Product and method for detecting bifidobacterium longum subspecies longum 6-1

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a product and a method for detecting bifidobacterium longum subsp. Longum 6-1. Bioinformatics analysis is carried out based on a whole genome sequence of the bifidobacterium longum subspecies longum 6-1, the primer pair and the primer probe combination are obtained through optimization, and the primer pair and the primer probe combination have good specificity and sensitivity in the aspect of detecting the bifidobacterium longum subspecies longum 6-1; the bifidobacterium longum subsp. Longum 6-1 and other strains (including other same subsp. Longum strains, other same strains and other strains) can be quickly and accurately distinguished, and the strain level quantitative detection of the strain in a complex biological sample (such as excrement) can be realized.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, specifically to a product and method for detecting Bifidobacterium longum subsp. 6-1. Background Technology

[0002] Bifidobacterium longum subsp. ( Bifidobacterium longum subsp. long Bifidobacterium longum subspecies 6-1 is a dominant strain of this species. Its accession number at the China Center for Type Culture Collection (CCTCC) is M 98003, at the American Center for Type Culture Collection (ATCC) is BAA-2753, and at the China Center for Medical Bacteriology Collection (CMCC) is P0001. Bifidobacterium longum subspecies 6-1 is one of the main active strains in the marketed drug Bifidobacterium longum and has a long history of clinical application.

[0003] With increased consumer awareness and advancements in testing technology, people are paying more attention to the colonization characteristics and metabolic capabilities of oral probiotic products. Given that Bifidobacterium has been proven to possess various probiotic functions, this genus has become a key focus for researchers and consumers.

[0004] Currently, there are two main methods for gut microbiota analysis: one is microbiome diversity analysis based on 16S rRNA gene sequencing or metagenomic sequencing, and the other is real-time quantitative PCR detection based on PCR amplification. The former can analyze the composition and relative abundance of the bacterial community, but only reflects the bacterial community structure and is difficult to achieve absolute quantification of the target species (strain); the latter amplifies the target gene by designing specific primers (or primer-probe combinations), which has the advantages of speed and sensitivity. However, the primers (or primer-probe combinations) reported in the existing literature are all limited to the species or subspecies level, and can only identify the whole subspecies of Bifidobacterium longum (Tannock GW, et al. Comparison of the compositions of the stool microbiota of infants fed goat milk formula, cow milk-based formula, or breast milk. Applied and Environmental Microbiology, 2013, 79(9): 3040-3048.; Ishikawa E, et al. Ethnic diversity of gut microbiota: species characterization of Bacteroides fragilisgroup and genus Bifidobacterium In healthy Belgianadults, and comparison with data from Japanese subjects. Journal of Bioscience and Bioengineering, 2013, 116(2): 265-270.). For example, Chinese invention patent CN118480615B discloses a triple absolute quantitative fluorescent PCR kit that can simultaneously detect Bifidobacterium longum subsp. longum, subsp. infantis, and Bifidobacterium shortum, but its limitation is that it cannot achieve precise quantification at the strain level.

[0005] In summary, since Bifidobacterium longum subsp. longum is a common member of the human gut microbiota, and current technologies lack rapid and accurate detection methods to distinguish Bifidobacterium longum subsp. longum 6-1 from other strains of the same subsp. longum, it is difficult to accurately assess the colonization and metabolism of the target active strain after taking the product in clinical studies. Summary of the Invention

[0006] This invention first provides a primer pair for detecting Bifidobacterium longum subsp. longum (Bifidobacterium longum subsp. longum) Bifidobacterium long subsp. long 6-1, the primer pair contains an upstream primer and a downstream primer; wherein, the nucleotide sequence of the upstream primer is as follows: 5'-GGTCACGCTTAAAGATGTGGCA-3' (SEQ ID No. 1); the nucleotide sequence of the downstream primer is as follows: 5'-TTCCGGCTTGACGATGTCTT-3' (SEQ ID No. 2).

[0007] The present invention also provides a primer-probe combination comprising: the primer pair and the probe; the nucleotide sequence of the probe is shown below: 5'-CATGTCAACAGCGTCGGCAGCAA-3' (SEQ ID No. 3).

[0008] The present invention also provides a reagent containing the primer pair or the primer-probe combination described herein.

