Primer and probe for detecting probiotics

Abstract

The present invention relates to a primer and a probe for detecting probiotics.

Description

Primers and probes for probiotic detection

[0001] The present invention relates to primers and probes for detecting probiotics.

[0002] Next-generation sequencing (NGS)-based 16S rRNA gene-based microbial community analysis is a powerful method for estimating microbial structures in diverse environments. This gene consists of conserved and variable regions, and the conserved regions are highly phylogenetically conserved, making them the most important gene for bacterial identification. While the 16S rRNA gene is a standard marker for bacterial identification, some bacteria share similar conserved region sequences within this gene within the same genus. Therefore, this gene may not be suitable for quantitatively analyzing single bacteria in fermented foods containing probiotic bacteria.

[0003] In Korea, the production of health functional foods using probiotic bacteria is required to include 19 specific probiotic bacteria species designated by the Ministry of Food and Drug Safety. Therefore, the rpoB gene can be an accurate method for identifying specific bacterial DNA within complex microbial communities. In this study, we designed a primer-probe assay targeting the rpoB gene in 19 probiotic bacteria species, which can be used to detect and quantify nucleic acids using droplet digital polymerase chain reaction (ddPCR).

[0004] The present invention was derived from the above-mentioned needs, and the inventors of the present invention completed the present invention by identifying primers and probes capable of detecting probiotics included in health functional foods and a method for detecting probiotics using the primers and probes.

[0005] In order to solve the above problem, the present invention provides a primer comprising: a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 3 for detecting Lactobacillus acidophilus; a forward primer of SEQ ID NO: 4, a reverse primer of SEQ ID NO: 5, and a probe of SEQ ID NO: 6 for detecting Lacticaseibacillus casei; a forward primer of SEQ ID NO: 7, a reverse primer of SEQ ID NO: 8, and a probe of SEQ ID NO: 9 for detecting Lactobacillus delbrueckiisubsp.bulgaricus; a forward primer of SEQ ID NO: 10, a reverse primer of SEQ ID NO: 11, and a probe of SEQ ID NO: 12 for detecting Limosilactobacillus fermentum; A forward primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14 and a probe of SEQ ID NO: 15 for the detection of Lactobacillus gasseri; a forward primer of SEQ ID NO: 16, a reverse primer of SEQ ID NO: 17 and a probe of SEQ ID NO: 18 for the detection of Lactobacillus helveticus; and a Lacticaseibacillus paracasei subsp. paracasei.A forward primer of SEQ ID NO: 19, a reverse primer of SEQ ID NO: 20, and a probe of SEQ ID NO: 21 for the detection of Lactiplantibacillus paracasei; a forward primer of SEQ ID NO: 22, a reverse primer of SEQ ID NO: 23, and a probe of SEQ ID NO: 24 for the detection of Lactiplantibacillus plantarum; a forward primer of SEQ ID NO: 25, a reverse primer of SEQ ID NO: 26, and a probe of SEQ ID NO: 27 for the detection of Lacticaseibacillus rhamnosus; a forward primer of SEQ ID NO: 28, a reverse primer of SEQ ID NO: 29, and a probe of SEQ ID NO: 30 for the detection of Limosilactobacillus reuterisubsp.reuteri; A forward primer of SEQ ID NO: 31, a reverse primer of SEQ ID NO: 32, and a probe of SEQ ID NO: 33 for the detection of Ligilactobacillus salivarius; a forward primer of SEQ ID NO: 34, a reverse primer of SEQ ID NO: 35, and a probe of SEQ ID NO: 36 for the detection of Bifidobacterium animalissubsp. lactis; a forward primer of SEQ ID NO: 37, a reverse primer of SEQ ID NO: 38, and a probe of SEQ ID NO: 39 for the detection of Bifidobacterium bifidum; a forward primer of SEQ ID NO: 40, a reverse primer of SEQ ID NO: 41, and a probe of SEQ ID NO: 42 for the detection of Bifidobacterium breve; a forward primer of SEQ ID NO: 41, and a probe of SEQ ID NO: 42 for the detection of Bifidobacterium longumsubsp.A forward primer of SEQ ID NO: 43, a reverse primer of SEQ ID NO: 44, and a probe of SEQ ID NO: 45 for the detection of Streptococcus longum; a forward primer of SEQ ID NO: 46, a reverse primer of SEQ ID NO: 47, and a probe of SEQ ID NO: 48 for the detection of Streptococcus thermophilus; a forward primer of SEQ ID NO: 49, a reverse primer of SEQ ID NO: 50, and a probe of SEQ ID NO: 51 for the detection of Lactococcus lactissubsp. lactis; a forward primer of SEQ ID NO: 52, a reverse primer of SEQ ID NO: 53, and a probe of SEQ ID NO: 54 for the detection of Enterococcus faecalis; Provided are primers and probes for simultaneous detection of 19 types of probiotics, including a forward primer of sequence number 55, a reverse primer of sequence number 56, and a probe of sequence number 57 for detection of Enterococcus faecium.

