Molecular markers of lactobacillus johnsonii

Abstract

This invention proposes a molecular marker for *Lactobacillus japonicus*, which was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26505. The molecular marker comprises a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99%, or higher homology to the nucleotide sequence shown in SEQ ID NO: 24; or (3) a nucleotide sequence having one or more nucleotide substitutions, deletions, or insertions in the nucleotide sequence shown in SEQ ID NO: 24. Using the molecular marker of this invention, not only can the target *Lactobacillus japonicus* be screened quickly and easily, but it can also highly specifically identify the target *Lactobacillus japonicus*, eliminating interference from other strains.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a molecular marker for Lactobacillus janniae. Background Technology

[0002] Lactobacillus japonicus is a Gram-positive, non-spore-forming, non-motile anaerobic or microaerophilic bacterium. As a type of lactobacillus, it is one of the most common and important beneficial bacteria colonizing the female reproductive tract and plays a key role in maintaining female vaginal health.

[0003] Currently, the main methods for identifying lactic acid bacteria include morphological observation and physiological and biochemical characteristic identification. Morphological observation is one of the most basic identification methods; by observing the morphological characteristics of colonies and cells under a microscope, the species of lactic acid bacteria can be preliminarily determined. Although morphological observation is simple to operate, its accuracy is relatively low, making it difficult to identify bacteria precisely at the species or subspecies level. Furthermore, it is easily affected by culture conditions. Therefore, it is usually necessary to combine it with other physiological and biochemical tests or molecular biology techniques to improve the accuracy and reliability of lactic acid bacteria identification. Physiological and biochemical identification refers to the species-level identification by measuring various physiological and biochemical indicators of lactic acid bacteria, such as sugar fermentation capacity, growth condition requirements, metabolite detection, and enzyme activity detection. This method can provide relatively detailed information on the characteristics of lactic acid bacteria, which helps to make more accurate species-level identification. However, it requires a lot of cultivation time and manpower and is relatively cumbersome to operate. For example, the sugar fermentation capacity test usually requires a cultivation time of 3-7 days, the growth condition test (measuring temperature, pH, salt concentration, etc.) requires a cultivation cycle of about 5-10 days, the metabolite detection (detection of products such as lactic acid and acetic acid) analysis requires 1-2 weeks, and the enzyme activity detection (involving the determination of multiple enzymes) requires 1-2 weeks. Moreover, the results of some indicators are easily affected by external factors, and the identification results may have a certain degree of uncertainty.

[0004] In their previous research, the inventors discovered a type of Lactobacillus jensenii that can maintain a slightly acidic environment in the vagina, inhibit the growth and reproduction of various pathogenic bacteria, thereby preventing vaginal infections and treating inflammation. In order to screen or identify this Lactobacillus jensenii more efficiently, quickly and accurately, it is urgent to develop a molecular marker and method for screening and identifying this Lactobacillus jensenii. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To rapidly and easily screen and identify *Lactobacillus japonicus*, the inventors employed whole-genome sequencing and genome analysis. Through these analyses, a specific nucleotide sequence was successfully screened, and primers were designed targeting this sequence. These primers can amplify specific products associated with the target nucleotide sequence, and these products can serve as markers for identifying the target *Lactobacillus japonicus*. This innovation provides a reliable tool and basis for the identification and detection of *Lactobacillus japonicus*, while greatly simplifying the screening process.

[0006] Therefore, in a first aspect, the present invention provides a molecular marker for *Lactobacillus jensenii*. According to an embodiment of the present invention, *Lactobacillus jensenii* (HY1335) was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26505, at the address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The molecular marker comprises a nucleotide sequence selected from one of the following:

[0007] (1) The nucleotide sequence shown in SEQ ID NO:24;

[0008] (2) A nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence shown in SEQ ID NO:24;

[0009] (3) A nucleotide sequence having one or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:24.

[0010] The molecular markers are designed based on the specific nucleic acid sequences of Lactobacillus japonicus. Therefore, by using the molecular markers of the present invention, not only can the target Lactobacillus japonicus be screened quickly and easily, but the target Lactobacillus japonicus can also be identified with high specificity, eliminating interference from other strains. In addition, the molecular marker method has highly reliable results in the screening and identification of Lactobacillus japonicus, increasing the accuracy and reliability of the screening and identification results, making it a reliable tool and method.

[0011] According to an embodiment of the present invention, the molecular marker has a nucleotide sequence as shown in SEQ ID NO:24.

[0012] In a second aspect, the present invention provides a primer set for amplifying the molecular marker described in the first aspect, characterized in that it comprises a forward primer and a reverse primer; the forward primer comprises the nucleotide sequence shown in SEQ ID NO:2; and the reverse primer comprises the nucleotide sequence shown in SEQ ID NO:3. Amplification experiments using the primer set of the present invention yield products that highly match the DNA sequence of the target *Lactobacillus jenny*, and the primer set specifically amplifies only in *Lactobacillus jenny*. This enables the primer set to accurately and reliably identify *Lactobacillus jenny*, excluding interference from other non-target strains.

[0013] In a third aspect, the present invention proposes the use of the molecular markers described in the first aspect for the identification and / or detection of *Lactobacillus jenny*, which was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26505. Using the molecular markers described in this invention for the identification and / or detection of *Lactobacillus jenny* not only offers simplicity, speed, specificity, and high sensitivity, but also saves significant time and labor, improving the efficiency of research and application.

[0014] In a fourth aspect, the present invention provides a kit. According to embodiments of the invention, the kit comprises the primer set described in the second aspect and optionally PCR amplification reagents. Therefore, this kit enables rapid and convenient screening and identification of target *Lactobacillus japonicus*, significantly improving screening and identification efficiency and saving time and labor costs.

[0015] In a fifth aspect, the present invention provides a method for identifying and / or detecting *Lactobacillus jenny*. According to an embodiment of the present invention, the method includes: amplifying the DNA of the strain to be tested using the primer set described in the second aspect or the kit described in the fourth aspect; comparing the amplification result with the molecular marker described in the first aspect; and determining, based on the comparison result, whether the strain to be tested is *Lactobacillus jenny*; wherein *Lactobacillus jenny* was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26505. Using the method described in this invention, *Lactobacillus jenny* of the present invention can be identified and detected efficiently and conveniently.

