Phytobacterium plantarum specific recognition primer, probe and detection method based on auxiliary gene regulatory protein B gene

Abstract

The invention discloses a specific recognition primer, a probe and a detection method for phytobacterium plantarum based on an auxiliary gene regulatory protein B. The primer comprises an upstream primer COG4512F1 and a downstream primer COG4512R1, and the probe is a fluorescent probe designed for a specific COG4512 gene segment of the phytobacterium plantarum. The specific COG4512 gene of the plant lactobacillus is screened as a target sequence, and the designed primer and probe have extremely high specificity and can accurately distinguish the plant lactobacillus from other more than 50 kinds of lactobacillus and non-lactobacillus strains in human intestinal tracts; a conventional PCR detection method and a real-time fluorescent quantitative PCR detection method established on the basis of the primer and the probe are simple and convenient to operate, high in specificity, high in sensitivity and good in accuracy, the blank of specific detection of the plant lactobacillus in the human intestinal environment in the prior art is filled, and the method is suitable for popularization and application. Important technical support is provided for related research, functional food development and micro-ecological intervention of the plant lactobacillus.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a specific recognition primer, probe, and detection method for *Lactobacillus plantarum* based on the helper gene regulatory protein B gene. Background Technology

[0002] *Lactiplantibacillus plantarum* is one of the most widely distributed and genomically plastic species in the lactic acid bacteria community. Since the systematic reconstruction of the genus *Lactobacillus* in 2020, the traditional *Lactobacillus* has been divided into several new genera, and *Lactiplantibacillus plantarum* has been classified into the genus *Lactiplantibacillus*. It stably exists in various ecological niches, including the human gut, oral cavity, female reproductive tract, fermented foods (dairy products, pickled vegetables, meat products, and alcoholic beverages), and plant surfaces. It possesses acid and bile salt tolerance, strong surface adhesion, and stress response capabilities, and is therefore widely used in the food industry, functional food development, and microecological intervention.

[0003] The probiotic functions of *Lactobacillus plantarum* have been extensively studied, including regulating the intestinal barrier and immunity, antagonizing pathogens and host infection, regulating metabolism, modulating the gut-brain axis, and improving functional gastrointestinal disorders. For example, *Lactobacillus plantarum* WCFS1 can regulate the TLR2 / TLR4 signaling of host epithelial cells through surface proteins and extracellular polysaccharides, upregulate tight junction proteins, and alleviate inflammation; *Lactobacillus plantarum* CCFM8610 can secrete bacteriocins and hydrogen peroxide to inhibit pathogen colonization and reduce intestinal inflammation and permeability.

[0004] The human gut microbiota contains hundreds to thousands of species with significant individual differences. According to the new classification after 2020, the traditional genus *Lactobacillus* has been reclassified into 25 new genera, encompassing 261 species. Fifty-one species of *Lactobacillus* have been detected in the human gut, with *Lactobacillus plantarum* being one of the most abundant. Although *Lactobacillus plantarum* often appears as the "dominant lactobacillus" in traditional culture and isolation systems, "culture dominance" does not equate to "molecular detection dominance." In molecular detection and quantification, "specific" primers and probes are required to accurately distinguish it from other gut bacteria.

[0005] 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 animal feed, where the types of *Lactobacillus* are limited, making targeted detection relatively simple; the other is molecular detection of targeted sequences of specific strains of *Lactobacillus plantarum*, whose functions have been clearly studied and have been applied in production. However, there are currently no patents for primers and probes specifically for the identification of *Lactobacillus plantarum* in the human gut. Existing technologies cannot meet the need for accurate detection of *Lactobacillus plantarum* in the context of the complex gut microbiota. Therefore, developing a highly specific and sensitive primer, probe, and detection method for the specific recognition of *Lactobacillus plantarum* is of significant practical importance. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a specific recognition primer, probe and detection method for *Lactobacillus plantarum* based on the helper gene regulatory protein B gene. The primer and probe have extremely high specificity and can accurately distinguish *Lactobacillus plantarum* from other lactobacillus and non-lactobacillus strains. The detection method established based on it is simple to operate, highly sensitive and accurate, and can realize rapid qualitative and accurate quantification of *Lactobacillus plantarum* in different samples.