[0006] In one example of the present invention, the primer set may be for use in real-time PCR, droplet digital PCR, or recombinase polymerase amplification.

[0007] In another example of the present invention, the probe is labeled with a fluorophore, a quencher or tetrahydrofuran (THF) at least one selected from the group consisting of a 5' end, a 3' end and an internal sequence, and the fluorophore is fluorescein, 6-carboxyfluorescein (FAM, 6-carboxyfluorescein), hexachloro-6-carboxyfluorescein (HEX, hexachloro-6-carboxyfluorescein), tetrachloro-6-carboxyfluorescein (TET, tetrachloro-6-carboxyfluorescein), 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC, 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein), At least one selected from the group consisting of 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE), 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid, coumarin and coumarin derivatives, cyanine-5 (Cy5), lucifer yellow, Texas red, tetramethylrhodamine, Yakima Yellow (YG), and Cal Fluor Red 610 (CFR), wherein the quencher is tetramethylrhodamine (TAMRA), 4-(4-dimethylaminophenylazo)benzoic acid, 4-dimethylaminophenylazophenyl-4-maleimide,It may be at least one selected from the group consisting of carboxytetramethylrhodamine and BHQ dyes.

[0008] The concentration of the above primer or probe can be selected and used in various ways by a person skilled in the art depending on the experimental conditions, and is preferably 1 to 1000 nM each, and more preferably 100 to 500 nM each, but is not limited thereto.

[0009] The step of performing the above real-time polymerase chain reaction may be performed under conditions of 30 to 50 cycles, 30 to 46 cycles, 30 to 44 cycles, 33 to 50 cycles, 33 to 46 cycles, 33 to 44 cycles, 36 to 50 cycles, 36 to 46 cycles, 36 to 44 cycles, 38 to 50 cycles or 38 to 46 cycles, for example, 38 to 44 cycles, but is not limited thereto.

[0010] The present invention relates to a method for quantifying Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus delbrueckiisubsp.bulgaricus, Limosilactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus paracaseisubsp. paracasei, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, comprising the above primers and probes. A composition for simultaneous detection of Limosilactobacillus reuterisubsp.reuteri, Ligilactobacillus salivarius, Bifidobacterium animalissubsp.lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longumsubsp.longum, Streptococcus thermophilus, Lactococcus lactissubsp.lactis, Enterococcus faecalis, and Enterococcus faecium is provided.

[0011] In one example of the present invention, the sample of the composition may be a probiotic product, and the product may be a food product.

[0012] In addition, the present invention provides a composition for selecting probiotic bacteria comprising the primer and probe.

[0013] The present invention comprises the steps of preparing an isolated RNA sample; amplifying the sample by polymerase chain reaction using the primers and probe; and a step of obtaining an amplification result; Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus delbrueckiisubsp.bulgaricus, Limosilactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus paracaseisubsp. paracasei, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteris subsp. reuteri, Ligilactobacillus salivarius, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, Streptococcus thermophilus, Lactococcus lactis subsp. lactis.lactis), Enterococcus faecalis and Enterococcus faecium are provided.

[0014] In addition, the present invention provides a method for selecting probiotic bacteria, comprising the steps of: preparing an isolated RNA sample; amplifying the sample using a polymerase chain reaction using the primers and probe; and obtaining an amplification result.

[0015] The primers and probes capable of detecting the probiotics of the present invention and the composition or method using the primers and probes successfully detected only a single signal from a DNA mixture containing positive DNA and 19 species, and thus can contribute to confirming the presence or absence of 19 specific probiotics designated by the Ministry of Food and Drug Safety of the Republic of Korea.