[0016] According to embodiments of the present invention, the method for identifying and / or detecting *Lactobacillus jenny* may further include at least one of the following additional technical features:

[0017] According to an embodiment of the present invention, the amplification result having at least 97%, 98%, 99% or higher identity with the molecular marker described in the first aspect is an indication that the strain to be detected is *Lactobacillus japonicus*.

[0018] According to an embodiment of the present invention, the amplification system comprises: 1.0-2.5 U / μL of Taq enzyme, 0.4-2.0 pmol / μL of the forward primer from the primer set, 0.4-2.0 pmol / μL of the reverse primer from the primer set, and DNA of the strain to be detected.

[0019] According to an embodiment of the present invention, the amplification reaction conditions are as follows: pretreatment: denaturation at 90-98℃ for 3-8 min; PCR reaction: denaturation at 90-98℃ for 10-20 s, annealing at 55-65℃ for 10-20 s, extension at 70-75℃ for 50-70 s, for a total of 30-35 cycles; extension: extension at 70-75℃ for 3-8 min.

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0022] Figure 1 This is a colony morphology diagram of Lactobacillus janniae HY1335 according to an embodiment of the present invention;

[0023] Figure 2 This is a cell morphology diagram of Lactobacillus janniae HY1335 according to an embodiment of the present invention;

[0024] Figure 3 This is a diagram showing the results of a hemolysis experiment using Lactobacillus japonicus HY1335 according to an embodiment of the present invention;

[0025] Figure 4 This is a diagram showing the animal experiment results of BV efficacy according to an embodiment of the present invention;

[0026] Figure 5 This is an electrophoretic pattern of PCR amplification of four Lactobacillus janniae using primer 1-p3 according to an embodiment of the present invention, wherein M is a marker, 1 is the supernatant after lysis of HY1335 as a template, 2 is the supernatant after lysis of HY00888 as a template, 3 is the supernatant after lysis of HY01360 as a template, and 4 is the supernatant after lysis of HY02445 as a template.

[0027] Figure 6This is an electrophoretic pattern of PCR amplification of four Lactobacillus janniae using primer 2-p3 according to an embodiment of the present invention, wherein M is a marker, 1 is the supernatant after lysis of HY1335 as a template, 2 is the supernatant after lysis of HY00888 as a template, 3 is the supernatant after lysis of HY01360 as a template, and 4 is the supernatant after lysis of HY02445 as a template. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0029] Terminology Definition

[0030] In this invention, the term "Lactobacillus jensenii" or simply "Lactobacillus jensenii" generally refers to a species of the genus Lactobacillus. This species is typically distinguished from other Lactobacilli based on the polynucleotide sequence of its 16S rDNA gene on the ribosome.

[0031] In this invention, the term "antibiotic sensitivity" refers to the weak resistance of bacteria to antibiotics, meaning that even trace amounts of the antibiotic can affect the normal growth of the bacteria. According to an embodiment of the present invention, the *Lactobacillus japonicus* HY1335 is sensitive to clindamycin and cefuroxime.

[0032] In this invention, the term "vagina" generally refers to the vaginal region or surrounding area, including the labia, vulva, cervix, uterus, fallopian tubes, ovaries, urethra, bladder, anus, and rectum, including their mucosal tissue.

[0033] In this invention, the term "pathogenic" (e.g., "pathogenic bacteria") generally refers to substances, microorganisms, or conditions capable of causing disease. In some contexts, pathogens also include microorganisms (e.g., bacteria) associated with a disease or symptom, but for which a causal relationship has not yet been established or is yet to be established (e.g., a direct causal relationship). In some embodiments, microorganisms that are not pathogens but may be symbiotic may cause or be associated with disease or dysbiosis, depending on various factors (e.g., site-specific immune status, abundance of microbial taxa, etc.). Such microorganisms are referred to as "pathogenic organisms."

[0034] In this invention, the terms "vaginal flora" or "vaginal microbiota" are used interchangeably and generally refer to the microorganisms that colonize the vagina.

[0035] In this invention, the term "inhibition" generally refers to the process of inhibiting or hindering the growth, reproduction, and activity of bacteria.

[0036] In this invention, the term "CFU (Colony-Forming Units)" usually refers to the total number of microbial communities such as bacteria, fungi, and yeast in a product, and is typically used for calculating the number of viable cells.

[0037] In this invention, the term "about" generally refers to a variation within a range of about 0.5% to 10% above or below a specified value, such as a variation within a range of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below a specified value.

[0038] In this invention, the terms “identity,” “homology,” or “similarity” are used to describe nucleic acid sequences relative to a reference sequence, employing conventional methods to determine the percentage of identical nucleotides between two nucleic acid sequences, see, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)). And the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0039] In this invention, the term "at least 97% homology" refers to at least 97% homology with each reference sequence, which can be 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 99%, 99.3%, 99.5%, 99.8%, or 99.9%. The term "at least 99% homology" refers to at least 99% homology with each reference sequence, which can be 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0040] This invention proposes molecular markers for Lactobacillus jenny and their uses, primer sets, kits, and methods for identifying and / or detecting Lactobacillus jenny, which will be described in detail below.

[0041] Molecular markers, primer set

[0042] This invention proposes a molecular marker for *Lactobacillus japonicus*. According to an embodiment of the invention, *Lactobacillus japonicus* was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26505. The molecular marker comprises a nucleotide sequence selected from one of the following:

[0043] (1) The nucleotide sequence shown in SEQ ID NO:24;

[0044] (2) A nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence shown in SEQ ID NO:24;

[0045] (3) A nucleotide sequence having one or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:24.

