Specific identification primer and probe for phytobacterium plantarum in human intestinal tract and application of specific identification primer and probe

Abstract

The invention discloses a group of primers for identifying the specificity of plant lactobacillus in human intestinal tracts. The group of primers comprises an upstream primer COG3419F1 and a downstream primer COG3419R1, the nucleotide sequence of the upstream primer COG3419F1 is 5 '-CGCCACAGCAAGTAACCAT-3', and the nucleotide sequence of the downstream primer COG3419F1 is 5 '-CGCCACAGCAAGTAACCAT-3 The nucleotide sequence of the downstream primer COG3419R1 is 5 '-GACCATACCCAAGTGCCAT-3', and the nucleotide sequence of the downstream primer COG3419R1 is 5 '-GACCATACCCAAGTGCCAT-3 The probe is a sequence designed aiming at the specific COG3419 gene segment of the plant lactobacillus; the specific COG3419 gene of the phytobacterium plantarum is screened as a target sequence, the primer and the probe with high specificity and sensitivity are designed, the phytobacterium plantarum and other intestinal bacteria can be accurately distinguished under the background of complex microorganisms of the human intestinal tract, and rapid qualitative detection and accurate quantitative analysis of the phytobacterium plantarum are achieved; the primer, the probe and the detection method can also be extensively applied to detection of the phytobacterium plantarum in various samples such as food, environment and the like.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to specific identification primers, probes and their applications for Lactobacillus plantarum in the human gut. Background Technology

[0002] Lactobacillus plantarum (formerly known as Lactobacillus plantarum) is a Gram-positive lactic acid bacterium widely distributed in nature, belonging to the family Lactobacillusaceae. It has an extremely wide range of habitats, mainly found in various fermented plant-based foods (such as kimchi, sauerkraut, fermented soy products, etc.), dairy products, animal intestines, and certain fermented meat products.

[0003] *Lactobacillus plantarum* possesses excellent environmental adaptability, growing over a wide pH and temperature range and tolerating certain salinity and gastrointestinal environments, making it an important probiotic. This strain can regulate the intestinal microecological balance, promote the proliferation of beneficial bacteria, inhibit the growth of harmful bacteria, and help maintain intestinal health. It can also enhance host immunity, participate in antioxidant reactions, lower cholesterol levels, improve lactose intolerance symptoms, prevent and alleviate diarrhea, and control some allergic reactions. Some strains can even produce substances with antibacterial activity (such as phytosin), used for food preservation and safety assurance. Furthermore, *Lactobacillus plantarum* has strong fermentation capabilities, efficiently fermenting various carbohydrates and producing significant amounts of lactic acid, thereby significantly improving the flavor and quality of food. Due to its high safety, low toxicity, and strong tolerability, it has been recognized as a "safe strain" by international food safety organizations (such as EFSA and FDA) and is widely used in functional foods and animal feed additives.

[0004] To date, there are nearly 30 patents related to primers and probes for *Lactobacillus plantarum*. These patents mainly fall into two categories: one is the identification and detection of *Lactobacillus plantarum* in dairy products, liquor brewing processes, and feed. These products contain a limited number of lactobacillus species, making targeted detection of *Lactobacillus plantarum* relatively simple. For example, patent CN105331723A discloses a rapid qualitative and quantitative detection kit, method, and application for *Lactobacillus plantarum* added to feed, specifically verifying its effectiveness against *Lactobacillus acidophilus*, *Lactobacillus casei*, and other bacteria that may be added to feed. Patent CN101712987B proposes a rapid qualitative and quantitative determination method for *Lactobacillus plantarum* in probiotic dairy products, designing species-specific primers based on the 16S rRNA gene sequence. The other category focuses on molecular detection of targeted sequences of specific strains of *Lactobacillus plantarum*. These strains often have well-defined functions and are already used in production. For example, patent CN117512152A proposes a rapid detection method for *Lactobacillus plantarum* Liang... The molecular marker combination, primer combination and detection method are disclosed in patent CN102533790B, which discloses a PCR detection method for detecting Lactobacillus plantarum ST-III, as well as its primers and kit.

[0005] However, the human gut microbiota is diverse and complex, and existing detection methods cannot accurately distinguish *Lactobacillus plantarum* from other gut bacteria at the species level, nor can they simultaneously achieve the characteristics of speed, sensitivity, and quantification. Therefore, developing a specific detection method for *Lactobacillus plantarum* suitable for the complex microbial background of the human gut has significant practical importance and application value. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a set of specific identification primers and probes for *Lactobacillus plantarum* in the human gut, as well as detection methods and applications based on these primers and probes, to achieve rapid, accurate, and quantitative detection of *Lactobacillus plantarum*.