[0009] The present invention also provides a kit containing the primer pair, or the primer-probe combination, or the reagent.

[0010] This invention also provides the primer pairs, or the primer-probe combinations, or the reagents, or the kits described herein for the detection of *Bifidobacterium longum* subsp. *longum* for diagnostic or non-diagnostic purposes. Bifidobacterium long subsp. long The application in 6-1, wherein the long subspecies of Bifidobacterium longum ( Bifidobacterium long subsp. long The accession number for 6-1 is CCTCC NO: M 98003.

[0011] This invention also provides the primer pairs, or the primer-probe combinations, or the reagents, or the kits described herein for the preparation of reagents for the detection of Bifidobacterium longum subsp. longum (Bifidobacterium longum subsp. longum). Bifidobacterium longum subsp. long Application in product 6-1, wherein the Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp. long The accession number for 6-1 is CCTCC NO: M 98003.

[0012] This invention also provides a method for detecting Bifidobacterium longum subsp. longum for diagnostic or non-diagnostic purposes. Bifidobacterium long subsp. long Method 6-1, wherein the Bifidobacterium longum subsp. ( Bifidobacterium long subsp. long The accession number of 6-1 is CCTCC NO: M 98003; the method includes: using the primer pair, or the primer-probe combination, or the reagent, or the kit, to amplify the nucleic acid of the sample to be tested, and analyzing the reaction results.

[0013] This invention utilizes bioinformatics analysis based on the whole genome sequence of Bifidobacterium longum subspecies 6-1 to optimize the primer pairs and primer-probe combinations, which exhibit good specificity and sensitivity in detecting Bifidobacterium longum subspecies 6-1. It can rapidly and accurately distinguish Bifidobacterium longum subspecies 6-1 from other strains (including other subspecies, other species, and other species), and can achieve strain-level quantitative detection of this strain in complex biological samples (such as feces). Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0015] Figure 1This is a strain-specific real-time PCR amplification curve of the primer and probe set in Example 2 of this invention. In this figure, a is the amplification curve of *Bifidobacterium longum* subsp. *longum* 6-1, and b is the amplification curve of 14 other *Bifidobacterium longum* subsp. *longum*, 1 *Bifidobacterium longum* subsp. *infant*, 1 *Bifidobacterium adolescentis*, 1 *Bifidobacterium breve*, 1 *Bifidobacterium bifidum*, 1 *Bifidobacterium pseudosporidis*, 1 *Bifidobacterium animalis* subsp. *mammary*, and the negative control (NTC).

[0016] Figure 2 This is a specific real-time PCR amplification curve of the control primer and probe set in Example 2 of the present invention. In this figure, a represents the amplification curves of 15 strains of *Bifidobacterium longum* (including *Bifidobacterium longum* subsp. 6-1) and 1 strain of *Bifidobacterium longum* subsp. infantis; b represents the amplification curves of 1 strain of *Bifidobacterium adolescentis*, 1 strain of *Bifidobacterium breve*, 1 strain of *Bifidobacterium bifidum*, 1 strain of *Bifidobacterium pseudosporidis*, 1 strain of *Bifidobacterium animalis* subsp. lactis, and the negative control (NTC).

[0017] Figure 3 This is a standard curve diagram of real-time quantitative PCR of Bifidobacterium longum subsp. 6-1 in Example 3 of the present invention.

[0018] Figure 4 This is a real-time PCR amplification curve of *Bifidobacterium longum* subsp. 6-1 and 6 fecal samples in Example 4 of this invention. In the graph, a (green line) is the amplification curve of the positive control sample (PC) of *Bifidobacterium longum* subsp. 6-1, b (blue line) is the amplification curve of the fecal samples from 6 healthy individuals, and c (red line) is the amplification curve of the amplified negative control sample (NTC) and the extracted negative control sample (NCS).

[0019] Figure 5 This is a real-time PCR amplification curve of the positive fecal sample containing *Bifidobacterium longum* subsp. 6-1 in Example 6. a, b, and c are the amplification curves of positive fecal sample 3, positive fecal sample 2, and positive fecal sample 1, respectively. Detailed Implementation

[0020] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0021] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0022] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B.