[0046] In their previous research, the inventors discovered a novel *Lactobacillus jensenii* HY1335. Extensive experiments revealed that this bacterium can produce large amounts of hydrogen peroxide (H2O2), which can directly kill pathogens and, through enzymatic catalysis, form halides with even stronger bactericidal effects. Furthermore, this bacterium exhibits strong inhibitory effects against pathogenic bacteria such as *Gardnerella vaginalis* (GV), *Escherichia coli* (EC), *Staphylococcus aureus* (SA), and *Prevotella bivia* (PB), and also shows some inhibitory effect against *Candida albicans* (CA). Therefore, this *Lactobacillus jensenii* has the potential to maintain a slightly acidic vaginal environment, thereby resisting the invasion of other pathogens and maintaining and improving vaginal homeostasis, preventing vaginal infections and treating inflammation.

[0047] To rapidly screen and identify *Lactobacillus japonicus*, the inventors used whole-genome sequencing and genome analysis to sequence a specific nucleotide sequence from the *Lactobacillus japonicus* gene. Primers were then designed using this sequence, including a forward primer and a reverse primer. The forward primer includes the nucleotide sequence shown in SEQ ID NO:2, and the reverse primer includes the nucleotide sequence shown in SEQ ID NO:3. Using these primers, specific products related to the target nucleotide sequence can be amplified. These products can serve as markers for identifying the target *Lactobacillus japonicus*, laying the foundation for accurate and reliable identification of *Lactobacillus japonicus* and eliminating interference from other non-target strains.

[0048] TACGGCCGTCTGGTAAAGTTAAAACCAAAACAAGATCATTTTTCACTTCGTAAGTTATCTATTGGTCTTATTTCTGTTACCATAGGTATTACCTTATACTTGGGAGTGGACAACTCCTATGTTATGGCTGATACGAATTTGAGGGATAATAACAATCAACAATTAGCCGGATCGGGTAATGGCACCAATATAATAAATGATAAAGTTAAAATTCCAACCGGTGCTTTTCAGT (SEQ ID NO: 24)

[0049] TACGGCCGTCTGGTAAAGTT(SEQ ID NO:2)

[0050] ACTGAAAAGCACCGGTTGGA(SEQ ID NO3)

[0051] Reagent test kit

[0052] This invention provides a kit. According to embodiments of the invention, the kit includes the aforementioned primer set and optionally PCR amplification reagents. The kit of this invention is used to identify and / or detect *Lactobacillus japonicus* HY1335. As mentioned above, the product amplified by the primer set serves as a marker to identify the target *Lactobacillus japonicus*. Therefore, this kit can rapidly and easily screen and identify the target *Lactobacillus japonicus*, greatly improving the efficiency of screening and identification, saving time and labor costs. Furthermore, this kit has the advantages of high sensitivity and high accuracy, avoiding false alarms and false positives, and increasing the accuracy and reliability of screening and identification results.

[0053] Uses and methods

[0054] This invention proposes the use of the aforementioned molecular markers in the identification and / or detection of *Lactobacillus jenny*, which was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26505. The use of the molecular markers described in this invention for the identification and / or detection of *Lactobacillus jenny* is not only simple and rapid, but also specific and highly sensitive. The molecular marker method not only saves a significant amount of time and labor, but also improves the efficiency of research and application.

[0055] This invention proposes a method for identifying and / or detecting *Lactobacillus jenny*. According to an embodiment of the invention, the method includes: amplifying the DNA of the strain to be tested using the aforementioned primer set or the aforementioned kit; comparing the amplification result with the aforementioned molecular markers; and determining, based on the comparison result, whether the strain to be tested is *Lactobacillus jenny*; wherein *Lactobacillus jenny* was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26505. The method described in this invention can efficiently and conveniently identify and detect *Lactobacillus jenny* of this invention, with high accuracy and reliability.

[0056] According to embodiments of the present invention, the method for identifying and / or detecting *Lactobacillus jenny* may further include at least one of the following additional technical features:

[0057] According to embodiments of the present invention, the amplification result has at least 97%, 98%, 99%, or higher identity with the aforementioned molecular marker, indicating that the strain to be detected is *Lactobacillus japonicus*. As mentioned above, this molecular marker can act as a specific marker, associating it with the target *Lactobacillus japonicus*, thereby enabling the screening and identification of the target *Lactobacillus japonicus*.

[0058] According to an embodiment of the present invention, the amplification system comprises: 1.0-2.5 U / μL of Taq enzyme, 0.4-2.0 pmol / μL of the forward primer from the primer set, 0.4-2.0 pmol / μL of the reverse primer from the primer set, and the DNA of the strain to be detected. The present invention does not specifically limit the concentration of the DNA of the strain to be detected in the amplification system, as long as it enables the primers in the system to amplify the DNA of the strain to be detected, and the amplified product can be sequenced and / or detected by agarose gel electrophoresis.

[0059] According to an embodiment of the present invention, the reaction conditions for the amplification process are as follows:

[0060] Pretreatment: Denaturation can be performed at 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, and 98℃ for 3 min, 4 min, 5 min, 6 min, 7 min, and 8 min, respectively.

[0061] PCR reaction: Denaturation can be performed at 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, and 98℃ for 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, and 20s respectively; annealing can be performed at 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, and 65℃ for 10s. The PCR reaction can be extended for 50s, 52s, 54s, 56s, 58s, 60s, 62s, 64s, 66s, 68s, and 70s at 70℃, 71℃, 72℃, 73℃, 74℃, and 75℃ respectively; the PCR reaction can be repeated for a total of 30, 31, 32, 33, 34, and 35 cycles.

[0062] Extension: Can be extended for 3 min, 4 min, 5 min, 6 min, 7 min, and 8 min at 70℃, 71℃, 72℃, 73℃, 74℃, and 75℃, respectively.

[0063] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0064] Example 1: Screening, isolation and identification of Lactobacillus janniae

[0065] 1. Collect samples

[0066] The samples were sourced from naturally fermented foods with regional characteristics, vaginal secretion samples collected by hospital gynecologists from volunteers who met the inclusion criteria, and human samples (feces and breast milk).

