Primer, probe and method for detecting lactobacillus casei in human intestinal tract

Abstract

The invention discloses a primer pair for detecting lactobacillus casei in human intestinal tracts, which comprises a group of primer pairs: a primer pair 1: an upstream primer COG0405F1 with a nucleotide sequence of 5 '-CCATGCAGCTTRATCCGAAT-3', and a downstream primer COG0405R1 with a nucleotide sequence of 5 '-CACCATATTACCATCCGAT-3'; the primer pair 2 comprises an upstream primer COG0405F2 of which the nucleotide sequence is 5 '-CACCACCAGATTGAAGCGAT-3', and a downstream primer COG0405R2 of which the nucleotide sequence is 5 '-GCAAGCAACTGATTCGGATT-3'; the primer and the probe are designed on the basis of a specific glutathione-like hydrolase YwrD precursor enzyme gene (COG0405) of the casei casei, so that the primer and the probe have high interspecific specificity, and the casei casei can be accurately and quickly identified under the background of complex flora in human intestinal tracts; the defect that in the prior art, specific detection is difficult to carry out in the coexistence environment of various kinds of lactobacillus is overcome. The method is easy and convenient to operate, high in sensitivity, good in repeatability and suitable for qualitative and quantitative analysis of the casei casei in human intestinal samples, dairy products and other environmental samples.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to primers, probes and methods for detecting Lactobacillus casei in the human gut. Background Technology

[0002] *Lactobacillus casei*, originally belonging to the genus *Lactobacillus*, was reclassified to the new genus *Lacticaseibacillus* in 2020 based on whole-genome phylogenetic reconstruction. *Lactobacillus casei* is widely found in the oral and gastrointestinal mucosa of humans and animals, and is also common in dairy products, plant-based fermented foods, and the natural environment. Its metabolic type is heterolactic fermentation, with moderate acid production and weak gas production. It grows in a microaerophilic to anaerobic manner and has a certain tolerance to acids and bile salts. Many strains have been reported to possess adhesion proteins and exfoliated polysaccharides on their surface. In recent years, increasing research has shown that *Lactobacillus casei* possesses a series of probiotic functions, exhibiting good effects in intestinal barrier regulation, immune regulation, and antagonism of pathogens. For example, *Lactobacillus casei* KBL382 can significantly alleviate skin symptoms in atopic dermatitis (AD) model mice, significantly reduce the production of Th1, Th2, and Th17 cytokines in the skin tissue of AD mice, and increase the production of anti-inflammatory cytokines IL-10 and transforming growth factor-β (TGF-β). Lactobacillus casei ATCC334 can inhibit joint swelling in arthritic rats, reduce arthritis severity scores, and decrease the expression of multiple pro-inflammatory cytokines in rat blood.

[0003] 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. Common lactobacilli in the human gut include *Lacticaseibacillus* (e.g., *L. casei*, *L. paracasei*, *L. rhamnosus*), *Lactiplantibacillus* (e.g., *L. plantarum*), *Limosilactobacillus* (e.g., *L. reuteri*, *L. fermentum*), and *Levilactobacillus* (e.g., *L. brevis*). In a previous study, we used *Lactobacillus* groEL gene sequencing to analyze the abundance and distribution of gut lactobacilli from 200 individuals from 10 cities in China, detecting a total of 51 species in the human gut. The most abundant lactobacilli in the human gut are, in descending order: *Lactobacillus mucosa*, *Lactobacillus plantarum*, *Lactobacillus salivarius*, *Lactobacillus rumenans*, *Lactobacillus gasseri*, *Lactobacillus rhamnosus*, *Lactobacillus curvatureii*, *Lactobacillus deuterans*, *Lactobacillus fermentum*, and *Lactobacillus oralis*. Furthermore, *Lactobacillus casei* ranks 12th in abundance among human gut lactobacilli, and its closest relative ranks 24th.