[0023] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0024] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0025] The numerical values ​​involved in this invention include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 1% can fluctuate within ±0.05%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 75% can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0026] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0027] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0028] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0029] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0030] In this invention, the *Bifidobacterium longum* subsp. *longum* (… Bifidobacterium longum subsp. long Bifidobacterium longum 6-1 has been disclosed in US06368591B2, and its accession number at the China Center for Type Culture Collection is CCTCC NO: M 98003. The original accession name of this strain was "Bifidobacterium longum 6-1 ( Bifidobacterium longum According to the "List of Microbial Strains that Can Be Used in Food" and the "List of Microbial Strains that Can Be Used in Infant Food" updated by the National Health Commission in 2022, the strain name has been adjusted and new taxonomic rules have been established, specifying the subspecies. The name is now standardized as "Bifidobacterium longum subspecies (…)". Bifidobacterium longum subsp. long The strain was labeled "6-1", but the strain itself remained unchanged. Furthermore, the accession number for this strain at the American Center for Type Culture Collection (ATCC BAA-2753) and at the China Center for Medical Bacteriology Collection (CMCC P0001) is 6-1.

[0031] This invention first provides a primer pair for detecting Bifidobacterium longum subsp. longum (Bifidobacterium longum subsp. longum) Bifidobacterium long subsp. long 6-1, the primer pair contains an upstream primer and a downstream primer; wherein, the nucleotide sequence of the upstream primer is as follows: 5'-GGTCACGCTTAAAGATGTGGCA-3' (SEQ ID No. 1); the nucleotide sequence of the downstream primer is as follows: 5'-TTCCGGCTTGACGATGTCTT-3' (SEQ ID No. 2). This invention has found that the above primer pair can highly specifically distinguish *Bifidobacterium longum* subsp. 6-1 from other strains (including other subsp. strains, other species strains, and other species strains).

[0032] This invention also provides a primer-probe combination comprising: the primer pair described above, and a probe; the nucleotide sequence of the probe is shown below: 5'-CATGTCAACAGCGTCGGCAGCAA-3' (SEQ ID No. 3). Using the above primer-probe combination, strain-level quantitative detection of *Bifidobacterium longum* subsp. 6-1 can be achieved in quantitative real-time detection.

[0033] In some embodiments, the 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

[0034] In some embodiments, the fluorescent group is selected from one of FAM (Fluorescein amidite), HEX (Hexachloro-fluorescein), TET (Tetrachlorofluorescein), Cyanine dye, ROX (Carboxy-X-rhodamine), and Texas Red.

[0035] In some embodiments, the quenching group is selected from one of BHQ1, BHQ2, BHQ3, Iowa Black, and BBQ.

[0036] In practical implementation, those skilled in the art can select specific fluorescent groups and quenching groups based on common sense.

[0037] In some specific embodiments, the fluorescent group is FAM and the quenching group is BHQ1.

[0038] The present invention also provides a reagent containing the primer pair or the primer-probe combination described herein.

[0039] In some specific embodiments, the reagent also contains a solvent for dissolving the primer pair or primer-probe combination as described above, such as TE buffer (Tris-EDTA buffer) or other solvents suitable for DNA stability.

[0040] The present invention also provides a kit containing the primer pair, or the primer-probe combination, or the reagent.

[0041] In some specific embodiments, the kit also provides DNA polymerase, dNTPs, and Mg-containing reagents in the form of one or more reagents (such as real-time PCR premix). 2+ The reaction buffer (3-5 mM), nucleic acid stabilizer, thermostable RNase H, and UNG (Uracil N-Glycosylase) are selected as one or more of these. In some specific embodiments, the real-time PCR premix is ​​KAPA PROBE FAST Universal 2X qPCR Master Mix or TakaraProbe qPCR Mix, with UNG (2X).

[0042] In some specific embodiments, the kit also contains nucleic acid extraction and purification components, such as bacterial lysis buffer (containing lysozyme and proteinase K), washing buffer (to remove PCR inhibitors), magnetic beads or silica gel columns (for DNA adsorption), and elution buffer. For complex samples such as feces, components specifically designed to remove interfering substances such as humic acid and bile salts can be added to the lysis buffer to ensure that the extracted DNA purity meets qPCR requirements (A260 / A280 ratio of 1.8-2.0).