[0067] 2. Strains Isolation

[0068] Vaginal secretions from healthy women of childbearing age were serially diluted tenfold with physiological saline. Diluted solutions of appropriate gradients were spread onto anaerobic blood agar plates (purchased from Huankai Microbiology) and incubated at 37°C for 48-72 hours in an anaerobic workstation. Single colonies of varying morphology were picked and streaked onto blood agar plates for purification. After further incubation, pure cultures were selected for bacterial identification (16S rDNA sequencing). Through identification and screening, a strain of *Lactobacillus janniae* was obtained and named *Lactobacillus janniae* HY1335.

[0069] After identifying the strain species, the pure culture (i.e., pure Lactobacillus janniae HY1335) was inoculated into MRS broth liquid medium for expansion. When the strain grew to a suitable concentration, it was mixed with an equal volume of liquid culture using sterile 50% glycerol solution and stored in a strain bank at -80℃.

[0070] 3. Strain identification

[0071] (1) Colony characteristics

[0072] Dip an inoculation loop into the bacterial culture tube and streak it onto an MRS plate. Incubate at 37°C for 48 hours anaerobically, then observe the colony morphology on the plate. Figure 1 As shown, after being cultured on MRS plates, this strain forms round, rough-edged, milky-white colonies with a raised center.

[0073] (2) Staining microscopy

[0074] Use an inoculation loop to pick up one loopful of sterile distilled water onto a clean glass slide. Pick a single colony from an MRS plate, mix it evenly with the distilled water, and spread it onto a glass slide. Stain according to the instructions of the Gram staining kit (purchased from Qingdao Haibo). Then observe the bacterial morphology under an electron microscope. Figure 2As shown, after staining, the strain was observed under an optical microscope, revealing that the bacterial cells were Gram-positive and short rod-shaped. Based on observation of colony characteristics and Gram staining identification, the isolated strain was preliminarily identified as *Lactobacillus*.

[0075] (3) Biochemical identification and analysis

[0076] ① Lactobacillus culture: The target strain was inoculated into MRS broth medium (purchased from Qingdao Haibo) and placed in an anaerobic incubator at 37°C for 24 hours.

[0077] ② Preparation of bacterial suspension: Centrifuge the bacterial fermentation broth at 4000 rpm for 5 min in a centrifuge, remove the supernatant, wash the bacterial sludge with physiological saline, centrifuge at 4000 rpm for 5 min, remove the supernatant, wash the bacterial sludge with physiological saline again, centrifuge at 4000 rpm for 5 min, remove the supernatant, add physiological saline and mix evenly with the bacterial sludge for later use.

[0078] ③ 1% sodium hippurate identification test: Take 50 μL of bacterial suspension, add it to a 1% sodium hippurate identification tube, seal the tube with sealing film, and incubate it in a 37℃ water bath for 2 h. Then, slowly add 200 μL of ninhydrin solution (3.5% ninhydrin solution: 0.175 g hydrated ninhydrin, 2.5 mL acetone, 2.5 mL butanol) along the tube wall without shaking. After placing the tube in a 37℃ water bath for 10 min, read the results.

[0079] ④ Other identification experiments: Take 50 μL of bacterial suspension and add it to a biochemical identification tube (Note: After adding the bacterial suspension to the esculin identification tube, cover the liquid surface with sterile liquid paraffin), seal with sealing film, and place in an anaerobic incubator. Incubate at 37℃ for 48 h, then interpret the results. The interpretation results are shown in Table 1:

[0080] Table 1. Interpretation results of Lactobacillus janniae identification tubes

[0081]

[0082] Note: + represents positive; - represents negative; +w represents weak positive.

[0083] Biochemical identification results showed that Lactobacillus janniae can utilize esculin, cellobiose, maltose, salicin, sucrose, and inulin as carbon sources.

[0084] (4) 16S rDNA identification

[0085] The *Lactobacillus jensenii* strain described above was amplified and sequenced using 16S rDNA. The sequence was then compared using BLAST in the NCBI database. The 16S rDNA alignment results showed that the strain was *Lactobacillus jensenii*, and it was named *Lactobacillus jensenii* HY1335. The 16S rDNA gene sequence is as follows:

[0086] AGTCGAGCGAGCTTGCCTATAGAAATTCTTCGGAATGGACATAGATACAAGCTAGCGGCGGAT

[0087] GGGTGAGTAACGTGGGTAACCTGCCCTTAAGTCTGGGATACCATTTGGAAACAGATGCTAATAC

[0088] CGGATAAAAGCTACTTTCGCATGAAAGAAGTTTAAAAGGCGGCGTAAGCTGTCGCTAAAGGATGG

[0089] ACCTGCGATGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTG

[0090] AGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGG

[0091] AATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATC

[0092] GTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAAC

[0093] CAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGG

[0094] ATTTATTGGGCGTAAAGCGAGCGCAGGCGGATTGATAAGTCTGATGTGAAAGCCTTCGGCTCAACC

[0095] GAAGAACTGCATCAGAAACTGTCAATCTTGAGTGCAGAAGAGGAGAGGTGGAACTCCATGTGTAGC

[0096] GGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACG

[0097] CTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATG

[0098] AGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGG

[0099] GGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCAT

[0100] GTGGTTTAATTCGAAGCAACGGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAAGA

[0101] TTAGGTTTTCCCTTCGGGGACAAAGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA

[0102] TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTAATAGTTGCCAGCATTAAGTTGGGCACTCT

[0103] ATTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGA

[0104] CCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAACCTGTGAAGGCAAGCGGAT

[0105] CTCTTAAAGCTGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGCTGGAATCGCTAG

[0106] TAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA

[0107] TGAGAGNTTGTAACACCCAAAGTCGGTGAGGTAACCNTTGGAGCCAGCCGCCTAA (SEQ ID NO: 1)

[0108] Example 2: Characteristics of the strain

[0109] (1) Antibiotic susceptibility test

[0110] Lactobacillus japonicus HY1335 was cultured in MRS broth, and the bacterial suspension was then evenly spread on MRS plates. After the suspension was absorbed and dried, antibiotic susceptibility testing discs were attached, and the plates were anaerobically incubated at 37°C for 48 hours. The diameter of the inhibition zone was measured using calipers. The antibiotic susceptibility of the strain was determined by the diameter of the inhibition zone on the antibiotic susceptibility testing discs. The results are shown in Table 2. This strain was resistant to metronidazole, norfloxacin, ofloxacin, ciprofloxacin, and kanamycin, but sensitive to clindamycin and cefuroxime.