[0016] Figure 1 shows the results of aligning the sequences of primers and probes according to one embodiment of the present invention.

[0017] Figure 2 shows the results of aligning the rpoB gene sequences of 19 types of probiotic bacteria according to one embodiment of the present invention.

[0018] Figure 3 shows the results of verifying the specificity of a primer-probe analysis for 19 types of probiotic bacteria according to one embodiment of the present invention.

[0019] FIG. 4 shows the proportion of bacteria present in 12 probiotic products commercially available in Korea based on the quantified copy numbers obtained from ddPCR according to one embodiment of the present invention.

[0020] Hereinafter, preferred embodiments of the present invention will be described in detail. Furthermore, the following description includes numerous specific details, such as specific components, but these are provided to facilitate a more comprehensive understanding of the present invention. It will be apparent to those skilled in the art that the present invention can be practiced without these specific details. Furthermore, in describing the present invention, detailed descriptions of known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention.

[0021] <Example 1> Primer-probe analysis sequence

[0022] Primer-probe assays for ddPCR of 19 probiotic bacteria were synthesized by SFC Co., Ltd. (Cheongju, Korea). All probes in this study were labeled with the fluorescent dye 6-carboxyfluorescein (FAM) at the 5'-end and the SFCQ1 quencher at the 3'-end. The sequences of the primers and probes are listed in Table 1.

[0023]

[0024] <Example 2> Preparation of samples for inspection verification

[0025] Nineteen probiotics designated by the Ministry of Food and Drug Safety of the Republic of Korea were obtained from the Korean Collection for Type Cultures (KCTC). The strains are listed in Table 2 below. Lactobacillus, Streptococcus, Lactococcus, and Enterococcus were cultured in MRS medium (Difco, USA) at 37°C under aerobic conditions. Lactobacillus lactis was grown in MRS medium at 30°C under aerobic conditions. Bifidobacterium was grown in MRS medium at 37°C under anaerobic conditions according to the following criteria: H2, 4%; CO2, 10%; and N2, balance. Eight strains were cultured in 2 mL of MRS medium for 48 h according to the instructions for each culture collection. 2 mL of culture medium was centrifuged at 13,000 rpm for 5 min to remove cells. Genomic DNA (gDNA) from 19 probiotic bacteria and 12 commercial products was extracted using the GenElute Bacterial Genomic DNA Kit (Sigma Aldrich) according to the manufacturer's instructions. All gDNA from the probiotic bacteria was used for validation of primer-probe assays.

[0026] NumberTaxonomyKCTC (Type)1Lactobacillus acidophilus(Lap)31642Lactobacillus casei(Lca)31093Lactobacillus delbrueckiisubsp.bulgaricus(Ldb)36354Limosilactobacillus fermentum(Lfe)31125Lactobacillus gasseri(Lga)31636Lactobacillus helveticus(Lhe)35457Lacticaseibacillus paracaseisubsp. paracasei(Lpa)35108Lactiplantibacillus plantarum(Lpl)31089Lacticaseibacillus rhamnosus(Lrm)323710Limosilactobacillus reuterisubsp.reuteri(Lrt)359411Ligilactobacillus salivarius(Lsa)4313312Bifidobacterium animalissubsp.lactis(Bal)320213Bifidobacterium bifidum(Bbi)322014Bifidobacterium breve(Bbr)312815Bifidobacterium longumsubsp.longum(Bll)585416Streptococcus thermophilus(Sth)2117317Lactococcus lactissubsp.lactis(Lll)376918Enterococcus faecalis(Efa)519119Enterococcus faecium(Efi)13225

[0027] <Example 3> Alignment of primer and probe sequences

[0028] The rpoB gene of each bacterium was obtained from Ezbiocloud and NCBI. Sequences of subspecies within each species were obtained and aligned using MAFT (v7.520). The rpoB sequences of each of the 19 strains were aligned using MAFT to confirm their specificity.