[0043] In some specific embodiments, the kit also contains one or more of a positive quantitative standard, a positive control, and a negative control.

[0044] In some specific embodiments, the kit also contains an internal reference gene detection system.

[0045] In some specific embodiments, the kit also contains a fluorescence correction component. For example, if the qPCR premix is ​​a ROX-free version, the kit may additionally provide high and / or low concentrations of ROX reference dye, which users can flexibly add according to their real-time quantitative PCR instrument model to achieve fluorescence signal normalization. In some specific embodiments, the ROX reference dye is KAPA 50X ROX High Reference Dye or Takara ROX Reference Dye (50X).

[0046] In some specific embodiments, the kit also contains one or more of nuclease-free water, mineral oil, or a hot-start sealing film.

[0047] In some specific embodiments, the kit also includes: an instruction manual providing detailed operating procedures, reaction conditions, usage guidelines, and precautions.

[0048] All other components in the kit mentioned in this invention are commercially available and can be provided individually or in combination in multiple or one reagent.

[0049] This invention also provides the primer pairs, or the primer-probe combinations, or the reagents, or the kits described herein for the detection of *Bifidobacterium longum* subsp. *longum* for diagnostic or non-diagnostic purposes. Bifidobacterium long subsp. long The application in 6-1, wherein the long subspecies of Bifidobacterium longum ( Bifidobacterium long subsp. long The accession number for 6-1 is CCTCC NO: M 98003.

[0050] This invention also provides the primer pairs, or the primer-probe combinations, or the reagents, or the kits described herein for the preparation of reagents for the detection of Bifidobacterium longum subsp. longum (Bifidobacterium longum subsp. longum). Bifidobacterium longum subsp. long Application in product 6-1, wherein the Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp. long The accession number for 6-1 is CCTCC NO: M 98003.

[0051] This invention also provides a method for detecting Bifidobacterium longum subsp. longum for diagnostic or non-diagnostic purposes. Bifidobacterium long subsp. long Method 6-1, wherein the Bifidobacterium longum subsp. ( Bifidobacterium long subsp. long The accession number of 6-1 is CCTCC NO: M 98003; the method includes: using the primer pair, or the primer-probe combination, or the reagent, or the kit, to amplify the nucleic acid of the sample to be tested, and analyzing the reaction results.

[0052] In some embodiments, the sample to be tested is derived from food, health products, pharmaceuticals, feces, or intestinal contents. The method of the present invention enables accurate quantitative detection of Bifidobacterium longum subsp. 6-1 in complex samples.

[0053] In some implementations, the concentration of the nucleic acid during amplification is greater than or equal to 3.26 × 10⁻⁶. 2 copies / μL.

[0054] In some specific embodiments, the method further includes: extracting nucleic acid (DNA) from the sample to be tested, and using the obtained nucleic acid as a template for amplification.

[0055] In some specific embodiments, each 20 μL reaction system contains 0.4–0.6 μL (e.g., 0.4 μL, 0.5 μL, or 0.6 μL) of upstream primer and 0.4–0.6 μL (e.g., 0.4 μL, 0.5 μL, or 0.6 μL) of downstream primer, with the concentrations of both upstream and downstream primers being 8–12 μM (e.g., 8 μM, 9 μM, 10 μM, 11 μM, or 12 μM).

[0056] In some specific embodiments, the amplification is quantitative real-time PCR.

[0057] In some specific embodiments, each 20 μL reaction system also contains 0.4~0.6 μL (e.g. 0.4 μL, 0.5 μL or 0.6 μL) of probe, and the concentration of the probe is 8~12 μM (e.g. 8 μM, 9 μM, 10 μM, 11 μM or 12 μM).

[0058] Those skilled in the art can, based on common sense, set the concentrations and amounts of the upstream primers, downstream primers, and probes in the reaction system within the above-mentioned range (or slightly deviating from the above-mentioned range, especially greater than or equal to the upper limit), or adjust the concentrations and amounts of the upstream primers, downstream primers, and probes so that their final concentrations in the reaction system are still substantially within the above-mentioned range (or slightly deviating from the above-mentioned range, especially greater than or equal to the upper limit), which also falls within the protection scope of this invention.