[0007] The objective of this invention is achieved as follows: a primer pair for species-specific recognition of Lactobacillus plantarum, comprising an upstream primer COG4512F1 and a downstream primer COG4512R1;

[0008] The nucleotide sequence of the upstream primer COG4512F1 is: 5'-CTATGGACGTCACTTGRAAT-3';

[0009] The nucleotide sequence of the downstream primer COG4512R1 is: 5'-CGGCTAATGCCATCGCAATT-3';

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

[0011] The present invention also provides a detection kit for *Lactobacillus plantarum*, comprising the above-mentioned primer pairs and / or probes, and may also include reagents required for conventional PCR or qPCR reactions such as Taq mix, 2×SYBR Green qPCR Master Mix, double-distilled water, and DNA extraction reagents.

[0012] A method for detecting *Lactobacillus plantarum*, characterized by using the above-mentioned primer pair and probe combination, or the above-mentioned detection kit, comprising the following steps:

[0013] (1) Extract total bacterial DNA from the sample to be tested;

[0014] (2) Using the extracted total bacterial DNA as a template, perform PCR amplification using the primer pair, or perform real-time quantitative qPCR amplification using the primer pair and probe;

[0015] (3) Determine whether the sample to be tested contains *Lactobacillus plantarum* based on the amplification results. If it is a quantitative detection, calculate the content of *Lactobacillus plantarum* using a standard curve.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention screened the COG4512 gene, unique to *Lactobacillus plantarum*, as a target sequence. This gene encodes a helper gene regulatory protein B in *Lactobacillus plantarum*, and its sequence differs significantly from homologous sequences of other lactobacilli and non-lactobacilli. Primers and probes designed based on this target sequence exhibit extremely high specificity. Experimental verification shows that this primer pair can specifically amplify only the COG4512 gene fragment of *Lactobacillus plantarum*, and cannot amplify genes from 13 other common lactobacilli and 5 non-lactobacilli. It can accurately distinguish *Lactobacillus plantarum* from other intestinal flora, filling the gap in existing technologies for the specific detection of *Lactobacillus plantarum* in the human intestinal environment.

[0018] The primer and probe combination provided by this invention is more targeted than existing technologies, and can meet the detection needs in the context of complex human gut microbiota. It can also be applied to the detection of food and other environmental samples, and has a wide range of applications.

[0019] The conventional PCR detection method and real-time quantitative PCR detection method established based on the primers and probes of this invention are simple, rapid and efficient to operate; and have high detection sensitivity and good accuracy. The amplification curve of real-time quantitative PCR detection shows a typical S-shape, and the melting curve is single and sharp without impurity peaks, which can realize accurate qualitative and quantitative analysis of Lactobacillus plantarum.

[0020] The detection kit of the present invention can be directly used to detect Lactobacillus plantarum in different types of samples. It does not require complicated sample pretreatment, is easy to use, has low cost, and is easy to promote and apply. It provides an important technical tool for related research on Lactobacillus plantarum, development of functional foods, quality control of microecological preparations, and clinical microecological intervention. Attached Figure Description

[0021] 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.

[0022] Figure 1 The gel electrophoresis image (COG4512F1 / COG4512R1) was validated using specific primers.

[0023] Figure 2 This is a qPCR amplification curve (parallel sample) of Lactobacillus plantarum.

[0024] Figure 3 This is the qPCR melting curve of Lactobacillus plantarum (parallel samples). Detailed Implementation

[0025] 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.

[0026] Example 1: Screening of species-specific sequences of Lactobacillus plantarum

[0027] Genomic data of 51 lactobacilli species found in the human gut were downloaded from the NCBI (National Center for Biotechnology Information) website (strains from human and animal gut sources were prioritized). For strains with fewer than 10 strains in the database, the genomes of all available strains were downloaded, resulting in 420 genome data points for 51 lactobacilli species. Among them, 10 strains were identified as *Lactobacillus plantarum*. The strain information and sources are shown in Table 1 below.