[0111] Table 2 Results of antibiotic susceptibility testing

[0112]

[0113] Note: S: Sensitive (15-20mm highly sensitive; >20mm extremely sensitive) I: Intermediate (10-14mm moderately sensitive) R: Tolerant (<10mm insensitive)

[0114] (2) Toxicity test

[0115] ① Hemolysis test

[0116] The bacterial culture was streaked onto an anaerobic blood agar plate using a frozen tube of bacterial culture. The plate was then incubated anaerobically at 37°C for 48 hours. The color change around the colonies on the blood agar plate was observed. The hemolysis test results are as follows: Figure 3 As shown, small grayish-white colonies appeared in the culture medium around the colonies, and no hemolytic zone appeared around the colonies, indicating that Lactobacillus janniae HY1335 is γ-hemolytic, i.e., non-hemolytic.

[0117] ② Mouse toxicity test

[0118] Five mice weighing 18-22g were used. Each mouse was orally administered 0.5ml of fresh bacterial solution (no less than 1.0×10 CFU / 0.5ml) once daily for three consecutive days. Observations were conducted from day 1 to day 7. All mice were expected to survive and gain weight. The results showed that mice administered fresh Lactobacillus japonicus HY1335 bacterial solution via gavage all survived and gained weight.

[0119] (3) Determination of metabolite content

[0120] ①D-lactic acid detection

[0121] The D-lactic acid production of Lactobacillus supernatant was detected using a D-lactic acid assay kit (purchased from Sigma-Aldrich). The method employed by this kit involves the oxidation of D-lactic acid by a specific D-lactic acid hydrogenase, resulting in a colorimetric reaction that is proportional to the D-lactic acid concentration. The absorbance at 450 nm was measured.

[0122] ②L-lactic acid detection

[0123] The supernatant was filtered using a 0.22 μm sterile filter membrane. The L-lactic acid concentration was determined using a biosensor.

[0124] ③ Hydrogen peroxide detection

[0125] 100 μl of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mM hydrogen peroxide standard solutions were prepared using PBS. 200 μl of enzyme reagent (0.5 mM / L 4-aminoantipyrrolidone (4-AA), 0.2 mM / L peroxidase) and 200 μl of phenol solution (19.66 mM / L) were added, and the mixture was thoroughly mixed. The mixture was incubated at 37°C for 20 min. 200 μl of each solution was then evenly transferred to a 96-well plate, and the absorbance of each well was measured at 505 nm. A standard curve for hydrogen peroxide solutions was established based on the results.

[0126] Inoculate Lactobacillus onto MRS medium at a 0.5% inoculum and incubate anaerobically at 37°C for 24 hours. After 24 hours of culture, centrifuge at 4000 rpm for 20 minutes at 4°C, discard the supernatant, wash with PBS buffer, and resuspend the cells in 0.5% glucose solution to OD0.05. 600 =1.0. Incubate at 37℃ and 170 rpm for 5 hours, centrifuge at 10,000 rpm for 5 min, collect the supernatant, and filter sterilize through a 0.22 μm filter. Take 100 μl of the supernatant, add 200 μl each of enzyme reagent and phenol reagent, mix thoroughly, and incubate at 37℃ for 20 min. Evenly transfer 200 μl to a 96-well plate, and measure the absorbance of each well at a wavelength of 505 nm.

[0127] Table 3 shows the results of D-lactic acid, L-lactic acid, and hydrogen peroxide detection in *Lactobacillus japonicus* HY1335 cultured in MRS broth for 24 hours. The D-lactic acid content in the metabolites of *Lactobacillus japonicus* HY1335 was 5.23 g / L, and the L-lactic acid content was 0 g / L. The total acid production of *Lactobacillus japonicus* HY1335 was 5.23 g / L, which is basically the same as that of *D. japonicus* (DJS-Lactobacillus dendriticus) (5.48 g / L). H2O2 can directly kill pathogens and can also... The enzyme-catalyzed oxidation forms halides with stronger bactericidal effects. As shown in Table 3, *Lactobacillus japonicus* HY1335 can produce 3170 μM hydrogen peroxide, while *Lactobacillus delbrueckii* (DJS-Lactobacillus) produces only 80.67 μM hydrogen peroxide. The ability of *Lactobacillus japonicus* HY1335 to produce hydrogen peroxide is much higher than that of *Lactobacillus delbrueckii* (DJS-Lactobacillus). Therefore, *Lactobacillus japonicus* HY1335 has the potential to kill pathogens, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections, and treat inflammation.

[0128] Table 3 Results of metabolite content determination

[0129]

[0130] Example 3: Application Function Analysis

[0131] (1) Antibacterial test

[0132] ① Preparation of working bacterial culture: Inoculate Lactobacillus into MM medium (MRS broth modified medium, composition: peptone 10g / L, beef extract 5.0g / L, yeast extract 4.0g / L, glucose 15g / L, K2HPO4 2.0g / L, triammonium citrate 1.0g / L, sodium acetate 2.5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L) at a anaerobic workstation and culture it. Six pathogenic bacteria (Gardnerella vaginalis ATCC14018, GV; Escherichia coli ATCC 25922, EC; Staphylococcus aureus ATCC25923, SA; Prevotella bivia NCTC11156, PB; and Candida albicans ATCC 10231, CA) were cultured in suitable media. After culturing the target strains, the supernatant was collected by centrifugation and filtered through a 0.22 μm filter to obtain a cell-free supernatant, which was used immediately or stored at -80°C. OD was measured after culturing the pathogenic bacteria. 600 Value, diluted to approximately OD 600 The value was 0.005 (viable bacteria count maintained at 5.0 × 10⁻⁵). 5CFU / mL ~5.0×10 6 CFU / mL)(WS / T 650—2019 Evaluation Method for Antibacterial and Bacteriostatic Effects).