[0059] In some specific implementations, each 20 μL reaction system also contains 1-5 μL of the nucleic acid to be tested (such as 1 μL, 2 μL, 3 μL, 4 μL, or 5 μL), 10 μL of real-time PCR premix (the amount can be adjusted according to actual needs), and 0.4 μL of ROX reference dye (the amount can be adjusted according to actual needs), with the remainder being water (such as nuclease-free pure water).

[0060] In some specific embodiments, the amplification reaction procedure is as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 15 s; (3) annealing / extension (collecting fluorescence) at 60°C for 30 s; (2) to (3) are performed for 45 cycles.

[0061] In practice, the reaction system, reagents, and amplification procedures can be adjusted based on common sense and actual needs.

[0062] In some specific implementations, the fluorescence Ct value of the sample to be tested can be used to distinguish Bifidobacterium longum subspecies 6-1 from other strains (including other subspecies, other species, and other species strains).

[0063] In some specific implementations, the content of Bifidobacterium longum subsp. 6-1 in the test sample can be calculated based on the standard curve and the fluorescence Ct value of the test sample.

[0064] Example The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0065] All strains involved in the following examples were obtained through commercial channels.

[0066] In the following examples, the 2X qPCR Master Mix is ​​either KAPA PROBE FAST Universal 2X qPCR Master Mix or Takara Probe qPCR Mix, with UNG (2X).

[0067] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0068] Example 1: Design and Synthesis of Primer-Probe Sets Bioinformatics analysis was performed on the whole genome sequence of *Bifidobacterium longum* subsp. *longum* 6-1 to identify specific gene sequences of this strain. These sequences were compared with the NCBI database to confirm specificity. Based on the target gene region, a strain-level specific primer-probe set was designed, and its sequences are shown in Table 1. The BL6-1_UP probe was labeled with the fluorescent group FAM at its 5' end and the quencher group BHQ1 at its 3' end.

[0069] Table 1. Designed primer-probe sets

[0070] Example 2: Verification of strain-level specificity Fourteen different strains of Bifidobacterium longum subsp. (Bifidobacterium longum) were selected. Bifidobacterium longum subsp. long ), 1 strain of Bifidobacterium longum infantis ( Bifidobacterium longum subsp. child ), 1 strain of Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), 1 strain of Bifidobacterium breve ( Bifidobacterium breve ), 1 strain of Bifidobacterium bifidum ( Bifidobacterium bifidum ), 1 strain of Bifidobacterium pseudosporidis ( Bifidobacterium pseudo-chained ), 1 strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animal subsp. milkThe control strain was *Bifidobacterium longum* subsp. *longum* 6-1, and the positive control was used. A quantitative real-time PCR experiment was performed to verify the strain-level specificity of the primer-probe set designed in this invention. DNA was extracted from the above strains using a commercially available bacterial genomic DNA extraction kit, and diluted uniformly to approximately 10 ng / μL. The same amount of DNA was loaded onto the sample, and the specificity of the reaction was determined by comparing the differences in Ct values. Each strain was tested in triplicate, and a negative control (NTC) was set up using nuclease-free pure water as a template. The reaction system is shown in Table 2, and the amplification program is shown in Table 3. The obtained amplification curves are shown in Table 3. Figure 1 As shown, the average Ct value of Bifidobacterium longum subsp. 6-1 was 13.73, while the detection results of 20 control strains were all negative or Ct>35. The Ct values ​​were significantly different from those of the target strain, indicating that the primer-probe set has strain-level detection specificity and good specificity within 35 cycles.

[0071] In addition, as a control, the same experiment was performed using the primer and probe set (lon / pF, lon / pR, lon-P) for *Bifidobacterium longum* subsp. *longum* described in Chinese invention patent publication number CN118480615B. The resulting amplification curves are shown below. Figure 2 As shown, the detection results for Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium pseudomicrobium, and Bifidobacterium animalis subsp. lactis were all negative or Ct>34. All of Bifidobacterium longum subsp. longis and Bifidobacterium longum subsp. infantis were detected, and the average Ct values ​​were similar (13.50-14.93), indicating that this primer-probe set can only achieve species-level specificity and cannot distinguish different strains within the same species.