[0007] The objective of this invention is achieved as follows: a set of primers for the specific identification of Lactobacillus plantarum in the human gut, characterized in that they include an upstream primer COG3419F1 and a downstream primer COG3419R1;

[0008] The nucleotide sequence of the upstream primer COG3419F1 is: 5'-CGCCACAGCAAGTAACCAT-3';

[0009] The nucleotide sequence of the downstream primer COG3419R1 is: 5'-GACCATACCCAAGTGCCAT-3'.

[0010] The probe sequence is: 5'-TCACACCCAATTACAGCGCC-3', wherein the 5' end is labeled with the FAM fluorescent group and the 3' end is labeled with the BHQ1 quencher group.

[0011] The present invention also provides a kit containing the above primers and / or probes, and its application in the qualitative and quantitative detection of Lactobacillus plantarum.

[0012] As a further preferred embodiment of the present invention, the primers are used in the detection of *Lactobacillus plantarum*, wherein the detection includes qualitative and / or quantitative detection; and the samples to be detected include human intestinal samples, food samples, or environmental samples.

[0013] As a further preferred embodiment of the present invention, the probe is used in the quantitative detection of *Lactobacillus plantarum*, wherein the quantitative detection adopts the real-time fluorescence quantitative qPCR method; the samples to be detected include human intestinal samples, food samples, or environmental samples.

[0014] A qualitative detection method for *Lactobacillus plantarum* includes the following steps:

[0015] (1) Extract bacterial DNA from the sample to be tested;

[0016] (2) Using the DNA extracted in step (1) as a template, PCR amplification was performed using the primers described in claim 1; the PCR reaction system was as follows: Taq mix 12.5 μL, upstream primer COG3419F1 1 μL, downstream primer COG3419R1 1 μL, DNA template 1 μL, double-distilled water 9.5 μL, and total system 25 μL;

[0017] The PCR reaction procedure is as follows: pre-denaturation at 95℃ for 5 min; followed by 30-35 cycles, each cycle including denaturation at 95℃ for 10-30 s, annealing at 63℃ for 30 s, extension at 72℃ for 15-25 s; after the cycle, extension at 72℃ for 5 min, and cooling to 12℃ to finish.

[0018] (3) The PCR products were detected by 1% agarose gel electrophoresis. If a single amplified band appeared at the 162bp position, the sample to be tested was determined to contain Lactobacillus plantarum; otherwise, it was not.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention screens out the COG3419 gene, which is unique to Lactobacillus plantarum, as a target sequence. This gene is present in 10 strains of Lactobacillus plantarum and has a highly consistent sequence, while it is not present in 50 other Lactobacillus species, thus ensuring the specificity of the detection.

[0020] Primers and probes designed based on the COG3419 gene, verified by PCR and qPCR, can specifically amplify the target sequence of *Lactobacillus plantarum* without cross-reactivity with other lactobacilli and non-lactobacilli, demonstrating high detection specificity.

[0021] The detection method of the present invention is simple and rapid to operate. Qualitative detection can be completed by conventional PCR combined with gel electrophoresis, and quantitative detection can be achieved by qPCR. It has high detection sensitivity and accurate and reliable results.

[0022] The primers, probes, and detection methods of this invention have a wide range of applications. They can be used not only for the detection of *Lactobacillus plantarum* in human intestinal samples, but also for various other samples such as food and the environment. This provides a powerful tool for research on *Lactobacillus plantarum*, the development of functional foods, intestinal health assessment, and quality control. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the technical description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The gel electrophoresis image (COG3419F1 / COG3419R1) was validated using specific primers.

[0025] Figure 2 This is a qPCR amplification curve (parallel sample) of Lactobacillus plantarum.

[0026] Figure 3 This is the qPCR melting curve of Lactobacillus plantarum (parallel samples). Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0028] Example 1: Validation of Specific Primers

[0029] Validation strain:

[0030] Thirteen lactobacilli, five non-lactobacilli, and three *Lactobacillus plantarum* strains were selected as validation strains, as detailed below:

[0031] Thirteen types of lactobacilli: *Ligilactobacillus salivarius* 27-2, *Lactaseibacillus rhamnosus* 16-1, *Lactobacillus crispatus* 16-3, *Limosilactobacillus fermentum* 27-3, *Limosilactobacillus vaginalis* 4-9, *Lactaseibacillus casei* 6-5, *Limosilactobacillus reuteri* 16-5, *Latilactobacillus sakei* 1-3, *Lactaseibacillus paracasei* 5-1, *Loigolactobacillus coryniformis* 4-7, and *Latilactobacillus* Lactobacillus curvatus 1-10, Lactobacillus buchneri 36-3, Lactobacillus brevis 7-10;

[0032] Five non-lactobacterial species: Weissella confuse 4-4, Weissella cibaria 4-2, Prdiococcus acidilactici 30-1, Leuconostoc mesenteroides 13-1, and Leuconostoc lactis 11-4;

[0033] Three species of Lactiplantibacillus plantarum: Lactiplantibacillus plantarum 1-7, Lactiplantibacillus plantarum 23-2, and Lactiplantibacillus plantarum 24-1.