[0004] To date, there are 27 patents related to primers and probes for *Lactobacillus casei*. These patents mainly target the identification and detection of *Lactobacillus casei* in food products such as dairy products and beer, and some of these patents focus on the identification of *Lactobacillus casei* groups (mainly referring to *Lactobacillus casei* / *paracasei* and *Lactobacillus rhamnosus*). For example, patent CN112029884B proposes molecular markers, detection primers, and detection methods for identifying *Lactobacillus casei* groups, which include *Lactobacillus casei* / *paracasei* and *Lactobacillus rhamnosus*. CN114736976B proposes a specific primer and kit for identifying *Lactobacillus casei*. For the detection of *Lactobacillus casei* in beer, the validation strains used are: *Lactobacillus casei*, *Lactobacillus brevis*, *Lactobacillus plantarum*, *Lactobacillus delbrueckii*, *Lactobacillus acidophilus*, *Bifidobacterium longum*, *Lactococcus lactis*, *Streptococcus thermophilus*, *Bacillus subtilis*, *Pediococcus pentosaceus*, *Leuconostoc mesenteroides*, *Pseudomonas aeruginosa*, and *Escherichia coli*. Patent CN101712989A proposes a rapid method for the identification and quantification of *Lactobacillus casei* in probiotic dairy products. Primers are designed based on the DNA-specific gene fragments in the 16S rRNA of each probiotic, applicable to single strains containing only *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus bulgaricus*, *Lactobacillus faecalis*, *Lactobacillus fermentum*, *Lactobacillus salivarius*, and *Bacillus natto*. While identifying *Lactobacillus casei* in samples with a limited number of lactobacillus species is generally feasible, there is currently no patent specifically designed to identify *Lactobacillus casei* in the context of the complex gut microbiota, encompassing 51 lactobacillus species. This patent designs species-specific primers and probes for *Lactobacillus casei* based on the *Lactobacillus casei* glutathione hydrolase YwrD precursor enzyme gene, and provides reaction systems and conditions for PCR and real-time quantitative PCR. Summary of the Invention

[0005] The purpose of this invention is to provide a primer pair, probe, and detection method for the specific detection of Lactobacillus casei in the human gut.

[0006] The object of this invention is achieved as follows: In a first aspect, this invention provides a primer pair for detecting Lactobacillus casei in the human gut, comprising a set of primer pairs:

[0007] Primer pair 1: Upstream primer COG0405F1, nucleotide sequence 5'-CCATGCAGCTTRATCCGAAT-3', downstream primer COG0405R1, nucleotide sequence 5'-CACCATATTACCATCCCGAT-3';

[0008] Primer pair 2: Upstream primer COG0405F2, nucleotide sequence 5'-CACCACCAGATTGAAGCGAT-3', downstream primer COG0405R2, nucleotide sequence 5'-GCAAGCAACTGATTCGGATT-3'.

[0009] Secondly, the present invention provides a probe for use with the primers, the nucleotide sequence of which is: 5'-ACCCGCAAGCCATGCAG-3', wherein the 5' end is labeled with a FAM fluorescent reporter group and the 3' end is labeled with a BHQ1 fluorescent quencher group.

[0010] Thirdly, the present invention provides a kit for detecting Lactobacillus casei in the human gut containing at least one primer pair and / or probe.

[0011] Fourthly, the present invention provides a method for detecting Lactobacillus casei in the human gut using primer pairs.

[0012] As a further preferred embodiment of the present invention, the PCR reaction program includes: pre-denaturation at 95°C for 5 min; 30-35 cycles of denaturation at 95°C for 10-30 s, annealing at 58°C for 30 s, extension at 72°C for 15-20 s; and final extension at 72°C for 5 min.

[0013] As a further preferred embodiment of the present invention, the kit comprises one or more of Taq mix, 2×SYBR Green qPCRMaster Mix, double-distilled water, and DNA extraction reagent.

[0014] Compared with existing technologies, the beneficial effects of this invention are as follows: The primers, probes, and detection methods provided by this invention solve the problem that existing technologies cannot specifically identify *Lactobacillus casei* in the context of complex human gut microbiota. It is highly specific, easy to operate, and widely applicable, enabling rapid qualitative and precise quantitative detection of *Lactobacillus casei* in human gut samples, food samples, and environmental samples. This provides important technical support for research on the probiotic functions of *Lactobacillus casei*, gut microbiota analysis, and quality control of related products. Attached Figure Description

[0015] 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.