[0072] Table 2. General Reaction System

[0073] Table 3. Amplification Procedure

[0074] Example 3: Construction of Standard Curve and Determination of Limit of Quantitation The synthesized plasmid standard powder was dissolved in nuclease-free pure water and then serially diluted to obtain a concentration of 3.26 × 10⁻⁶. 2 copies / μL - 3.26 × 10 10 Plasmid standards in copies / μL. At 3.26 × 10⁻⁶. 2 copies / μL - 3.26 × 10 8A standard curve was established using plasmid standards at seven dilutions (copies / μL) as templates. Each dilution was tested in triplicate. A negative control (NTC) was set up using nuclease-free purified water as template. The reaction system and amplification program are shown in Tables 2 and 3 of Example 2, respectively. The obtained standard curve results are as follows: Figure 3 As shown, the X-axis represents the copy number of the plasmid standard (copies / μL), the Y-axis represents the Ct value, and the equation of the standard curve is Y = 3.394lgX + 39.141, correlation coefficient R 2 =1, amplification efficiency E = 97.07%. The results show that there is a good linear relationship between different concentrations of standards, which is in line with expectations.

[0075] With a concentration of 3.26 × 10 2 Using plasmid standards at a concentration of 10 copies / μL as templates, 10 replicates were performed to determine the limit of quantitation (LOQ) of this method. The results showed that the coefficient of variation for the calculated copy number between 10 replicates at this concentration was 10.99%, and the deviation was 1.70% of the labeled value, which meets the requirements. Therefore, the LQ of this detection system is confirmed to be 3.26 × 10⁻⁶. 2 copies / μL.

[0076] Example 4: Specificity and anti-interference experiment against fecal sample background The established real-time quantitative PCR method was used to detect fecal samples from six healthy individuals without *Bifidobacterium longum* subsp. *longum* 6-1 to evaluate the specificity of the primer-probe set. The experiment included amplified negative control samples (NTC), extracted negative control samples (NCS), and positive control samples (PC) using *Bifidobacterium longum* subsp. *longum* 6-1 as a template. DNA templates were diluted to approximately 10 ng / μL before loading. The amplification curves and results are shown below. Figure 4 As shown in Table 4, the Ct values ​​of the fecal samples from all 6 healthy individuals were greater than the limit of quantitation Ct value (30.71), indicating that the detection system is specific to Bifidobacterium longum subsp. 6-1.

[0077] The diluted fecal samples from six healthy individuals were mixed in equal volumes, and two reaction systems were prepared according to Table 5 and Table 2 in Example 2 for interference resistance experiments. The experimental results are shown in Table 6. The average Ct values ​​of all mixed template samples and corresponding single plasmid template samples were similar, and the linear relationship between different concentrations of standards remained good under this mixed sample background, with a correlation coefficient R0. 2 =0.999, amplification efficiency E=97.98%, indicating that the detection system of Bifidobacterium longum subsp. longum 6-1 is not affected by complex biological samples and has good stability.

[0078] Table 4. Specific experimental results in the context of fecal samples

[0079] Note: In the table, "ND" (Undetermined) means "not detected", that is, no fluorescence signal above the background level was detected in the sample within the set number of cycles.

[0080] Table 5. Reaction system against the background of mixed fecal samples DNA

[0081] Table 6. Results of interference resistance experiments against a mixed fecal sample DNA background

[0082] Note: In the table, "ND" (Undetermined) means "not detected", that is, no fluorescence signal above the background level was detected in the sample within the set number of cycles.

[0083] Example 5: Precision and accuracy experiment against a fecal sample background Three concentrations of standard qualitymids (low, medium, and high) were used as template DNA. Reaction systems were prepared according to Table 5 in Example 4, with three replicates for each concentration. The coefficient of variation (COP) between copy numbers and the deviation from the labeled value were used as evaluation indicators to examine the intra-batch precision and accuracy of the detection system in complex biological sample backgrounds. In addition, two experimenters repeated the same experiment three times over three days to examine inter-batch precision and accuracy. The precision test results are shown in Table 7, with intra-batch COPs all less than 3.50% and inter-batch COPs all less than 6.50%. The accuracy test results are shown in Table 8, with intra-batch deviations all less than ±1.00% and inter-batch deviations all less than ±7.50%, indicating that the detection method has good repeatability and accuracy.