[0034] All the validation strains used were deposited in the strain bank of the College of Marine Food and Bioengineering, Jiangsu Ocean University.

[0035] Strain activation:

[0036] The above-mentioned strains were streaked into MRS solid medium at an inoculum of 1% and cultured anaerobically at 37°C for 48 hours. Single colonies on the solid medium were selected and inoculated into MRS liquid medium. After enrichment culture at 37°C for 24 hours for activation, 1 mL of bacterial solution was centrifuged to collect bacterial sludge. 2 mL of sterile physiological saline was added and vortexed to mix. The bacterial sludge was collected by centrifugation and finally 5 mL of sterile water was added and vortexed to mix, which served as a DNA template.

[0037] PCR amplification and gel electrophoresis verification:

[0038] The PCR reaction system is shown in Table 1.

[0039] name Dosage increase (μL) Taq mix 12.5 12.5 upstream primer F 1 0.5-1 Downstream primer R 1 0.5-1 DNA template 1 0.5-1 Double distilled water 9.5 Add 25 μL

[0040] The PCR reaction program is as follows: pre-denaturation at 95℃ for 5 min, followed by 30-35 cycles. The program for each cycle is as follows: denaturation at 95℃ for 10-30 s; annealing at 63℃ for 30 s; extension at 72℃ for 15-25 s; extension at 72℃ for 5 min after the cycle; and cooling to 12℃ to finish.

[0041] The PCR products were verified by 1% agarose gel electrophoresis. The results showed that, except for the standard strain of *Lactobacillus plantarum*, the other control strains did not have specific amplification bands, proving that the primers have strong specificity for amplifying *Lactobacillus plantarum* in the human intestinal environment.

[0042] Example 2: Primer-based validity verification (qPCR verification)

[0043] Glycerol tubes of bacterial strains were obtained from the bacterial strain bank of the College of Marine Food and Bioengineering, Jiangsu Ocean University. Single bacteria were purified and then detected by qPCR.

[0044] The qPCR reaction system is shown in Table 2.

[0045] name Dosage increase (μL) 2× SYBR Green qPCR Master Mix‌ 10 10 upstream primer F 0.4 0.4-1 Downstream primer R 0.4 0.4-1 DNA template 1 0.5-2 Double distilled water 8.2 Add 20 μL

[0046] The qPCR reaction program uses a three-step method: the first stage is pre-denaturation at 95℃ for 5 min; the second stage is 40 cycles of amplification reaction, each cycle including denaturation at 95℃ for 10 s, annealing at 63℃ for 30 s, and extension at 72℃ for 30 s; the third stage is melting curve analysis, using the instrument's default program to acquire signals.

[0047] The results showed that the samples exhibited typical S-shaped amplification curves, indicating that the qPCR reaction system was functioning normally and had high amplification efficiency. The melting curves showed a single and sharp peak, indicating that the amplification products had high specificity and that the binding between the primers and the template was specific. No non-specific amplification or primer dimers were observed. The Tm value was uniformly 83.78℃, confirming that the qPCR reaction had high amplification efficiency and good specificity, enabling accurate quantitative detection of *Lactobacillus plantarum*.

[0048] Example 3: Detection method of Lactobacillus plantarum in human intestinal samples and other environmental samples

[0049] Conventional PCR-gel electrophoresis detection method (qualitative detection):

[0050] For human intestinal samples and other environmental samples, bacterial DNA was first extracted from the samples using a fecal DNA or environmental DNA extraction kit. Using the extracted DNA as a template, amplification was performed using the PCR reaction system and procedure described above. After the PCR reaction, 6 μL of the PCR product was analyzed by 1% agarose gel electrophoresis. The presence of a single amplified band at the 162 bp position was used to determine whether *Lactobacillus plantarum* was present in the sample.

[0051] Real-time quantitative PCR detection method (quantitative detection):

[0052] Bacterial DNA was extracted from the sample as described above. A *Lactobacillus plantarum* DNA standard was prepared, and a standard curve was plotted. Using this DNA as a template, amplification was performed using the primer pair COG3419F1 / COG3419R1 and the corresponding primer probes, following the qPCR reaction system and conditions described above. Finally, qualitative and quantitative detection of *Lactobacillus plantarum* in the sample was performed using the standard curve and the Ct value of the sample.