[0029] <Example 4> Droplet digital PCR

[0030] The copy number of genomic DNA was determined using the QX200 Droplet Digital PCR (ddPCR) system (Bio-Rad, USA) according to the manufacturer's instructions. 5 μl of template, 10 μl of 2X QX200 ddPCR Supermix (Bio-Rad, USA) for probe, 1 μl of forward primer, 1 μl of reverse primer, 1 μl of probe, and 2 μl of distilled water were mixed. 45 μl of droplet template was prepared using the QX200 Droplet Generation Generator (Bio-Rad, USA) with a total of 20 μl of the mixture and 70 μl of Droplet Generation Oil (Bio-Rad, USA) for probe. Amplification was then performed through initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, primer annealing, and 60 °C for 1 min, followed by amplification at 98 °C for 10 min. All thermal cycling experiments were performed on a Veriti Thermal Cycler (Thermofisher, USA) using a ramp rate of 2°C / s. Fluorescence was initially analyzed using QuantaSoft software (Bio-Rad, USA).

[0031] <Experimental Example 1> Alignment of primer and probe sequences

[0032] The present invention developed 19 primer-probe assays for the specific quantification of 19 probiotic bacterial species approved by the Ministry of Food and Drug Safety. The rpoB gene sequence of each species was aligned with subspecies sequences obtained from NCBI and EzBioCloud. All assays were designed within conserved regions and enabled quantification within each species (Fig. 1).

[0033] <Test Example 2> Specificity of primer and probe sequences

[0034] To confirm the specificity of the primer-probe, the rpoB gene sequences of 19 probiotic bacteria were aligned (Fig. 2). The forward primer, reverse primer, and probe for Lactobacillus acidophilus species were designed specifically for this strain without cross-reactivity with other strains. Similarly, the forward primer, reverse primer, and probe for Lacticaseibacillus casei species were designed to ensure strain-specific detection. The forward primer, reverse primer, and probe for Lactobacillus delbrueckii subsp. bulgaricus species were designed specifically for this strain. The forward primer, reverse primer, and probe for Limosilactobacillus fermentum species were designed to be strain-specific. The forward primer, reverse primer, and probe for Lactobacillus gasseri species were designed to ensure strain specificity while preventing cross-reactivity with other species. The forward primer, reverse primer, and probe for Lactobacillushelveticus species were designed to specifically detect each strain. Similarly, forward primers, reverse primers and probes for the species Lacticaseibacillus paracaseisubsp. paracasei were designed to ensure strain-specific detection. For the species Lactiplantibacillus plantarum, forward primers, reverse primers and probes were carefully designed to be specific for their strains. For the species Lacticaseibacillus rhamnosus, forward primers, reverse primers and probes were developed to be specific for their strains. For the species Limosilactobacillus reuteri, forward primers, reverse primers and probes were designed to ensure specificity and avoid cross-reactivity.Forward primers, reverse primers, and probes for Ligilactobacillus salivarius species were tuned for strain-specific detection, and those for Bifidobacterium animalis subsp. lactis species were carefully designed to ensure specificity. Forward primers, reverse primers, and probes for Bifidobacterium bifidum species were designed specifically for their strains. Similarly, for Bifidobacterium breve species, the forward primers, reverse primers, and probes were designed to prevent cross-reactivity with other strains. For Bifidobacterium longum subsp. longum species, the forward primers, reverse primers, and probes were developed to be specific for their strains. For Streptococcus thermophilus species, the forward primers, reverse primers, and probes were carefully designed to be strain specific. For Lactococcus lactis subsp. lactis, the forward primers, reverse primers, and probes were designed to ensure specificity and avoid cross-reactivity. For Enterococcus faecalis species, the forward primers, reverse primers, and probes were carefully designed to detect only their specific strains. Finally, for Enterococcus faecium species, the forward primers, reverse primers, and probes were developed to be specific for their strains and not cross-react with other strains.

[0035] <Experimental Example 3> Verification of the specificity of primer-probe assays for 19 bacterial species

[0036] The present invention developed a primer-probe assay targeting the rpoB gene across 19 bacterial species. To verify the specificity of the assay, validation tests were performed using droplet digital PCR (ddPCR) with four different template conditions. The experimental conditions included:

[0037] (1) Distilled water (DW) as a control group without template

[0038] (2) A mixture of gDNA from 18 non-target bacterial species

[0039] (3) Mixed gDNA of all 19 bacterial species

[0040] (4) gDNA of target species with specificity for analysis (Fig. 3).

