LNA-qPCR accurate detection method of bifidobacterium longum subsp.infantis CICC 6069, and primer, probe, kit and application thereof

Abstract

The invention relates to the technical field of microbiological detection, in particular to an LNA-qPCR accurate detection method for bifidobacterium longum subsp.infantis CICC 6069, a specific primer, a probe and a kit for the method and application of the specific primer, the probe and the kit. The invention provides application of a nucleotide sequence as shown in SEQ ID NO: 1 in precise identification and quantitative detection of a bifidobacterium longum subsp.infantis CICC 6069 strain. The method comprises the following steps: constructing a bifidobacterium longum generic genome, excavating a specific SNP (Single Nucleotide Polymorphism) site of CICC 6069, designing a locked nucleic acid (LNA) modified specific probe SEQ ID NO: 14 and primers SEQ ID NO: 4 and SEQ ID NO: 5, and combining a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) technology, so that the rapid and accurate quantitative detection of the strain on the strain level is realized. The method has the advantages of strong specificity, high sensitivity (the quantitation limit is about 500 CFU / mL), short detection period and the like, and is suitable for quality control and quantitative analysis of samples such as foods, daily chemicals, complex microbial inoculants and the like containing CICC 6069.

Description

Technical Field

[0001] The present invention belongs to the field of microbial detection technology, and specifically relates to a method for precise quantification of Bifidobacterium longum subspecies infantis CICC 6069 at the strain level, as well as supporting specific primers, LNA probes, detection kits, and their applications in the fields of food, daily chemicals, feed, and medicine. Background Art

[0002] Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis ) as an intestinal probiotic, has the beneficial properties of metabolizing human milk oligosaccharides (HMO), regulating intestinal flora, enhancing immune function, reducing intestinal barrier damage, and promoting epithelial barrier repair. Among them, CICC 6069 T As a model strain of Bifidobacterium longum infantis subspecies, it exhibits significant efficacy in breast milk oligosaccharide utilization, anti-inflammation, and antioxidant activities. It has broad application prospects in the development of functional foods and disease prevention, especially in the field of infant intestinal health related to breastfeeding.

[0003] The beneficial properties of probiotics have significant strain specificity. Achieving accurate identification at the strain level is a core technical challenge in the probiotics industry. Traditional detection methods can only identify to the species level. Although high-throughput technologies such as whole genome sequencing can meet the needs of strain identification, they are time-consuming, costly, and require high technical skills from operators, making them difficult to be widely used in the industrial production of probiotics. Common counting methods cannot accurately count target strains at the strain level in compound probiotic products. Therefore, the development of fast, accurate, and cost-effective strain-level quantification technology is of great significance to ensuring the efficacy of probiotic products, supporting personalized product development, and protecting consumer health. The development of fast, accurate, and economical strain-level detection methods is crucial to the industrial application and quality control of CICC 6069. Summary of the Invention

[0004] To address the above-mentioned technical problems, the present invention aims to provide a precise LNA-qPCR quantitative detection method for Bifidobacterium longum subsp. infantis CICC 6069. By designing highly specific primers and LNA probes, combined with real-time fluorescence quantitative PCR technology, this method can achieve rapid strain-level quantification of this strain in complex samples, solving the problems of insufficient detection accuracy and time-consuming and labor-intensive detection in existing technologies, and providing efficient technical support for the intellectual property protection, quality control, and commercial application of the strain.

[0005] The purpose of the present invention is achieved through the following technical solutions: The present invention proposes the use of the nucleotide sequence shown in SEQ ID NO: 1 in the precise quantification of Bifidobacterium longum subsp. infantis CICC6069 strain levels.

[0006] Optionally, the 39th, 627th and 927th bases of the nucleotide sequence shown in SEQ ID NO: 1 are specific polymorphic sites, and the specific bases of Bifidobacterium longum subsp. infantis CICC 6069 at these three sites are T, T and A, respectively.