[0133] ② Interaction: Take equal volumes of supernatant and pathogenic bacterial suspension and mix thoroughly. Immediately transfer 100 μL of the well-mixed bacterial suspension into a blank 96-well plate and measure the OD. 600 The remaining culture medium was placed at 37°C and cultured anaerobically or aerobically, depending on the culture conditions of the pathogen. After 48 hours of culture, 100 μL of the well-mixed bacterial solution was transferred into a blank 96-well plate to measure the OD. 600 Set up a blank control group. Each sample has two replicates. Calculate the inhibition rate of Lactobacillus against pathogenic bacteria using the formula below.

[0134] Antibacterial rate = (AB) / A * 100%

[0135] A: The increase in OD in the positive control group (i.e., blank culture medium) within 48 hours 600 value;

[0136] B: Increased OD in the experimental group within 48 hours 600 value.

[0137] Two different strains of the same species, along with the positive control drug Dingjunsheng and the screened Lactobacillus japonicus HY1335, were selected for antibacterial performance testing. The results are shown in Table 4. This strain exhibited strong antibacterial rates against GV, PB, EC, SA, and CA, and its antibacterial performance against GV, PB, EC, and SA was superior to both the control group of the same species and the positive control group. This strain can simultaneously exert a strong inhibitory effect on these four pathogenic bacteria, an effect that neither the positive control nor the control group of different strains of the same species could achieve.

[0138] Table 4 Results of antibacterial experiment

[0139]

[0140] Note: The strains numbered HY02405 and HY01361 in Table 4 are Lactobacillus japonicus obtained by the inventors from the samples collected in Example 1, in order to verify the inhibition rate of Lactobacillus japonicus HY1335 and two strains of the same species against different pathogenic bacteria.

[0141] (2) Experiment on the interaction between Lactobacillus and GV co-culture

[0142] Lactobacillus culture: Take Lactobacillus japonicus glycerol tubes and inoculate them into MM medium (MRS broth modified medium, composition: peptone 10g / L, beef extract 5.0g / L, yeast extract 4.0g / L, glucose 15g / L, K2HPO4 2.0g / L, triammonium citrate 1.0g / L, sodium acetate 2.5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L). Concentrate or dilute the cultured bacterial solution to approximately 1×10⁻⁶. 9 The concentration of CFU / mL was used as the working culture solution for Lactobacillus.

[0143] GV culture: GV glycerol tubes were inoculated into BHI liquid medium containing 10% fetal bovine serum and cultured anaerobically at 37°C. After culture, the bacterial culture was centrifuged to remove the supernatant, and then the bacterial concentration was adjusted to approximately 1.0 × 10⁻⁶ using fresh medium. 7 CFU / mL was used as the working bacterial culture for GV.

[0144] 400 μL of Lactobacillus and GV working bacterial suspensions were inoculated separately into 40 ml of BHI liquid medium containing 10% fetal bovine serum. GV was inoculated alone as a blank control group. Each group was repeated in two replicates. The cultures were placed in an anaerobic workstation, and samples were taken 27 h after incubation. The viable count of GV was detected using quantitative real-time qPCR with GV-specific probe primers. Lactobacillus janniae and GV were co-cultured for 27 h, and the viable count of GV was also detected. The experimental results are shown in Table 5. After co-culturing Lactobacillus janniae HY1335 with GV for 27 h, the inhibition rate reached 89%, indicating that this strain has a strong inhibitory effect on the growth of GV.

[0145] Table 5 Antibacterial experiment of co-culture of Lactobacillus and GV.

[0146]

[0147] (3) Biofilm clearance experiment

[0148] 1) Experiment on GV biofilm clearance by Lactobacillus

[0149] Adjust the GV culture solution to 1.0 × 10⁻⁶. 7 CFU / mL was inoculated into 96-well plates, with two groups and four replicates per group. Equal volumes of blank MM liquid medium (MRS modified broth medium, composition: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K₂HPO₄ 2.0 g / L, triammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and Lactobacillus japonicus HY1335 culture supernatant were added to each group. The plates were incubated at 37℃ in an anaerobic environment for 24 h.

[0150] After cultivation, the inhibitory effect of *Lactobacillus japonicus* HY1335 on GV biofilm formation was determined using the crystal violet staining method in microplates. The results are shown in Table 6. The supernatant of *Lactobacillus japonicus* HY1335 showed a strong inhibitory effect on GV biofilm formation, reaching 91.4%. Therefore, it can effectively inhibit GV growth.

[0151] Table 6. Results of the experiment on the removal of GV biofilm by Lactobacillus.

[0152] strain Inhibition rate of Lactobacillus supernatant on GV biofilm formation Lactobacillus japonicus HY1335 91.4％±2.4％

[0153] 2) Lactobacillus biofilm clearance experiment on CA

[0154] Biofilm inhibition assay: The CA culture solution was adjusted to 1.0 × 10⁻⁶. 7 CFU / mL was inoculated into 96-well plates, with two groups and four replicates per group. Equal volumes of Sabouraud dextrose broth and Lactobacillus supernatant were added to each group, respectively. The plates were incubated at 37°C in an anaerobic environment for 24 hours. After incubation, the inhibitory effect of Lactobacillus on CA biofilm formation was determined using crystal violet staining in microplates.

[0155] Biofilm removal experiment: The bacterial culture of CA was adjusted to 1.0 × 10⁻⁶. 7 CFU / mL was inoculated into 96-well plates, with two groups and four replicates per group. The plates were incubated at 37℃ in an anaerobic environment for 24 hours. After 24 hours, the mixture in the wells was discarded, and the plates were washed with sterile PBS. Equal volumes of MM medium (MRS modified broth medium, composition: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K₂HPO₄ 2.0 g / L, triammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and Lactobacillus supernatant were added to each group sequentially, and the plates were incubated at 37℃ for 24 hours. After incubation, the effect of the Lactobacillus supernatant on the removal of mature CA biofilm was determined using the crystal violet staining method in microplate microplates.