[0041] The ddPCR results of the present invention demonstrated that no assay signal was detected in either the template-free control (Condition 1) or the non-target bacterial mixture (Condition 2), confirming the absence of non-specific amplification under these conditions. In contrast, specificity was observed in the mixed gDNA of all 19 species (Condition 3) and the target species gDNA (Condition 4). This confirmed the specificity of each assay for its respective bacterial target within a complex bacterial mixture. These findings demonstrate that the developed primer-probe assay is highly specific for each bacterial target and that no cross-reactivity with non-target species was detected under the tested conditions.

[0042] <Experimental Example 4> Quantification of probiotic products using specific primer-probe analysis

[0043] Genomic DNA (gDNA) was extracted from commercially available probiotic products in Korea. The bacterial proportions in 12 different gDNA samples derived from these products were quantitatively assessed using specific primer-probe assays (Table 3). Using this assay, the proportion of bacteria present in each product was calculated based on the quantified copy numbers obtained by ddPCR. This method enabled accurate measurement of the bacterial composition of probiotic products, facilitating an accurate assessment of their microbiota composition (Fig. 4).

[0044] Product 1BacteriaDilution factorcopy / ulCopy meanLap10-3 174.4167 ± 6164.4163.6Lfe10 -3 78.483 ± 486.483.6Lpl10 -4 307.6301 ± 9305.2290.8Bal10 -3 90.887 ± 581.288.4Bbi10 -4 26.826 ± 124.426Sth10 -4 3437 ± 238.438Product 2BacteriaDilution factorcopy / ulCopy meanLap10 -2 232231 ± 6236.4224Lpl10 -5 4647 ± 147.248.4Lrt10 -4 85.691 ± 59096.4Bal10 -3 8483 ± 380.485.6Bbi10 -4 3635 ± 13434.4Sth10 -4 4243 ± 438.847.2Product 3BacteriaDilution factorcopy / ulCopy meanLap10 -2 201.6198 ± 13183.6208.4Lpl10 -4 216220 ± 8228.4214.4Lrt10 -4 9294 ± 391.697.6Bal10 -4 2423 ± 221.225.2Bbi10 -4 43.642 ± 338.842.4Sth10 -4 6870 ± 271.269.6Product 4BacteriaDilution factorcopy / ulCopy meanLap10 -3 52.852 ± 348.855.2Lpa10 -3 176.8178 ± 1178179.6Lpl10 -4 27.632 ± 436.430.8Bal10 -3 122.8118 ± 4117.2114Bbi10 -4 103.6108 ± 5113.6108Sth10 -424.424 ± 125.622.8Lll10 -3 3031 ± 131.231.6Efi10 -4 410.4396 ± 20403.6373.6Product 5BacteriaDilution factorcopy / ulCopy meanLap10 -4 64.861 ± 457.260.4Lpl10 -4 39.641 ± 343.638.4Lrt10 -3 165.2174 ± 8174.4181.6Bal10 -4 26.827 ± 0.427.227.6Bbi10 -2 233.6238 ± 4240241.6Sth10 -2 41.240 ± 337.242.8Product 6BacteriaDilution factorcopy / ulCopy meanLap10 -2 112.4107 ± 5104104Lpl10 -3 84.882 ± 477.284.8Bal10 -2 180.4177 ± 6170.4180.4Bbi10 -2 163.6160 ± 8151.2165.6Sth10 -3 43.241 ± 337.642.8Product 7BacteriaDilution factorcopy / ulCopy meanLap10 -2 216.4221 ± 5225.6222Lpa10 -3 172.4173 ± 5168.8178Lpl10 -2 55.653 ± 252.851.6Lrt10 -2 7679 ± 38280Bal10 -3 66.466 ± 0.46665.6Bbi10 -4 8084 ± 893.679.6Sth10 -3 8287 ± 586.892.4Lll10 -2 123.6129 ± 5130.8132.8Efi10 -42626 ± 127.624.8Product 8BacteriaDilution factorcopy / ulCopy meanLap10 -2 219.2219 ± 3215.6222Lpa10 -2 3.883 ± 0.483.22.96Lpl10 -4 2423 ± 224.821.6Lrm10 -2 18.418 ± 118.417.2Bal10 -2 15.615 ± 11614.4Bbi10 -2 174.4185 ± 10188192.8Sth10 -3 26.424 ± 419.626.419.626.4Product 9BacteriaDilution factorcopy / ulCopy meanLap10 -3 61.264 ± 759.671.6Lpa10 -2 14.812 ± 39.211.6Lpl10 -4 199.2203 ± 5209.2201.6Lrm14.524 ± 0.064.484.4Lrt10 -4 74.470 ± 465.669.2Bal10 -4 50.447 ± 549.642Bbi10 -4 32.831 ± 229.231.6Sth10 -4 356.4366 ± 20352.8389.2Product 10BacteriaDilution factorcopy / ulCopy meanLap10 -4 417.2407 ± 9404399.2Lrm10 -4 162153 ± 9144.8153.6Bal10 -4 153.6161 ± 7165.6164.8Bbi10 -4 163.2168 ± 5173.2168.8Bll10 -4 155.2160 ± 10154171.2Sth10 -4 194.4202 ± 8202.4210.4Product 11BacteriaDilution factorcopy / ulCopy meanLap10-3 4443 ± 143.241.6Lpl10 -4 313.2296 ± 15286288.4Lrm10 -4 156.8164 ± 6168166.4Lrt10 -4 52.853 ± 250.455.2Bal10 -4 102.4103 ± 3106.8101.2Bbr10 -3 120.4110 ± 9103.6105.2Lll10 -3 79.277 ± 373.278Product 12BacteriaDilution factorcopy / ulCopy meanLap10 -4 81.686 ± 586.490.8Lpa10 -3 60.862 ± 364.459.6Lrm10 -4 25.226 ± 124.427.2Bal10 -3 178169 ± 8162.8166.8Bll10 -4 27.227 ± 228.825.2