[0007] The present invention provides a precise quantitative method for Bifidobacterium longum subspecies infantis CICC 6069, characterized by comprising at least the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Designing primers and probes for the nucleotide sequence shown in SEQ ID NO: 1, wherein the amplification regions of the primers cover the polymorphic sites 39, 627, and 927 of SEQ ID NO: 1, and the probes comprise nucleotide sequences corresponding to the sites and are LNA-modified; S3. Real-time fluorescence quantitative PCR amplification using the primers and probes described in S2; S4. Calculate the concentration of CICC 6069 in the sample based on the standard curve.

[0008] Optionally, in S2, the primer sequences are as shown in SEQ ID NO:4 and SEQ ID NO:5, the amplified product length is 190 bp, and the amplified region covers the 627th specific SNP site of SEQ ID NO:1; the probe sequence is as shown in SEQ ID NO:14, the sequence length is 20 bp, contains the 627th polymorphic site of SEQ ID NO:1 and is LNA-modified, the 5' end is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a quencher group BHQ1.

[0009] Optionally, in S3, the qPCR reaction system is: 10 μL of 2× TaqMan qPCR premix, 0.4 μL of upstream and downstream primers with a working concentration of 10 μmol / L, 0.4 μL of probe with a working concentration of 10 μmol / L, 2 μL of DNA template, and sterile water to 20 μL; the qPCR reaction conditions are: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 63°C for 32 s, and extension at 72°C for 25 s, for a total of 40 cycles.

[0010] The present invention provides a kit for the precise quantitative detection of Bifidobacterium longum subspecies infantis CICC 6069, which comprises at least a specific primer pair of SEQ ID NO: 4 and SEQ ID NO: 5 and an LNA probe of SEQ ID NO: 14, and is used for the "strain" level identification and quantitative detection of CICC6069 in probiotic products, fermented dairy products, cosmetics, feed and intestinal microbial samples.

[0011] The technical solution provided by the embodiment of the present invention has the following advantages compared with the existing technology: The present invention screens specific SNP sites of Bifidobacterium longum infantis subspecies CICC 6069 and designs highly specific LNA probes. Through single-base mismatch recognition, it generates a specific signal only for CICC 6069, eliminating interference from other strains in complex situations, and constructs an accurate, sensitive, and efficient CICC 6069 quantitative detection system.

[0012] In the preferred technical solution, the primers and LNA probes of the present invention have high specificity, a limit of quantification of about 500 CFU / mL, and can detect the presence of interfering bacteria at concentrations as high as 10 8 The difference between the measured value and the theoretical value at CFU / mL is ≤|±0.5 Log 10 | (CFU / mL), suitable for different matrix samples such as bacterial powder, compound probiotic products, intestinal microorganisms, etc., and can meet the precise quality control needs of the strain in the entire process of research and development, production, and supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1The following are electrophoretic diagrams for primer exclusive amplification verification: (a) is the primer pair of SEQ ID NO: 2 and SEQ ID NO: 3; (b) is the primer pair of SEQ ID NO: 4 and SEQ ID NO: 5; (c) is the primer pair of SEQ ID NO: 6 and SEQ ID NO: 7; (d) is the primer pair of SEQ ID NO: 8 and SEQ ID NO: 9; (e) is the primer pair of SEQ ID NO: 10 and SEQ ID NO: 11; and (f) is the primer pair of SEQ ID NO: 12 and SEQ ID NO: 13. Lanes in the figure: M is DNA Marker, 1 is CICC 6069, 2 is CICC 6186, 3 is CICC 6070, 4 is CICC 6250, 5 is CICC 24210, 6 is CICC 6071, 7 is CICC 6079, 8 is CICC 6081, 9 is CICC 25191, 10 is CICC 6103, 11 is CICC 25164, 12 is CICC 24878, 13 is CICC 24208, 14 is CICC 6252, 15 is CICC 25167, 16 is CICC 6117, 17 is CICC 6224, 18 is CICC24209, 19 is CICC 6132, 20 is CICC 6240, 21 is CICC 25161, 22 is CICC 25190, 23 is CICC6245, 24 is CICC 6222, 25 is CICC 6246, 26 is CICC 24337, 27 is CICC 25163, 28 is CICC24923, 29 is CICC 25070, 30 is CICC 25245, 31 is CICC 25166, 32 is CICC 25165, 33 is CICC25162, 34 is CCUG 33636, 35 is CICC 25192, 36 is CCUG 15605, and CK is blank.