[0016] Figure 1 The gel electrophoresis image (COG0405F1 / COG0405R1) was validated using specific primers.

[0017] Figure 2 Gel electrophoresis image validated with specific primers (COG0405F2 / COG0405R2).

[0018] Figure 3 qPCR amplification curves of Lactobacillus casei (COG0405F1 / COG0405R1) (parallel samples).

[0019] Figure 4 qPCR melting curves of Lactobacillus casei (COG0405F1 / COG0405R1) (parallel samples).

[0020] Figure 5 qPCR amplification curve of Lactobacillus casei (COG0405F2 / COG0405R2).

[0021] Figure 6 qPCR melting curve of Lactobacillus casei (COG0405F2 / COG0405R2). Detailed Implementation

[0022] 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.

[0023] Example 1

[0024] Screening of species-specific sequences of Lactobacillus casei

[0025] We initially detected 51 species of lactobacilli in the human gut, and the abundance ranking of these lactobacilli in the human gut is shown in Table 1. Subsequently, we downloaded the genomes of 10 strains of each species from the NCBI (National Center for Biotechnology Information) website (prioritizing strains from human and animal gut sources). For databases with fewer than 10 strains, we downloaded the genomes of all available strains. Ultimately, we obtained 420 genome data points from 51 lactobacilli species, including 10 strains of *Lactobacillus casei*, whose strain information and origin are shown in Table 2.

[0026] Table 1 Ranking of Lactobacillus abundance in the human gut

[0027] ranking bacteria ranking bacteria 1 Limosilactobacillus mucosae 27 Ligilactobacillus acidipiscis 2 Lactiplantibacillus plantarum 28 Limosilactobacillus gastricus 3 Ligilactobacillus salivarius 29 Loigolactobacillus coryniformis 4 Lactobacillus ruminans 30 Latilactobacillus curvatus 5 Lactobacillus rumen 31 Schleiferilactobacillus harbinensis 6 Lactobacillus rhamnosus 32 Companilactobacillus mindensis 7 Lactobacillus crispatus 33 Lactobacillus acetotolerans 8 Lactobacillus delbrueckii 34 Lentilactobacillus buchneri 9 Limosilactobacillus fermentum 35 Lactobacillus kefiranofaciens 10 Limosilactobacillus oris 36 Limosilactobacillus bread 11 Limosilactobacillus vaginalis 37 Lentilactobacillus parafaraginis 12 Lactobacillus casei 38 Levilactobacillus brevis 13 Limosilactobacillus reuteri 39 Lentilactobacillus diolivorans 14 Lactobacillus amylovorus 40 Lapidilactobacillus dextrinicus 15 Lactobacillus sakei 41 Limosilactobacillus ingluviei 16 Lactobacillus johnsonii 42 Lactobacillus intestinalis 17 Lactobacillus acidophilus 43 Lentilactobacillus parabuchneri 18 Lactobacillus helveticus 44 Ligilactobacillus agilis 19 Furfurilactobacillus rossiae 45 Lentilactobacillus hilgardii 20 Lactiplantibacillus pentosus 46 Lacticaseibacillus manihotivorans 21 Limosilactobacillus frumenti 47 Limosilactobacillus secaliphilus 22 Lactobacillus gallinarum 48 Latilactobacillus fuchuensis 23 Limosilactobacillus pontis 49 Ligilactobacillus murinus 24 Lacticaseibacillus paracasei 50 Levilactobacillus spicheri 25 Lactobacillus iners 51 Paucilactobacillus vaccinostercus 26 Fructilactobacillus sanfranciscensis

[0028] Table 2. Information on 10 Lactobacillus casei strains from which the genome originated.

