34 cycles, followed by extension at 16℃. Verification results are shown in [link to verification results]. Figure 1 . Figure 1 This is a diagram validating the optimal reaction system for ERIC-PCR. The results show the ERIC-PCR validation using 1×TBE as the starting material, ethidium bromide staining, and 0.8% agarose gel electrophoresis at 120 V for 25 min. M: 2K Plus II DNA marker. Lane 1: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 1 min, 42℃ annealing for 40 s, 72℃ extension for 1 min, for 31 cycles; Lane 2: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 1 min, 42℃ annealing for 40 s, 72℃ extension for 2 min, for 31 cycles; Lane 3: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 1 min, 42℃ annealing for 40 s, 72℃ extension for 3 min, for 31 cycles; Lane 4: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 1 min, 45℃ annealing for 40 s, 72℃ extension for 1 min, for 31 cycles; Lane 5: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 1 min, 45℃ annealing for 40 s, 72℃ extension for 2 min, for 31 cycles; Lane 6: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 1 min, 45℃ annealing for 40 s, 72℃ extension for 2 min, for 31 cycles; Lane 1: Pre-denaturation at 95℃ for 1 min, denaturation at 45℃ for 40 s, extension at 72℃ for 3 min, for 31 cycles; Lane 2: Pre-denaturation at 95℃ for 1 min, denaturation at 95℃ for 1 min, annealing at 50℃ for 40 s, extension at 72℃ for 1 min, for 31 cycles; Lane 3: Pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 1 min, annealing at 50℃ for 40 s, extension at 72℃ for 2 min, for 31 cycles; Lane 4: Pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 1 min, annealing at 50℃ for 40 s, extension at 72℃ for 3 min, for 31 cycles. Figure 1 It can be seen that the ERIC-PCR reaction system in lane 4 is the optimal condition for HP-B1083 Lactobacillus rhamnosus genome; the reaction system is: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 1 min, 45℃ annealing for 40 s, 72℃ extension for 1 min, for 31 cycles. Example 3

[0040] Recovery of genomic DNA fragments after ERIC-PCR reaction.

[0041] After ERIC-PCR amplification, the sample was stained with ethidium bromide using 1×TBE and electrophoresed on a 0.8% agarose gel at 120 V for 25 min. Following the agarose gel DNA extraction kit, two single-target DNA bands were excised and added to a 1-volume buffer PG solution. The gel was incubated at 50°C until completely dissolved (the centrifuge tube was gently inverted every 2-3 minutes until the solution turned yellow, ensuring complete dissolution of the gel). The solution was then transferred to an adsorption column equilibrated with 200 μL of buffer PS and incubated at room temperature for two minutes, followed by electrophoresis at 12500 rpm·min. -1 Centrifuge for 1 min, discard the waste liquid in the collection tube; add 450 μL of Buffer PW containing anhydrous ethanol to the adsorption column, and centrifuge at 12500 rpm·min. -1 Centrifuge for 1 min, discard the waste liquid; add 450 μL of Buffer PW containing anhydrous ethanol, centrifuge at 12500 rpm·min. -1 Centrifuge for 1 min and discard the waste liquid; place the adsorption column into a new EP tube and dry at 55℃ for 20 min; after removing it, place the adsorption column into a new 1.5 ml EP tube, add 30 μL of sterilized double-distilled water dropwise to the center of the adsorption membrane, let stand for 2 min, and then centrifuge at 12500 rpm·min. -1 Centrifuge for 1 min; mix 3 μL of DNA solution with 2 μL of Loading Buffer, and perform 0.8% agarose gel electrophoresis for verification. Store at -20℃. Fragments of approximately 600bp, 1200bp, 1800bp, and 2300bp have been successfully recovered by gel electrophoresis. See [link to details]. Figure 2 . Example 4

[0042] The ERIC fragment was ligated into a T vector for sequencing.

[0043] Preparation of competent cells Pick a small amount of Escherichia coli GB05-dir and incubate overnight in liquid LB medium; add 40 μL of culture medium to a 1.5 mL EP tube containing 1.3 mL liquid LB medium, and incubate at 30℃ and 950 rpm·min. -1 Cultured for 2 h; then induced for 45 min with 35 μL arabinose at 9300 rpm·min. -1 Centrifuge for 30 seconds, discard the supernatant; add 1 mL of double-distilled water, centrifuge at 9700 rpm·min -1 Centrifuge for 30 seconds; add 1 mL of double-distilled water again, centrifuge at 11000 rpm·min. -1 Centrifuge for 30 seconds, discard the supernatant, and keep about 50 μL of liquid.