[0014] Figure 2The following electropherograms demonstrate inclusive amplification validation of primers. (a) shows the primer pair SEQ ID NO: 2 and SEQ ID NO: 3; (b) shows the primer pair SEQ ID NO: 4 and SEQ ID NO: 5; (c) shows the primer pair SEQ ID NO: 6 and SEQ ID NO: 7; and (d) shows the primer pair SEQ ID NO: 12 and SEQ ID NO: 13. Lanes M represents DNA marker; lane 1 represents CICC 6069; lane 2 represents CICC 6203; lane 3 represents CICC 6202; lane 4 represents CICC 6199; lane 5 represents CICC 6197; lane 6 represents CICC 6196; lane 7 represents CICC 25033; lane 8 represents CICC 6186; lane 9 represents CICC 6198; and lane CK represents blank.

[0015] Figure 3 The following are amplification curves verifying probe specificity and primer compatibility. (a) shows the specificity for SEQ ID NO: 14, (b) shows the specificity for SEQ ID NO: 15, (c) shows the compatibility between the primer pair SEQ ID NO: 2 and SEQ ID NO: 3 and the probe SEQ ID NO: 14, (d) shows the compatibility between the primer pair SEQ ID NO: 4 and SEQ ID NO: 5 and the probe SEQ ID NO: 14, (e) shows the compatibility between the primer pair SEQ ID NO: 6 and SEQ ID NO: 7 and the probe SEQ ID NO: 14, and (f) shows the compatibility between the primer pair SEQ ID NO: 12 and SEQ ID NO: 13 and the probe SEQ ID NO: 14.

[0016] Figure 4 This is a verification of the amplification efficiency of the probe shown in SEQ ID NO: 14 and two pairs of primers under different annealing temperature conditions.

[0017] Figure 5 This is the result of verifying the genetic stability of specific loci in strain CICC 6069. Lanes in the figure: M represents DNA marker, 1 to 10 represent the passage numbers of CICC 6069, and CK represents blank. DETAILED DESCRIPTION

[0018] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features therein can be combined with each other.

[0019] The following description sets forth numerous specific details to facilitate a thorough understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. The examples in this specification are intended to be a partial, rather than exhaustive, description of the present invention. The experimental reagents used in the following examples are all commercially available products. The strains involved in this invention are all existing strains deposited with the China Industrial Culture Collection (CICC). The public may obtain access to these strains through formal CICC channels, subject to compliance with scientific research ethics and legal procedures. Example 1

[0020] This example is used to illustrate the primer and probe design and simulation verification process.

[0021] The present invention provides a conserved gene sequence specific to Bifidobacterium longum for use in strain-level detection of Bifidobacterium longum subsp. infantis CICC 6069. The nucleotide sequence is shown in SEQ ID NO: 1: The sequence information of SEQ ID NO: 1 is as follows: Bifidobacterium longum genome sequences were retrieved from the China Industrial Culture Collection (CICC) and NCBI GenBank databases. After data quality control, a total of 513 genomes were subjected to comparative genomic analysis. A conserved cell division protein, FtsQ, was identified in the CICC 6069 genome, with the nucleotide sequence shown as SEQ ID NO:1. A homology search of the NCBI database revealed 192 homologous hits. Within B. longum, 127 homologous sequences were identified, with sequence coverage ranging from 96% to 100% and similarities exceeding 96%. Sequence coverage for non-B. longum species was below 10%. The results indicate that the sequence shown in SEQ ID NO:1 is a conserved and ubiquitous characteristic sequence within B. longum.