[0029] strain number source Lacticaseibacillus casei 867_LCAS Human source Lacticaseibacillus casei BCRC 80156 Human source (saliva) Lacticaseibacillus casei BIO5773 Human source Lacticaseibacillus casei DS1_13 Human source Lacticaseibacillus casei DS13_13 Human source Lacticaseibacillus casei LC130 Human source (feces) Lacticaseibacillus casei LMG 24099 Human source (blood) Lacticaseibacillus casei LMG 24102 Human-derived (heart tissue) Lacticaseibacillus casei N87 Human source (feces) Lacticaseibacillus casei UD1001 Human source

[0030] Subsequently, we compared these genomes with the COG (Clusters of Orthologous Groups) database and found that the gene annotated as COG0405 is only present in *Lactobacillus casei*. This gene expresses a glutathione hydrolase-like precursor enzyme YwrD, and the sequences of this gene in these 10 *Lactobacillus casei* strains are highly identical. Combined with NCBI-BLAST alignment, the specificity and accuracy of this target gene sequence were confirmed. The COG0405 gene sequences of the 10 *Lactobacillus casei* strains are as follows:

[0031] >casei867_LCAS_01870 Glutathione hydrolase-like YwrD proenzyme

[0032]

[0033] >caseiBCRC 80156_01626 Glutathione hydrolase-like YwrD proenzyme

[0034]

[0035] >caseiBIO5773_01237 Glutathione hydrolase-like YwrD proenzyme

[0036]

[0037] >caseiDS1_13_01581 Glutathione hydrolase-like YwrD proenzyme

[0038]

[0039] >caseiDS13_13_01617 Glutathione hydrolase-like YwrD proenzyme

[0040]

[0041] >caseiLC130_00149 Glutathione hydrolase-like YwrD proenzyme

[0042]

[0043] >caseiLMG24099_00180 Glutathione hydrolase-like YwrD proenzyme

[0044]

[0045] >caseiLMG24102_00179 Glutathione hydrolase-like YwrD proenzyme

[0046]

[0047] >caseiN87_00259 Glutathione hydrolase-like YwrD proenzyme

[0048]

[0049] >caseiUD1001_02144 Glutathione hydrolase-like YwrD proenzyme

[0050]

[0051] Example 2

[0052] Design of specific primers and probes

[0053] We used SnapGene software for primer selection and design, and Oligo7 software for probe design. We ultimately selected two primer pairs: Primer 1: upstream primer COG0405F1, 5'-CCATGCAGCTTRATCCGAAT-3', Tm 51°C; downstream primer COG0405R1, 5'-CACCATATTACCATCCCGAT-3', Tm 53°C, sequence length 155 bp; Primer 2: upstream primer COG0405F2, 5'-CACCACCAGATTGAAGCGAT-3', Tm 53°C; downstream primer COG0405R2, 5'-GCAAGCAACTGATTCGGATT-3', Tm 53°C, sequence length 98 bp. A specific probe was designed using Oligo7 software targeting the COG0405 gene fragment of Lactobacillus casei and primer 2. The probe sequence is 5'-ACCCGCAAGCCATGCAG-3', where the 5' end is labeled with the FAM reporter fluorescent group and the 3' end is labeled with the BHQ1 quencher fluorescent group.

[0054] Example 3

[0055] Specific primer PCR, gel electrophoresis verification

[0056] Validation strains: 13 Lactobacillus species: 1. *Lactiplantibacillus plantarum* 1-7; 2. *Ligilactobacillus salivarius* 27-2; 3. *Lactaseibacillus rhamnosus* 16-1; 4. *Lactobacillus crispatus* 16-3; 5. *Limosilactobacillus fermentum* 27-3; 6. *Limosilactobacillus vaginalis* 4-9; 7. *Limosilactobacillus reuteri* 16-5; 8. *Latilactobacillus sakei* 1-3; 9. *Lactaseibacillus paracasei* 5-1; 10. * *Loigolactobacillus scoryniformis* 4-7; *Latilactobacillus curvatus* 1-10; *Lentilactobacillus buchneri* 36-3; *Levilactobacillus brevis* 7-10;

[0057] Five non-lactobacterial species: 14 *Weissella confuse* 4-4; 15 *Weissella cibaria* 4-2; 16 *Prdiococcus acidilactici* 30-1; 17 *Leuconostoc mesenteroides* 13-1; 18 *Leuconostoclactis* 11-4;

[0058] 19 Lacticaseibacillus casei 6-5; 20 Lacticaseibacillus casei 8-4; 21 Lacticaseibacillus casei 16-4.