[0044] Connecting T-carrier Add 1 μL of the gel-recovered target fragment and 1 μL of the T vector to competent cells, mix by pipetting and aspiration, and incubate for 6 min; electrolyze at 1350 V, incubate for 6 min; wash out the cells with LB liquid, and recover at 37℃ and 950 rpm for 1 h; electrolyze at 8000 rpm·min -1 Centrifuge for 1 min; retain approximately 200 μL of the liquid and spread it onto a kanamycin-resistant LB agar plate, then incubate overnight at 37°C inverted. T vector model: T5 Zero Cloning Kit (Beijing TransGen Biotech Co., Ltd.).

[0045] Submit for testing Single colonies from kanamycin-resistant LB agar plates were transferred to kanamycin-resistant liquid LB medium and incubated at 37°C and 950 rpm·min. -1 The culture was incubated for 2 hours; the bacterial culture was then sent to Genewiz for gene sequencing using T7 / T7-Term universal primers. Database comparisons showed that the sequence is highly conserved in *Lactobacillus rhamnosus* and can be used as a template for specific molecular design. The sequence is listed in the nucleic acid sequence listing. Example 5

[0046] Four pairs of specific primers were designed based on the sequencing results.

[0047] The target ERIC gene fragment from *Lactobacillus rhamnosus* HP-B1083 was recovered, and four fragments were successfully recovered and sequenced (see SEQUENCE LISTING). Based on the sequencing results from Genewiz Suzhou, primers were designed using Primer Premier 5 and Oligo 7 software, resulting in the following four pairs of specific primer sequences: 1083-1-dF: 5'-CGTCCTTAGTATCACCCACCAGC-3', 1083-1-dR: 5'-GATGTGAATCAGATTACCGCATTGCCG-3'; 1083-2-aF: 5'-GACAATGGTCAACTGAATATGAGGC-3', 1083-2-aR: 5'-CACCACCATGACGAGCATTG-3'; 1083-3-ERIC-F:5'-GCCTGCTATTGTCGTGGCGATTATAAC-3', 1083-3-ERIC-R: 5'-GTTCACTGTTGCATCGTTGCCAGC-3'; 1083-4-F: 5'-CACTCAATCCATCAGTCAGAATGTGG-3', 1083-4-R: 5'-CAGCTATCAAGCCGTTCATCCATC-3'. Example 6

[0048] Design and testing of four primer pairs for Lactobacillus rhamnosus HP-B1083 The obtained four primer pairs were used for PCR verification. The reaction system was as follows: 92-98℃ pre-denaturation for 8-12 min, 92-98℃ denaturation for 1 min, 54-56℃ annealing for 50-60 s, 65-78℃ extension for 120 s, for 30-34 cycles, followed by a final extension at 16℃. 5 μl of the PCR product was then subjected to agarose gel electrophoresis for verification. Finally, the amplification product using *Lactobacillus rhamnosus* HP-B1083 genomic DNA as a template was successfully obtained using the four primer pairs. (See [link to relevant documentation]). Figure 3 . Example 7

[0049] Specificity test of Lactobacillus rhamnosus HP-B1083 primers Eight commonly added probiotic strains from the "List of Microbial Strains that Can Be Used in Food" were selected: *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus casei*, *Pediococcus lactis*, *Lactobacillus fermentum*, *Lactobacillus acidophilus*, *Lactobacillus helveticus*, and *Streptococcus thermophilus*. PCR verification was performed on these eight strains using four pairs of designed primers. The reaction system was as follows: pre-denaturation at 92-98℃ for 8-12 min, denaturation at 92-98℃ for 1 min, annealing at 54-56℃ for 35-45 s, extension at 65-78℃ for 120 s, for 30-34 cycles, followed by a final extension at 16℃. 5 μl of each PCR product was then subjected to agarose gel electrophoresis for verification. See [link to details]. Figure 4 ,from Figure 4 It can be seen that the four primer pairs have good selectivity for Lactobacillus rhamnosus. Example 8

[0050] Universality test of Lactobacillus rhamnosus HP-B1083 primers Extracts were taken from Lactobacillus rhamnosus HP-B1083, Lacticaseibacillus rhamnosus ATCC 11443, Lactobacillus rhamnosus DSM 14870, Lactobacillus rhamnosus ATCC 8530, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei DSM 20011, Lactobacillus paracaseiATCC 334 genomic DNA was used for PCR validation using four pairs of designed primers. The reaction system was as follows: 92-98℃ pre-denaturation for 8-12 min, 92-98℃ denaturation for 1 min, 54-56℃ annealing for 35-45 s, 65-78℃ extension for 120 s, for 30-34 cycles, followed by a final extension at 16℃. 5 μL of each product was then subjected to agarose gel electrophoresis for validation. (See attached image) Figure 5 ,from Figure 5 It can be seen that the four primer pairs have universality in detecting Lactobacillus rhamnosus. Example 9