[0084] Table 7. Precision Experiment Results

[0085] Table 8. Accuracy Experiment Results

[0086] Example 6: Detection of positive fecal samples containing Bifidobacterium longum subsp. 6-1 The established real-time quantitative PCR method was used to detect the fecal samples of three subjects who had taken Bifidobacterium longum subsp. longum 6-1 powder. Approximately 0.2g of feces was collected, and total DNA was extracted from the fecal samples using a commercially available fecal genomic DNA extraction kit. After diluting the DNA to an appropriate concentration, the reaction system was prepared and the amplification program was set according to Tables 2 and 3 in Example 2, using the DNA as a template. Simultaneously, a standard curve was established using serially diluted plasmid standards as templates. The content of Bifidobacterium longum subsp. longum 6-1 in the test samples was calculated based on the standard curve and the Ct value of the test samples. The resulting amplification curves are shown below. Figure 5 As shown in Table 9, the test results were successfully quantified in all three fecal samples as Bifidobacterium longum subsp. 6-1, with a content of 1.14 × 10⁻⁶. 6 -1.80×10 7 copies / g of feces.

[0087] Table 9. Detection results of Bifidobacterium longum subsp. 6-1 in positive fecal samples

[0088] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A primer pair for detecting Bifidobacterium longum subsp. longum (Bifidobacterium longum subsp. longum) Bifidobacterium longum subsp. longum 6-1, the primer pair contains an upstream primer and a downstream primer; in, The long subspecies of Bifidobacterium longum ( Bifidobacterium longum subsp. longum The accession number for 6-1 is CCTCC NO: M 98003; The nucleotide sequence of the upstream primer is shown below: 5'-GGTCACGCTTAAAGATGTGGCA-3'; The nucleotide sequence of the downstream primer is shown below: 5'-TTCCGGCTTGACGATGTCTT-3'.

2. A primer-probe combination comprising: The primer pair as described in claim 1, and probe; The nucleotide sequence of the probe is shown below: 5'-CATGTCAACAGCGTCGGCAGCAA-3'.

3. The primer-probe combination according to claim 2, wherein, The probe is modified with a fluorescent group at its 5' end and a quenching group at its 3' end.

4. The primer-probe combination according to claim 3, wherein, The fluorescent group is selected from one of FAM, HEX, TET, Cyanine dye, ROX, and Texas Red; and / or, The quenching group is selected from one of BHQ1, BHQ2, BHQ3, Iowa Black, and BBQ.

5. A reagent comprising the primer pair of claim 1 or the primer-probe combination of any one of claims 2 to 4.

6. A kit comprising the primer pair of claim 1, or the primer-probe combination of any one of claims 2 to 4, or the reagent of claim 5.

7. The primer pair of claim 1, or the primer-probe combination of any one of claims 2-4, or the reagent of claim 5, or the kit of claim 6, for non-diagnostic purposes in detecting *Bifidobacterium longum* subsp. *longum* (…). Bifidobacterium longum subsp. longum The application in 6-1, where, The long subspecies of Bifidobacterium longum ( Bifidobacterium longum subsp. longum The accession number for 6-1 is CCTCC NO: M 98003.

8. The primer pair of claim 1, or the primer-probe combination of any one of claims 2-4, or the reagent of claim 5, or the kit of claim 6, in the preparation of a reagent for detecting *Bifidobacterium longum* subsp. *longum* (… Bifidobacterium longum subsp. longum Applications in products 6-1, among which, The long subspecies of Bifidobacterium longum ( Bifidobacterium longum subsp. longum The accession number for 6-1 is CCTCC NO: M 98003.

9. A non-diagnostic method for detecting Bifidobacterium longum subsp. (Long) Bifidobacterium longum subsp. longum The method of 6-1, in which, The long subspecies of Bifidobacterium longum ( Bifidobacterium longum subsp. longum The accession number of 6-1 is CCTCC NO: M 98003; the method includes: using the primer pair of claim 1, or the primer-probe combination of any one of claims 2 to 4, or the reagent of claim 5, or the kit of claim 6, to amplify the nucleic acid of the sample to be tested, and to analyze the reaction results.

10. The method according to claim 9, wherein, The test samples are derived from food, health products, medicines, feces, or intestinal contents.

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