[0059] Strain activation: The above strains were streaked into MRS solid medium at an inoculum of 1% and cultured anaerobically at 37 ℃ for 48 h. Single colonies on the solid medium were selected and inoculated into MRS liquid medium. The cultures were enriched and cultured anaerobically at 37 ℃ for 24 h for activation. 1 mL of bacterial solution was centrifuged to collect bacterial sludge. 2 mL of sterile physiological saline was added and shaken to mix. The bacterial sludge was collected by centrifugation and finally 5 mL of sterile water was added and shaken to mix. This mixture was used as a DNA template.

[0060] The polymerase chain reaction (PCR) system is shown in Table 3. 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 58℃ for 30 s; extension at 72℃ for 15-20 s; after the cycle, extension at 72℃ for 5 min; and cooling to 12℃ to finish.

[0061] Table 3 PCR reaction system

[0062] 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

[0063] The PCR products obtained above were verified by 1% agarose gel electrophoresis. Except for the standard strain of *Lactobacillus casei*, the other control strains did not show any specific amplification bands (primer 1, COG0405F1, COG0405R1, 155 bp and COG0405F2, COG0405R2, 98 bp). A total of 18 control strains were used in this experiment, representing 18 species, including 13 lactobacilli and 5 non-lactobacilli. These are all common intestinal bacteria in humans, and *Lactobacillus rhamnosus* and *Lactobacillus casei* are closely related species to *Lactobacillus casei*. Figure 1 and Figure 2 The results fully demonstrate that our two primer pairs are highly specific for amplifying Lactobacillus casei in the human intestinal environment.

[0064] Example 4

[0065] Primer effectiveness verification based on Lactobacillus casei

[0066] 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 subjected to qPCR detection. The reaction system is shown in Table 4. The qPCR reaction program adopted a three-step method: the first stage was pre-denaturation at 95℃ for 5 min; the second stage was 40 cycles of amplification reaction, each cycle including denaturation at 95℃ for 10 s, annealing at 58℃ for 30 s, and extension at 72℃ for 30 s; the third stage was melting curve analysis, using the instrument's default program to acquire signals and verify the specificity of the amplified products. The results are as follows: Figure 5 and Figure 6 .

[0067] Table 4 qPCR 20μL reaction system

[0068] 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

[0069] from Figure 3 and Figure 5 As can be seen from the amplification curve, the sample exhibits a typical S-shaped curve with a clear inflection point. This indicates that the qPCR reaction is efficient, the primers bind to the template efficiently, the DNA polymerase activity is normal, and the proportions of each component in the reaction system are appropriate, enabling effective amplification. Furthermore, as the number of cycles increases, the fluorescence signal accumulates significantly, providing a reliable basis for quantitative analysis. Figure 4 and Figure 6 The melting curves showed that all reaction products exhibited single, sharp peaks with consistent Tm values, and no primer dimers or other non-specific amplification product peaks, indicating product homology and good primer specificity. This prevented binding to non-target sequences, ensuring the specificity and accuracy of the reaction. Overall, the amplification and melting curves showed a high degree of consistency, demonstrating that the primers possess excellent specificity, amplification efficiency, and reaction uniformity in complex sample environments, enabling precise quantitative detection of *Lactobacillus casei*.

[0070] Example 5

[0071] Methods for detecting Lactobacillus casei in human intestinal samples and other environmental samples

[0072] Conventional PCR and gel electrophoresis detection method: For human intestinal samples and other environmental samples, bacterial DNA is first extracted from the samples using a fecal DNA or environmental DNA extraction kit. Using the extracted DNA as a template, the PCR reaction system consists of: 12.5 μL Taq mix, 1 μL upstream primer F, 1 μL downstream primer R, 1 μL DNA template, and 9.5 μL double-distilled water. The PCR reaction program is as follows: pre-denaturation at 95℃ for 5 min, followed by 30-35 cycles. Each cycle consists of: denaturation at 95℃ for 10-30 s; annealing at 58℃ for 30 s; extension at 72℃ for 15-20 s; final extension at 72℃ for 5 min; and cooling to 12℃ to complete the reaction. After the PCR reaction was completed, 6 μL of the PCR product was analyzed by electrophoresis on a 1% agarose gel. The presence of a single amplified band at the positions of primer 1 (upstream primer COG0405F1, downstream primer COG0405R1, sequence length 155 bp) and primer 2 (upstream primer COG0405F2, downstream primer COG0405R2, sequence length 98 bp) was used to determine whether Lactobacillus casei was present in the sample.