[0022] Further comparison and analysis of 513 Bifidobacterium longum genomes revealed that bases 39, 627, and 927 in the nucleotide sequence shown in SEQ ID NO:1 are polymorphic sites unique to CICC 6069: CICC 6069 has a T at base 39 (other Bifidobacterium longum has a C), a T at base 627 (other Bifidobacterium longum has a C), and an A at base 927 (other Bifidobacterium longum has a G). This specific SNP site can be used to accurately detect CICC 6069 at the strain level. Primer and probe design were performed based on the nucleotide sequence shown in SEQ ID NO:1 and its polymorphic site at position 627, resulting in six pairs of specific primers and two LNA probes. Specific sequence information is shown in Table 1. The target product designs for the six primer pairs ranged in length from 87 bp to 190 bp and covered the SNP site unique to strain CICC 6069. Two sets of specific LNA-modified Taqman probes were also developed, with SEQ ID NO:14 having one modified locked nucleic acid site and SEQ ID NO:15 having three modified sites. The probes were 20 bp and 21 bp in length, respectively, and covered the sites unique to strain CICC 6069 on the template gene.

[0023] Table 1: Specific primers and probe information for CICC 6069

[0024] Note: The bases marked with * are the CICC 6069-specific SNP sites on the probe sequence; the bases with capital letters are LNA-modified bases.

[0025] The NCBI primer tool Primer-BLAST was used to search for bacteria. <taxid:2>) Six primer pairs were validated genome-wide for specificity and site exclusivity, allowing for up to four base mismatches per primer and amplified fragments no longer than 2000 bp. Primers with strong specificity for Bifidobacterium longum were screened. Results demonstrated that all six primer pairs exhibited good specificity and strong exclusivity for the specific sites on the probes, enabling accurate identification of CICC 6069 at the strain level. Example 2

[0026] This example is used to illustrate the specificity verification and optimization process of primers and probes.

[0027] To systematically evaluate the intraspecies inclusiveness and interspecies exclusivity of the primers, model strains of 36 species, including Bifidobacterium longum subsp. infantis CICC6069, were selected as experimental verification strains for primer exclusivity verification. A total of 9 strains of Bifidobacterium longum, including CICC6069, were selected as experimental verification strains for primer inclusiveness verification. The strain information is shown in Table 2. PCR amplification results showed that primers SEQ ID NO: 2 / NO: 3, SEQ ID NO: 4 / NO: 5, SEQ ID NO: 6 / NO: 7, and SEQ ID NO: 12 / NO: 13 could specifically amplify Bifidobacterium longum strains (CICC 6069 and CICC 6186), and had no amplification signals for strains outside the species, showing good exclusivity (such as Figure 1 Inclusiveness verification showed that these four primer pairs all produced bright target bands, could effectively amplify Bifidobacterium longum, and had good inclusiveness at the species level (such as Figure 2 Based on the above results, these four primer pairs were selected for subsequent quantitative system development.

[0028] To validate the probe's specificity at the strain level, 12 strains of Bifidobacterium longum (including CICC 6069) and five strains of the same genus but different species (Bifidobacterium adolescentis CICC 6070, Bifidobacterium animalis subsp. animalis CICC 6250, Bifidobacterium animalis subsp. lactis CICC 24210, Bifidobacterium bifidum CICC 6071, and Bifidobacterium breve CICC 6079) were selected for qPCR experiments. The results showed that the probe represented by SEQ ID NO: 14 can accurately distinguish CICC 6069 from other Bifidobacteria (including different strains of the same species and different species, such as Figure 3 a); while the probe shown in SEQ ID NO: 15 detected non-specific amplification signals other than CICC 6069, as shown in Figure 3 Therefore, the more specific probe SEQ ID NO: 14 is preferably used for the construction of the subsequent detection system.

[0029] The compatibility between primers and probes was verified by combining the four preferred primer pairs and the probe SEQ ID NO: 14. The specificity verification results showed that the two primer pairs SEQ ID NO: 4 / NO: 5 and SEQ ID NO: 6 / NO: 7 had high specificity during the amplification process and could accurately detect the target strain CICC 6069 without non-specific amplification signals ( Figure 3 d, 3e); and SEQ ID NO: 2 / NO: 3 and SEQ ID NO: 12 / NO: 13 ( Figure 3 c, 3f) These two primer pairs produced obvious non-specific amplification signals during the amplification process, affecting the accuracy of the results. Further, the amplification efficiency of the primers SEQ ID NO: 4 / NO: 5 and SEQ ID NO: 6 / NO: 7 combined with the probe SEQ ID NO: 14 was verified at different annealing temperatures. By optimizing the gradient annealing temperature (e.g. Figure 4 ), 63°C was determined to be the optimal annealing temperature. At this time, the amplification efficiency of the SEQ ID NO:4 / NO:5 primer pair reached 98.39%, significantly better than the 91.57% at 62°C; while the amplification efficiency of the SEQ ID NO:6 / NO:7 primer pair was less than 90%.