[0028] Table 1. Information on 10 Lactobacillus plantarum strains with genomic origins.

[0029] Strain number Origin Lactiplantibacillus plantarum HOM2217 Human (milk) Lactiplantibacillus plantarum LZ95 Human (infant feces) Lactiplantibacillus plantarum SRCM100442 Human (feces) Lactiplantibacillus plantarum VHProbi E15 Human Lactiplantibacillus plantarum VHProbi O04 Human Lactiplantibacillus plantarum VHProbi O10 Human Lactiplantibacillus plantarum VHProbi P32 Human Lactiplantibacillus plantarum VHProbi V22 Human Lactiplantibacillus plantarum ZFM9 Human (feces) Lactiplantibacillus plantarum ZX028 Human

[0030] Alignment analysis of the above genomes with the COG (Clusters of Orthologous Groups) database revealed that the gene annotated as COG4512 was present only in 10 strains of *Lactobacillus plantarum*, 2 strains of *Lentilactobacillus diolivorans*, and 9 strains of *Lactobacillus sakei*. Furthermore, this gene sequence showed significant differences between *Lactobacillus plantarum* and the other two strains. Combined with NCBI-BLAST alignment, the specificity and accuracy of this target gene sequence were confirmed, identifying COG4512 as a specific target sequence for *Lactobacillus plantarum*.

[0031] Example 2: Design of Specific Primers and Probes

[0032] Primer selection and design were performed using SnapGene software, and probe design was performed using Oligo7 software. The design principles are as follows:

[0033] (1) The primer length is 18-25bp, the Tm value is between 50-60℃, and the difference in Tm value between the upstream and downstream primers does not exceed 3℃;

[0034] (2) Avoid primers from forming hairpin structures, dimers, and complementary pairings;

[0035] (3) The probe length is 20-30 bp, and the Tm value is 5-10℃ higher than the primer Tm value to avoid complementary pairing with the primer;

[0036] (4) Avoid using G at the 5' end of the probe and avoid using C at the 3' end.

[0037] The final primer and probe sequences obtained are as follows:

[0038] Upstream primer COG4512F1: 5'-CTATGGACGTCACTTGRAAT-3', Tm is 53℃;

[0039] Downstream primer COG4512R1: 5'-CGGCTAATGCCATCGCAATT-3', Tm is 55℃;

[0040] Probe: 5'-TCCGATTCCAAGTGACGTCCA-3', 5' end labeled with FAM reporter fluorescent group, 3' end labeled with BHQ1 quencher fluorescent group.

[0041] The primer pair amplified a 305bp fragment of the Lactobacillus plantarum COG4512 gene.

[0042] Example 3: Specific primer PCR and gel electrophoresis verification

[0043] Validation strains included 13 Lactobacillus species (Lactobacillus salivarius 27-2, Lactobacillus rhamnosus 16-1, Lactobacillus curvatureus 16-3, Lactobacillus fermentans 27-3, Lactobacillus vaginalis 4-9, Lactobacillus casei 6-5, Lactobacillus reuteri 16-5, Lactobacillus sacchariformis 1-3, Lactobacillus caseiformis 5-1, Lactobacillus corynebacterium putrefaction 4-7, Lactobacillus curvatureus 1-10, Lactobacillus brücken 36-3, Lactobacillus brevis 7-10), 5 non-Lactobacillus species (Westernella fusionis 4-4, Westernella esculenta 4-2, Pediococcus lactis 30-1, Leuconostoc mesenteroides 13-1, Leuconostoc lactis 11-4), and 3 Lactobacillus plantarum strains (1-7, 23-2, 24-1). All strains were deposited in the strain bank of the College of Marine Food and Bioengineering, Jiangsu Ocean University.

[0044] Strain activation: The above strain was streaked into MRS solid medium at an inoculum of 1% and cultured anaerobically at 37°C for 48 h. Single colonies on the solid medium were selected and inoculated into MRS liquid medium. After enrichment culture at 37°C for 24 h, the strain was activated. 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. This mixture was used as a DNA template.