Claims

1. A forward primer of sequence number 1, a reverse primer of sequence number 2, and a probe of sequence number 3 for detection of Lactobacillus acidophilus; Forward primer of SEQ ID NO: 4, reverse primer of SEQ ID NO: 5 and probe of SEQ ID NO: 6 for detection of Lacticaseibacillus casei; Forward primer of SEQ ID NO: 7, reverse primer of SEQ ID NO: 8 and probe of SEQ ID NO: 9 for detection of Lactobacillus delbrueckiisubsp.bulgaricus; Forward primer of SEQ ID NO: 10, reverse primer of SEQ ID NO: 11 and probe of SEQ ID NO: 12 for detection of Limosilactobacillus fermentum; Forward primer of SEQ ID NO: 13, reverse primer of SEQ ID NO: 14 and probe of SEQ ID NO: 15 for detection of Lactobacillus gasseri; Forward primer of SEQ ID NO: 16, reverse primer of SEQ ID NO: 17 and probe of SEQ ID NO: 18 for detection of Lactobacillus helveticus; Forward primer of SEQ ID NO: 19, reverse primer of SEQ ID NO: 20 and probe of SEQ ID NO: 21 for detection of Lacticaseibacillus paracaseisubsp. paracasei; Forward primer of SEQ ID NO: 22, reverse primer of SEQ ID NO: 23 and probe of SEQ ID NO: 24 for detection of Lactiplantibacillus plantarum; Forward primer of SEQ ID NO: 25, reverse primer of SEQ ID NO: 26 and probe of SEQ ID NO: 27 for detection of Lacticaseibacillus rhamnosus; Forward primer of SEQ ID NO: 28, reverse primer of SEQ ID NO: 29 and probe of SEQ ID NO: 30 for detection of Limosilactobacillus reuterisubsp.reuteri; Forward primer of SEQ ID NO: 31, reverse primer of SEQ ID NO: 32 and probe of SEQ ID NO: 33 for detection of Ligilactobacillus salivarius; Forward primer of SEQ ID NO: 34, reverse primer of SEQ ID NO: 35 and probe of SEQ ID NO: 36 for detection of Bifidobacterium animalissubsp.lactis; A forward primer of SEQ ID NO: 37, a reverse primer of SEQ ID NO: 38 and a probe of SEQ ID NO: 39 for the detection of Bifidobacterium bifidum; A forward primer of SEQ ID NO: 40, a reverse primer of SEQ ID NO: 41 and a probe of SEQ ID NO: 42 for the detection of Bifidobacterium breve; Forward primer of SEQ ID NO: 43, reverse primer of SEQ ID NO: 44 and probe of SEQ ID NO: 45 for detection of Bifidobacterium longumsubsp.longum; Forward primer of SEQ ID NO: 46, reverse primer of SEQ ID NO: 47 and probe of SEQ ID NO: 48 for detection of Streptococcus thermophilus; Forward primer of SEQ ID NO: 49, reverse primer of SEQ ID NO: 50 and probe of SEQ ID NO: 51 for detection of Lactococcus lactis subsp. lactis; A forward primer of SEQ ID NO: 52, a reverse primer of SEQ ID NO: 53, and a probe of SEQ ID NO: 54 for detection of Enterococcus faecalis; and Primers and probes for simultaneous detection of 19 types of probiotics, including a forward primer of sequence number 55, a reverse primer of sequence number 56, and a probe of sequence number 57 for detection of Enterococcus faecium; 2. In the first paragraph, the primers and probes are primers and probes for simultaneous detection of 19 types of probiotics for use in real-time PCR, droplet digital PCR, or recombinase polymerase amplification.