[0073] Real-time quantitative qPCR detection method: Bacterial DNA extraction from the sample is the same as above. Prepare *Lactobacillus casei* DNA standards and plot a standard curve. Using this DNA as a template, employ primer pair COG0405F2 / COG0405R2 and the corresponding primer probes. The reaction system is: 10 μL of 2×SYBR Green qPCR Master 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 a final volume of 20 μL. The three-step qPCR reaction conditions are: the first stage is 95℃ pre-denaturation for 5 min; the second stage is 40 cycles of amplification, each cycle including 95℃ pre-denaturation for 10 s, 58℃ 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 and verify the specificity of the amplified products. Perform qPCR amplification under the same conditions as the above PCR reactions. Finally, qualitative and quantitative detection of Lactobacillus casei in the samples was performed using a standard curve and the Ct value of the test samples.

[0074] The primers and probes provided by this invention can not only perform qualitative and quantitative analysis of Lactobacillus casei in the human gut, but also be applied to the qualitative and quantitative detection of Lactobacillus casei in food, such as dairy products, and other environments.

[0075] 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 primer pair for detecting Lactobacillus casei in the human intestine, characterized by The primer pair comprises: The primer pair 1: an upstream primer COG0405F1 with a nucleotide sequence of 5'-CCATGCAGCTTRATCCGAAT-3', and a downstream primer COG0405R1 with a nucleotide sequence of 5'-CACCATATTACCATCCCGAT-3'; The primer pair 2: an upstream primer COG0405F2 with a nucleotide sequence of 5'-CACCACCAGATTGAAGCGAT-3', and a downstream primer COG0405R2 with a nucleotide sequence of 5'-GCAAGCAACTGATTCGGATT-3'.

2. A probe for detecting Enterococcus casei in the intestinal tract of a human, characterized by, The nucleotide sequence is 5'-ACCCGCAAGCCATGCAG-3', wherein the 5' end is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ1 fluorescent quenching group.

3. A kit for detecting L. casei in the human intestine, characterized in that, The probe comprises at least one pair of primers according to claim 1 and / or the probe according to claim 2.

4. The kit of claim 3, wherein The probe further comprises one or more of Taq mix, 2x SYBR Green qPCR Master Mix, double distilled water, and a DNA extraction reagent.

5. A method for detecting Lactobacillus casei in the human intestine using the primer pair according to claim 1, characterized by, The method comprises the following steps: extracting genomic DNA from a sample to be tested; performing PCR or qPCR amplification using the primer pair; detecting the amplification product, and determining that the sample contains Lactobacillus casei if specific bands of 155 bp or 98 bp are present.

6. The method of claim 5, wherein, The PCR reaction procedure comprises: 95°C pre-denaturation for 5 min; 30-35 cycles of 95°C denaturation for 10-30 s, 58°C annealing for 30 s, and 72°C extension for 15-20 s; and 72°C final extension for 5 min.

7. The method of claim 5, wherein: The sample is derived from human intestinal tract, dairy products, or other environmental samples. The method comprises the following steps: extracting genomic DNA from a sample to be tested; performing PCR or qPCR amplification using the primer pair; detecting the amplification product, and determining that the sample contains Lactobacillus casei if specific bands of 155 bp or 98 bp are present. The PCR reaction procedure comprises: 95°C pre-denaturation for 5 min; 30-35 cycles of 95°C denaturation for 10-30 s, 58°C annealing for 30 s, and 72°C extension for 15-20 s; and 72°C final extension for 5 min. The sample is derived from human intestinal tract, dairy products, or other environmental samples.

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