[0053] The primers and probes provided by this invention can not only perform qualitative and quantitative analysis of Lactobacillus plantarum in the human gut, but also be applied to the qualitative and quantitative detection of Lactobacillus plantarum in food (such as dairy products) and other environments.

[0054] Example 4

[0055] from Figure 2 The amplification curves showed a typical S-shaped curve, indicating that the qPCR reaction system was functioning normally and the amplification efficiency was high. As the cycle number increased, the fluorescence signal intensity (Rn) gradually increased and then stabilized, indicating a smooth amplification process and reliable data quality, providing a good foundation for subsequent quantitative analysis. Figure 3The melting curves of both samples show a single, sharp peak, indicating high specificity of the amplification products and specific primer-template binding. No non-specific amplification or primer dimers were observed. The Tm value was uniformly 83.78℃, further confirming product homology and reaction specificity. In summary, these two figures demonstrate that the qPCR reaction exhibits high amplification efficiency and good specificity, enabling accurate quantitative detection of *Lactobacillus plantarum*.

[0056] The above description is merely a specific embodiment of the present invention, but the scope of protection of the invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A set of primers for the specific identification of *Lactobacillus plantarum* in the human gut, characterized in that, Including upstream primer COG3419F1 and downstream primer COG3419R1; The nucleotide sequence of the upstream primer COG3419F1 is: 5'-CGCCACAGCAAGTAACCAT-3'; The nucleotide sequence of the downstream primer COG3419R1 is: 5'-GACCATACCCAAGTGCCAT-3'.

2. A probe for the specific identification of *Lactobacillus plantarum* in the human gut, characterized in that, The nucleotide sequence of the probe is: 5'-TCACACCCAATTACAGCGCC-3'; the 5' end of the probe is labeled with the FAM reporter fluorescent group, and the 3' end is labeled with the BHQ1 quencher fluorescent group.

3. The application of the primers of claim 1 in the detection of *Lactobacillus plantarum*, characterized in that, The detection includes qualitative and / or quantitative detection; the samples tested include human intestinal samples, food samples, or environmental samples.

4. The application of the probe according to claim 2 in the quantitative detection of *Lactobacillus plantarum*, characterized in that, The quantitative detection is performed using real-time quantitative qPCR; the samples tested include human intestinal samples, food samples, or environmental samples.

5. A qualitative detection method for *Lactobacillus plantarum*, characterized in that, Includes the following steps: (1) Extract bacterial DNA from the sample to be tested; (2) Using the DNA extracted in step (1) as a template, PCR amplification was performed using the primers described in claim 1; the PCR reaction system was as follows: Taq mix 12.5 μL, upstream primer COG3419F1 1 μL, downstream primer COG3419R1 1 μL, DNA template 1 μL, double-distilled water 9.5 μL, and total system 25 μL; The PCR reaction procedure is as follows: pre-denaturation at 95℃ for 5 min; followed by 30-35 cycles, each cycle including denaturation at 95℃ for 10-30 s, annealing at 63℃ for 30 s, extension at 72℃ for 15-25 s; after the cycle, extension at 72℃ for 5 min, and cooling to 12℃ to finish. (3) The PCR products were detected by 1% agarose gel electrophoresis. If a single amplified band appeared at the 162bp position, the sample to be tested was determined to contain Lactobacillus plantarum; otherwise, it was not.

6. A method for quantitative detection of *Lactobacillus plantarum*, characterized in that, Includes the following steps: (1) Extract bacterial DNA from the sample to be tested; (2) Prepare *Lactobacillus plantarum* DNA standards and plot a standard curve; (3) Using the DNA extracted in step (1) as a template, real-time quantitative qPCR amplification was performed using the primers described in claim 1 and the probe described in claim 2; the qPCR reaction system was: 10 μL of 2×SYBR Green qPCRMaster Mix, 0.4-1 μL of upstream primer, 0.4-1 μL of downstream primer, 0.5-2 μL of DNA template, and double-distilled water to 20 μL; The qPCR reaction program was as follows: pre-denaturation at 95℃ for 5 min; followed by 40 cycles, each cycle consisting of denaturation at 95℃ for 10 s, annealing at 63℃ for 30 s, and extension at 72℃ for 30 s; finally, melting curve analysis was performed. (4) Calculate the content of Lactobacillus plantarum in the sample to be tested based on the standard curve and the Ct value of the sample to be tested.

7. A detection kit for *Lactobacillus plantarum*, characterized in that, It contains the primers of claim 1 and / or the probes of claim 2; it may also contain one or more of Taq mix, 2×SYBR Green qPCR Master Mix, double-distilled water, and DNA extraction reagent.

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