[0051] Lactobacillus rhamnosus HP-B1083 primer test for genomic DNA content limits Genomic DNA was extracted from *Lactobacillus rhamnosus* HP-B1083, *Bifidobacterium animalis* HP-B1124, *Lactobacillus casei* HP-B1142, *Pediococcus lactis* HP-B1099, *Lactobacillus fermentum* HP-B1153, *Lactobacillus acidophilus* HP-B1079, *Lactobacillus helveticus* HP-B1126, and *Streptococcus thermophilus* HP-B1134, and PCR was performed on each strain. Equal volumes of the extracted genomic DNA were mixed to prepare a 100% (absolute concentration 900 ng / mL) *Lactobacillus rhamnosus* HP-B1083 genomic DNA concentrator. -1 ), 70% (absolute content 630 ng·mL) -1 ), 30% (absolute content 270 ng·mL) -1 0% (absolute content 0 ng·mL) -1 A solution containing 100% Lactobacillus rhamnosus (absolute concentration 900 ng / mL) was prepared. -1 ), 70% (absolute content 630 ng·mL) -1 ), 30% (absolute content 270 ng·mL) -1 ), 3% (absolute content 27 ng·mL) -1 0% (absolute content 0 ng·mL) -1 5 μl of each solution was used for agarose gel electrophoresis verification, see [link to relevant documentation]. Figure 6 ,from Figure 6 It can be seen that the four primer pairs can specifically detect Lactobacillus rhamnosus in the presence of trace amounts of DNA. Example 10

[0052] Four pairs of primers were used to detect commercially available probiotic products that claimed to contain Lactobacillus rhamnosus. Three commercially available ready-to-eat probiotic products claiming to contain *Lactobacillus rhamnosus* were selected. Genomic DNA was extracted from the samples, and PCR verification was performed using four pairs of designed primers. The reaction system was as follows: 92-98℃ pre-denaturation for 8-12 min, 92-98℃ denaturation for 1 min, 54-56℃ annealing for 35-45 s, 65-78℃ extension for 120 s, for 30-34 cycles, followed by a final extension at 16℃. 5 μl of each PCR product was then subjected to agarose gel electrophoresis for verification. (See attached image) Figure 7 ,from Figure 7 The results show that the four primer pairs can specifically detect Lactobacillus rhamnosus in commercially available probiotic products, indicating a promising market application prospect.

[0053] Results Analysis: The four specific primers disclosed in this patent overcome the shortcomings of traditional detection methods, such as complex operation, large errors, and long processing times. Based on ERIC-PCR and gene alignment technology, this invention successfully developed four pairs of specific primers, enabling rapid, accurate, and specific detection of *Lactobacillus rhamnosus* in complex samples. These developed primers are specific, universal, and capable of detecting samples with extremely low concentrations of *Lactobacillus rhamnosus*. They can rapidly detect the presence of *Lactobacillus rhamnosus* in products, and have broad market and application prospects in pharmaceuticals, animal husbandry, and consumer goods.

[0054] Based on the foregoing description, those skilled in the art can make modifications and alterations to the above embodiments. Therefore, this invention is not limited to the specific embodiments disclosed and described above, and some modifications and alterations to this invention should also fall within the protection scope of the claims. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on this invention.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A primer set for detecting Lactobacillus rhamnosus, characterized in that, It contains four primer pairs, the sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.8 of the sequence listing, and the sequences of SEQ ID NO.1 to SEQ ID NO.8 are as follows: 1083-1-dF: 5'-CGTCCTTAGTATCACCCACCAGC-3'; 1083-1-dR: 5'-GATGTGAATCAGATTACCGCATTGCCG-3'; 1083-2-aF: 5'-GACAATGGTCAACTGAATATGAGGC-3'; 1083-2-aR: 5'-CACCACCATGACGAGCATTG-3'; 1083-3-ERIC-F: 5'-GCCTGCTATTGTCGTGGCGATTATAAC-3'; 1083-3-ERIC-R: 5'-GTTCACTGTTGCATCGTTGCCAGC-3'; 1083-4-F: 5'-CACTCAATCCATCAGTCAGAATGTGG-3'; 1083-4-R: 5'-CAGCTATCAAGCCGTTCATCCATC-3'.

2. A kit for detecting Lactobacillus rhamnosus, characterized in that, It includes the primer set as described in claim 1.

3. The use of the primer set described in claim 1 in the preparation of Lactobacillus rhamnosus detection reagents or kits.

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