[0156] The experimental results are shown in Table 7. Lactobacillus janniae HY1335 has a certain inhibitory or destructive effect on the formation of CA biofilm and the existing CA biofilm.

[0157] Table 7 Results of Lactobacillus's biofilm clearance experiment on CA

[0158]

[0159] (4) Cell adhesion experiment

[0160] ① Preparation of working bacterial solution: Culture Lactobacillus and detect OD of the bacterial solution. 600The Lactobacillus culture medium was centrifuged at 4000 rpm for 5 min at 4℃, the supernatant was discarded, and the bacteria were washed three times with PBS. Finally, the Lactobacillus was resuspended in MEM complete medium (purchased from Zhongqiao Xinzhou). An appropriate amount of the resuspended liquid was plated to detect the number of viable bacteria, which is L1.

[0161] ② Cell culture: HeLa cells were seeded in 24-well plates, with 1.5 × 10⁶ cells per well. 5 HeLa cells were cultured at a density of 1 cell / mL for 16 hours until the confluence reached 90%. The cells were washed in 24-well plates with serum-free MEM medium (purchased from Zhongqiao Xinzhou), and then counted and recorded as C1.

[0162] ③ Interaction: Lactobacillus was inoculated into cells at a ratio of 100:1 and incubated at 37°C with 5% CO2 for 1.5 h. The supernatant after centrifugation was added to the cells as a blank control. After 1.5 h, the culture medium in the wells was collected, and the cells in the wells were washed with MEM medium. Then, trypsin was added to each well for digestion, and the reaction was terminated by adding MEM complete medium. The suspension was collected, and a portion was taken to count the number of cells (C2). The viable number of Lactobacillus bacteria adhering to the cells (L2) was detected by plate spreading.

[0163] ④ Calculate the number of adherent cells and the adhesion rate: Average number of adherent Lactobacillus cells = L2 / C2.

[0164] Lactobacillus japonicus HY1335 showed a single-cell adhesion rate of 3.60 CFU to HeLa cells, indicating that this strain has good adhesion or colonization characteristics to vaginal epithelial cells.

[0165] (5) Efficacy test of animal model of BV (bacterial vaginosis).

[0166] Healthy SPF-grade female Balb / c mice, 6-8 weeks old, were used for modeling. After acclimatization culture, the animals were randomly divided into three groups of 8 mice each: a model group (M), an experimental group (Lactobacillus jenny HY1335), and a positive control group (DJS). Before inoculation with the pathogenic bacteria, all animals in each group were subcutaneously injected with estradiol benzoate, followed by vaginal administration of the same concentration of GV (20 μl) to establish the BV pathogenic bacteria model. After successful modeling, the experimental group received vaginal administration of Lactobacillus jenny HY1335 solution (1×10⁻⁶) for 5 consecutive days. 10 CFU / mL, 20μL), the positive control group received an equal volume of Lactobacillus delbrueckii solution (Dingjun Shengzhong strain, 1×10 CFU / mL, 20μL) vaginally for 5 consecutive days. 10The model group received an equal volume of saline (CFU / mL, 20 μL). After treatment, each animal was irrigated with 50 μL of PBS four times a day. The fluid was collected and placed in 1.5 mL Eppendorf tubes and stored at -80°C. The GV load in the irrigated fluid was detected by qPCR, and the differences in GV load in the irrigated fluid among the groups were compared.

[0167] Experimental results are as follows Figure 4 As shown in the figure. The experimental results showed that the use of Lactobacillus japonicus HY1335 to treat BV infection animal models significantly reduced the GV content in their vaginas, and the treatment effect was significantly better than that of the positive control drug (DJS), indicating that this strain has a good therapeutic effect on BV in mice.

[0168] Example 4: Molecular markers for Lactobacillus janniae HY1335

[0169] To rapidly screen or identify *Lactobacillus japonicus* HY1335 with excellent antibacterial activity and good therapeutic effect against BV, the inventors developed a molecular marker for *Lactobacillus japonicus* HY1335, the specific process of which is as follows:

[0170] a. Screening for specific nucleotide sequences: The whole genome of strain HY1335 was sequenced and analyzed. The genome sequences of Lactobacillus janniae strains included in the NCBI database were compared and analyzed to screen for specific nucleotide sequences of Lactobacillus janniae HY1335.

[0171] b. Primer Design: Primers were designed for the specific nucleotide sequences screened in step a. Primer fragments with predicted product lengths of approximately 200 to 800 bp were designed for the screened specific nucleotide sequences, and 11 primer pairs were designed as controls for other nucleotide sequences across the entire genome. Primer sequences are shown in Table 8.

[0172] Table 8 Primer List

[0173]

[0174]

[0175] c. Primer Screening: Lactobacillus japonicus HY1335 and three strains of the same species were used as control groups (specific strain information is shown in Table 9) for template preparation. Single colonies were picked from 50 μL of lysis buffer (TaKaRa), centrifuged briefly, lysed at 80℃ for 15 min, and centrifuged at 4000 rpm for 5 min. The supernatant was used as the template. 1-p3 was selected for PCR amplification through primer screening. The PCR amplification system consisted of 12.5 μL Taq enzyme, 1 μL F, 1 μL R, 1.5 μL template, and ddH2O to a final volume of 25 μL. The PCR reaction conditions are shown in Table 10. After the PCR experiment, 1.5 g of agarose was added to 100 mL of 1×TAE buffer, heated thoroughly to melt, and then 5 μL of Gel Red dye was added. The mixture was poured into a gel plate and allowed to solidify. 4 μL of the PCR product was then subjected to agarose gel electrophoresis. The electrophoresis conditions were as follows: voltage 130V, current 400mA, time 35min.