[0045] PCR reaction system (25μL): Taq mix 12.5μL, upstream primer COG4512F1 1μL, downstream primer COG4512R1 1μL, DNA template 1μL, double-distilled water 9.5μL.

[0046] PCR reaction procedure: pre-denaturation at 95℃ for 5 min; then 35 cycles, each cycle including denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 20 s; after the cycle, extension at 72℃ for 5 min, then cooling to 12℃ to finish.

[0047] Gel electrophoresis verification: 6 μL of PCR product was subjected to 1% agarose gel electrophoresis at 120V for 30 min, and the results were observed under UV light.

[0048] The results showed that only 3 strains of *Lactobacillus plantarum* showed a single specific amplification band at the 305bp position in their PCR products, while the remaining 13 lactobacilli and 5 non-lactobacilli did not show specific amplification bands. This indicates that the primer pair has extremely strong species specificity and can accurately distinguish *Lactobacillus plantarum* from other strains.

[0049] Example 4: Validation of primers and probes based on Lactobacillus plantarum (qPCR validation)

[0050] Strain preparation: Glycerol tubes of Lactobacillus plantarum 1-7 were obtained from the strain bank of the College of Marine Food and Bioengineering, Jiangsu Ocean University. After purification of the single strain, DNA templates were prepared according to the method in Example 3.

[0051] qPCR reaction system (20μL): 10μL 2×SYBR Green qPCR Master Mix, 0.4μL upstream primer COG4512F1, 0.4μL downstream primer COG4512R1, 0.3μL probe, 1μL DNA template, and 8.2μL double-distilled water.

[0052] qPCR reaction program: The first stage is 95℃ pre-denaturation for 5 min; the second stage is 40 cycles of amplification reaction, each cycle including 95℃ denaturation for 10 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s; the third stage is melting curve analysis, using the instrument's default program to acquire signals.

[0053] Results analysis: The qPCR amplification curves showed a typical S-shaped curve with clear inflection points, indicating good qPCR reaction efficiency, efficient primer-template binding, and reasonable proportions of components in the reaction system. The melting curves showed a single and sharp peak with consistent Tm values ​​(78.41℃) and no impurities from primer dimers or other non-specific amplification products, indicating homology of the products and good specificity of the primers and probes.

[0054] The above results demonstrate that the primers and probes possess excellent specificity, amplification efficiency, and reaction uniformity in complex sample backgrounds, enabling accurate quantitative detection of *Lactobacillus plantarum*.

[0055] Example 5: Detection of Lactobacillus plantarum in human intestinal samples

[0056] Sample processing: Collect fecal samples from healthy volunteers and extract total bacterial DNA from the samples using a fecal DNA extraction kit, following the instructions of the kit.

[0057] Routine PCR testing:

[0058] (1) The PCR reaction system and reaction procedure are the same as in Example 3;

[0059] (2) Gel electrophoresis verification: Take 6 μL of PCR product and perform 1% agarose gel electrophoresis. Observe whether a single specific amplification band appears at the 305bp position. If it appears, it indicates that the sample contains Lactobacillus plantarum; otherwise, it does not.

[0060] Real-time quantitative PCR detection:

[0061] (1) Standard curve plotting: The DNA templates of Lactobacillus plantarum 1-7 were serially diluted (10⁻⁶ oz / mL). 6 10 5 10 4 10³, 10², and 10¹ copies / μL were used as standards for qPCR amplification. A standard curve was plotted with the logarithm of the standard concentration on the x-axis and the Ct value on the y-axis.

[0062] (2) Sample detection: Using the extracted fecal DNA as a template, amplification was performed according to the qPCR reaction system and reaction procedure in Example 4. The concentration of Lactobacillus plantarum in the sample was calculated based on the standard curve and the sample Ct value.