3. In the first paragraph, the probe is a primer and probe for simultaneous detection of 19 types of probiotics, wherein at least one selected from the group consisting of the 5' end, the 3' end, and the sequence interior is labeled with a fluorophore, a quencher, or tetrahydrofuran (THF).

4. In the third paragraph, the fluorophore is fluorescein, 6-carboxyfluorescein (FAM, 6-carboxyfluorescein), hexachloro-6-carboxyfluorescein (HEX, hexachloro-6-carboxyfluorescein), tetrachloro-6-carboxyfluorescein (TET, tetrachloro-6-carboxyfluorescein), 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC, 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), Primers and probes for simultaneous detection of 19 kinds of probiotics, one or more selected from the group consisting of 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid, coumarin and coumarin derivatives, cyanine-5 (Cy5, Cyanine-5), lucifer yellow, Texas red, tetramethylrhodamine, Yakima Yellow (YG), and Cal Fluor Red 610 (CFR, Cal Fluor Red 610) 5. In the third paragraph, the quencher is at least one selected from the group consisting of tetramethylrhodamine (TAMRA), 4-(4-dimethylaminophenylazo)benzoic acid, 4-dimethylaminophenylazophenyl-4-maleimide, carboxytetramethylrhodamine, and BHQ dyes. Primers and probes for simultaneous detection of 19 types of probiotics.

6. Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus delbrueckiisubsp.bulgaricus, Limosilactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus paracaseisubsp. paracasei, Lactiplantibacillus plantarum, Lacticaseibacillus comprising any one of the primers and probes of clauses 1 to 5. Lacticaseibacillus rhamnosus, Limosilactobacillus reuteris subsp. reuteri, Ligilactobacillus salivarius, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Enterococcus faecalis, and Enterococcus Composition for simultaneous detection of Enterococcus faecium 7. In the 6th paragraph, the sample of the composition is a probiotic product, Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus delbrueckiisubsp.bulgaricus, Limosilactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus paracaseisubsp. paracasei, Lactiplantibacillus plantarum, Lacticaseibacillus Lacticaseibacillus rhamnosus, Limosilactobacillus reuteris subsp. reuteri, Ligilactobacillus salivarius, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Enterococcus faecalis, and Enterococcus Composition for simultaneous detection of Enterococcus faecium 8. A composition for selecting probiotic bacteria comprising the primer and probe of any one of claims 1 to 5.

9. In the 8th paragraph, the probiotic bacteria are Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus delbrueckiisubsp.bulgaricus, Limosilactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus paracaseisubsp. paracasei, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus rhamnosus), Limosilactobacillus reuteris subsp. reuteri, Ligilactobacillus salivarius, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Enterococcus faecalis and Enterococcus faecium. A composition for screening probiotic bacteria comprising one or more selected probiotic bacteria 10. In the 8th paragraph, the composition for selecting probiotic bacteria, wherein the sample of the composition is a probiotic product.

11. Step of preparing the isolated RNA sample; A step of amplifying by polymerase chain reaction using any one of the primers and probes of claims 1 to 5; and A step of obtaining an amplification result; comprising Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus delbrueckiisubsp.bulgaricus, Limosilactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus paracaseisubsp. paracasei, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, Simultaneous detection method for Limosilactobacillus reuteris subsp. reuteri, Ligilactobacillus salivarius, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Enterococcus faecalis, and Enterococcus faecium 12. Step of preparing the isolated RNA sample; A step of amplifying by polymerase chain reaction using any one of the primers and probes of claims 1 to 5; and A method for screening probiotic bacteria, comprising: a step of obtaining an amplification result;

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