[0176] Table 9. Strain Information

[0177] strain number Identification Name HY1335 Lactobacillus jensenii HY00888 Lactobacillus jensenii HY01360 Lactobacillus jensenii HY02445 Lactobacillus jensenii

[0178] Note: The strains numbered HY00888, HY01360 and HY02445 in Table 9 are Lactobacillus japonicus selected from naturally fermented foods with regional characteristics, vaginal secretion samples collected by hospital gynecologists from volunteers who met the inclusion criteria, and human samples (feces and breast milk). In order to verify the specificity of the above-mentioned specific nucleotide sequence for Lactobacillus japonicus HY1335, the above primers were used to perform PCR amplification on HY1335 and the three strains of the same species, respectively.

[0179] Table 10 PCR Reaction Conditions

[0180]

[0181] Among primers designed for specific nucleotide sequences, using 1-p3 as a primer in PCR amplification experiments, only HY1335 produced a specific amplification band, which was clear and highly concentrated with relatively few primer dimers. HY00888, HY01360, and HY02445, however, showed no amplification bands. Agarose gel electrophoresis patterns are shown below. Figure 5 However, when using primers designed with other nucleotide sequences for PCR amplification experiments, there were instances where control bacterial groups also amplified bands or exhibited a high number of primer dimers (for example, 2-p3; see agarose gel electrophoresis pattern for details). Figure 6 Therefore, primer 1-p3 is a molecular marker primer for HY1335, and the amplification product generated by this primer is a molecular marker of Lactobacillus japonicus HY1335. The amplification product of primer 1-p3 is 232 bp in size, and its nucleotide sequence is as follows:

[0182] TACGGCCGTCTGGTAAAGTTAAAACCAAAACAAGATCATTTTTCACTTCGTAAGTTATCTATTG

[0183] GTCTTATTTCTGTTACCATAGGTATTACCTTATACTTGGGAGTGGACAACTCCTATGTTATGGCTGATA

[0184] CGAATTTGAGGGATAATAACAATCAACAATTAGCCGGATCGGGTAATGGCACCAATATAATAAATGAT

[0185] AAAGTTAAAATTCCAACCGGTGCTTTTCAGT(SEQ ID NO:24)

[0186] This confirmed that the nucleotide sequence was part of the specific nucleotide sequence obtained in step a, further verifying that primer 1-p3 can be used as a specific primer for Lactobacillus janniae HY1335. The nucleotide sequence shown in SEQ ID NO:24 is a specific nucleotide sequence marker for Lactobacillus janniae HY1335, which provides a reliable tool and basis for identifying and detecting Lactobacillus janniae HY1335.

[0187] During the experiment, it was found that when using the 1-p3 primer to sequence multiple Lactobacillus janniae HY1335 samples, the amplified product sequences differed from the above nucleotide sequences in a few bases. However, compared with the control bacteria, they all had specific amplification bands, and the bands were clear and highly concentrated, with relatively few primer dimers.

[0188] In one experiment, the nucleotide sequence of the product amplified from a Lactobacillus japonicus HY1335 sample using the 1-p3 primer was shown in SEQ ID NO:25. The nucleotide sequence was found to be 99.57% identical to the nucleotide sequence shown in SEQ ID NO:24.

[0189] TACGGCCGTCTGGTAAAGTTAAAACCAAAACAAGATCATTTTTCCCTTCGTAAGTTATCTATTGGTCTTATTTCTGTTACCATAGGTATTACCTTATACTTGGGAGTGGACAACTCCTATGTTATGGCTGATACGAATTTGAGGGATAATAACAATCAACAATTAGCCGGATCGGGTAATGGCACCAATATAATAAATGATAAAGTTAAAATTCCAACCGGTGCTTTTCAG (SEQ ID NO: 25)

[0190] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0191] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A molecular marker for Lactobacillus jenny, characterized in that, The *Lactobacillus japonicus* was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26505. The molecular marker contains a nucleotide sequence selected from one of the following: (1) The nucleotide sequence shown in SEQ ID NO:24; (2) A nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence shown in SEQ ID NO:24; (3) A nucleotide sequence having one or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:

24.

2. The molecular marker according to claim 1, characterized in that, The molecular marker has a nucleotide sequence as shown in SEQ ID NO:

24.

3. A primer set for amplifying the molecular marker of claim 1, characterized in that, Including forward primers and reverse primers; The forward primer comprises the nucleotide sequence shown in SEQ ID NO:2; The reverse primer comprises the nucleotide sequence shown in SEQ ID NO:

3.

4. The use of the molecular marker according to claim 1 in the identification and / or detection of Lactobacillus janniae, which was deposited on February 7, 2023 at the China General Microbiological Culture Collection Center with accession number CGMCC No. 26505.

5. A reagent kit, characterized in that, Includes the primer set as described in claim 3 and optionally PCR amplification reagents.

6. A method for identifying and / or detecting Lactobacillus jenny, characterized in that, include: The DNA of the strain to be tested is amplified using the primer set described in claim 3 or the kit described in claim 5. The amplification results were compared with the molecular markers described in claim 1. Based on the comparison results, it was determined whether the strain to be tested was *Lactobacillus janniae*. The Lactobacillus janniae was deposited on February 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26505.

7. The method according to claim 6, characterized in that, The amplification result having at least 97%, 98%, 99% or higher identity with the molecular marker described in claim 1 is an indication that the strain to be detected is *Lactobacillus janniae*.

8. The method according to claim 6, characterized in that, The amplification process includes: 1.0-2.5 U / μL of Taq enzyme, 0.4-2.0 pmol / μL of the forward primer from the primer set, 0.4-2.0 pmol / μL of the reverse primer from the primer set, and the DNA of the strain to be tested.

9. The method according to claim 6, characterized in that, The reaction conditions for amplification treatment are: Pretreatment: Denaturation at 90–98℃ for 3–8 min; PCR reaction: denaturation at 90-98℃ for 10-20s, annealing at 55-65℃ for 10-20s, extension at 70-75℃ for 50-70s, for a total of 30-35 cycles; Extension: Extend at 70-75℃ for 3-8 minutes.

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