[0030] In summary, after rigorous specificity screening and condition optimization, the primer sets SEQ ID NO:4 and SEQ ID NO:5 with higher specificity and better amplification efficiency and the probe SEQ ID NO:14 were finally selected to construct a CICC 6069 strain-level precise detection system. This combination showed excellent performance in species identification and strain-specific recognition, ensuring the accuracy and reliability of the test results.

[0031] Table 2: Validation experiment strain information Example 3

[0032] This example is used to illustrate the universal applicability of polymorphic sites in identifying the CICC 6069 strain within the species Bifidobacterium longum.

[0033] To fully validate the universality of the polymorphic sites used as a standard for intraspecific identification of B. longum strain CICC 6069, additional gene-level validation was conducted based on 513 collected B. longum genomes. Sequence homologs of SEQ ID NO:1 were retrieved from all 513 genomes, and clustering was performed using a 100% sequence similarity threshold to eliminate redundancy. Ultimately, 177 homologous groups were identified. One representative nucleotide sequence from each group was compared with SEQ ID NO:1 for differential analysis. The results are shown in Table 3.

[0034] Results showed that the nucleotide sequence represented by SEQ ID NO:1 is 1400 bp in length, and a total of 133 SNPs were detected when compared with homologous genes. CICC 6069 showed specific differences at positions 39 (T / C), 627 (T / C), and 927 (A / G) compared with other homologous sequences of Bifidobacterium longum. These differences can serve as specific genetic markers for strain-level identification, and this discrimination criterion is universal across the species.

[0035] Table 3: Different sites of SEQ ID NO: 1 between CICC 6069 strain and other Bifidobacterium longum

[0036] Note: "." indicates the base sequence is consistent with the CICC 6069 reference sequence. Example 4

[0037] This example is used to illustrate the genetic stability of polymorphic sites in different generations of CICC 6069 strains.

[0038] Strain CICC 6069 was obtained from the CICC bacterial resource bank and subcultured using MRS medium under anaerobic conditions at 37°C to obtain passages 1 to 10. Genomic DNA from each passage was extracted and used as template for amplification using primer pairs SEQ ID NO:4 and SEQ ID NO:5. The PCR reaction system consisted of a 50 μL total volume: 2 μL DNA template, 1 μL of each primer at a working concentration of 10 μmol / L, 25 μL of 2× PCR Taqmix, and 21 μL of ddH2O. PCR amplification conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 35 s, and finally 72°C for 10 min. Amplified products were verified by 1% agarose gel electrophoresis and analyzed by Sanger sequencing.

[0039] The results showed that the PCR amplification products of each generation of strains all showed a target band of 190 bp ( Figure 5 Sequencing results showed a 100% match with the predicted sequence of SEQ ID NO:1. Bases at position 627 were all characteristic Ts, with no mutations or polymorphisms. This result confirms that the SNP at position 627 of SEQ ID NO:1 is highly genetically stable within the CICC6069 strain and can serve as a specific molecular marker for strain-level identification of this strain. Example 5

[0040] This example is used to illustrate the accuracy of the precise quantification method for Bifidobacterium longum subspecies infantis CICC 6069.

[0041] By simulating a complex bacterial background, the concentration of 10 8 CFU / mL of 15 interfering bacteria (including 10 different strains and 5 different strains of the same species of Bifidobacterium longum) and CICC 6069 bacterial suspension (concentration 10 8 The established qPCR method was used for repeatability testing. The accuracy of the method was evaluated by the difference between the measured value and the theoretical plate count value. The results are shown in Table 4.