[0063] The results showed that some fecal samples had a specific amplification band at the 305bp position, indicating that these samples contained *Lactobacillus plantarum*. The content of *Lactobacillus plantarum* in the samples could be accurately calculated by real-time quantitative PCR, realizing the qualitative and quantitative detection of *Lactobacillus plantarum* in human intestinal samples.

[0064] Example 6 Detection of Lactobacillus plantarum in food samples

[0065] Sample processing: Commercially available yogurt was selected as the food sample. 10g of yogurt sample was weighed and added to 90mL of sterile physiological saline. The mixture was shaken well to prepare a 1:10 dilution. 1mL of the dilution was taken to extract total bacterial DNA according to the bacterial DNA extraction kit instructions.

[0066] Conventional PCR detection and real-time quantitative PCR detection: Conventional PCR detection and real-time quantitative PCR detection were performed according to the method in Example 5.

[0067] The results showed that a specific amplification band appeared at the 305bp position in the yogurt sample, indicating that it contained *Lactobacillus plantarum*. The content of *Lactobacillus plantarum* in the yogurt could be accurately calculated by real-time quantitative PCR, proving that the detection method can be used to detect *Lactobacillus plantarum* in food samples.

[0068] 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 specific primer pair for detecting *Lactobacillus plantarum*, characterized in that, Its sequences are: COG4512F1: 5'-CTATGGACGTCACTTGRAAT-3'; COG4512R1: 5'-CGGCTAATGCCATCGCAATT-3'; The nucleotide sequence of the probe is: 5'-TCCGATTCCAAGTGACGTCCA-3', wherein the 5' end is labeled with the FAM reporter fluorescent group and the 3' end is labeled with the BHQ1 quencher fluorescent group.

2. A detection kit for *Lactobacillus plantarum*, characterized in that, It includes the primer pair and probe combination as described in claim 1.

3. The detection kit according to claim 2, characterized in that, It also contains one or more of the following: PCR reaction buffer, Taq enzyme, dNTPs, double-distilled water, and SYBR Green qPCR Master Mix.

4. A method for detecting *Lactobacillus plantarum*, characterized in that, Using the primer pair and probe combination of claim 1, or the detection kit of any one of claims 2-3, the method includes the following steps: (1) Extract total bacterial DNA from the sample to be tested; (2) Using the extracted total bacterial DNA as a template, perform PCR amplification using the primer pair, or perform real-time quantitative qPCR amplification using the primer pair and probe; (3) Determine whether the sample to be tested contains *Lactobacillus plantarum* based on the amplification results. If it is a quantitative detection, calculate the content of *Lactobacillus plantarum* using a standard curve.

5. The detection method according to claim 4, characterized in that, The PCR amplification reaction system described in step (2) is as follows: Taq mix 12.5 μL, upstream primer COG4512F1 0.5-1 μL, downstream primer COG4512R1 0.5-1 μL, DNA template 0.5-1 μL, and double-distilled water to make up to 25 μL.

6. The detection method according to claim 4, characterized in that, The PCR amplification reaction procedure in step (2) is as follows: pre-denaturation at 95℃ for 5 min; 30-35 cycles, each cycle including denaturation at 95℃ for 10-30 s, annealing at 60℃ for 30 s, extension at 72℃ for 15-25 s; after the cycle, extension at 72℃ for 5 min, and cooling down to 12℃ to finish.

7. The detection method according to claim 4, characterized in that, The system for the real-time quantitative qPCR amplification reaction described in step (2) is as follows: 10 μL of 2×SYBR Green qPCR Master Mix, 0.4-1 μL of upstream primer COG4512F1, 0.4-1 μL of downstream primer COG4512R1, 0.5-2 μL of DNA template, and double-distilled water to a final volume of 20 μL.

8. The detection method according to claim 4, characterized in that, The procedure for the real-time quantitative qPCR amplification reaction described in step (2) is as follows: pre-denaturation at 95℃ for 5 min; 40 cycles, each cycle including denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s; finally, melting curve analysis is performed, and the signal is acquired using the instrument's default program.

9. The detection method according to any one of claims 4-8, characterized in that, The samples to be tested include human intestinal samples, food samples, or environmental samples.

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