[0042] Experimental data showed that when 10 non-longum Bifidobacterium and 5 different longum Bifidobacterium of the same species were used as high concentration (10 8 CFU / mL) interfering bacteria, the difference between the measured value and the theoretical value of the target strain CICC 6069 was ≤|±0.5 Log 10 | (CFU / mL), which meets the acceptable error range of the ISO 16140-2 standard, and the test results are highly consistent. These results demonstrate that this qPCR method can achieve accurate identification through highly specific primer-probe combinations, confirming its efficient detection capability for CICC 6069 in complex bacterial matrices. This provides reliable technical support for the practical detection of CICC 6069 in complex samples such as probiotic products, fermented dairy products, cosmetics, feed, and intestinal microbial samples.

[0043] Table 4: CICC 6069 detection accuracy results under mixed bacteria conditions Example 6

[0044] This example is used to verify the applicability of the precise quantification method for Bifidobacterium longum subspecies infantis CICC 6069 in a composite bacterial preparation.

[0045] Five samples containing Bifidobacterium longum but not CICC 6069, as well as two samples containing CICC 6069, were collected for method applicability testing. The results, shown in Table 5, indicate that no Ct values were detected in the five samples without CICC 6069 (Products 1–5), demonstrating the method's high exclusion of non-target strains in complex probiotic products. However, the two complex probiotic products containing CICC 6069 (Products 6–7) successfully and accurately detected CICC 6069, with Ct values within an acceptable range, demonstrating the method's high inclusiveness and accuracy. Overall, these validation results demonstrate the high specificity and reliability of the established LNA-qPCR method for identifying the target strain CICC 6069T, making it suitable for the precise detection of target strains in complex bacterial strains.

[0046] Table 5: Applicability of CICC 6069 test in probiotic products Example 7

[0047] This example is used to illustrate the limit of quantification (LOQ) of the precise quantification method for Bifidobacterium longum subsp. infantis CICC 6069.

[0048] Based on the characteristics of TaqMan-qPCR technology, a Ct value of 35 was set as the upper limit of detection. Based on the standard curve data from 10 replicates, the corresponding CICC 6069 strain concentration at a Ct value of 35 was calculated to be approximately 500 CFU / mL (validated by plate count). To assess the consistency and accuracy of low-concentration detection, DNA samples were diluted to 500 CFU / mL and tested in 10 replicates. The results are shown in Table 6.

[0049] The experimental data showed that the logarithmic difference between the measured value and the theoretical value of plate count after 10 repeated tests was ≤|±0.5 Log 10 | (CFU / mL), which meets the acceptable error range of the ISO 16140-2 standard, and the target signal was consistently detected across all replicates. This result confirms that the method has a limit of quantification of 500 CFU / mL and exhibits excellent repeatability and accuracy even in a low concentration range, meeting the requirements for detecting the low-abundance target strain CICC 6069 in the product.

[0050] Table 6: Verification of method quantification limit

[0051] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments described herein, but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. The use of the nucleotide sequence shown in SEQ ID NO: 1 in the precise quantification of Bifidobacterium longum subsp. infantis CICC 6069 at the strain level, characterized in that: The 39th, 627th and 927th bases of the nucleotide sequence shown are specific polymorphic sites.

2. The use according to claim 1, characterized in that At the corresponding positions of the nucleotide sequence shown in SEQ ID NO: 1, the 39th, 627th and 927th bases are T, T and A, and the test strain is identified as Bifidobacterium longum subsp. infantis CICC 6069.

3. A precise quantitative method for Bifidobacterium longum subspecies infantis CICC 6069, characterized in that: At least the following steps are included: S1. Extract genomic DNA from the sample to be tested; S2. Designing primers and probes for the nucleotide sequence shown in SEQ ID NO: 1, wherein the amplification region of the primers covers the polymorphic site at base 39, base 627, or base 927 of SEQ ID NO: 1, and the probe comprises a nucleotide sequence corresponding to the polymorphic site and is LNA-modified; S3. Real-time fluorescence quantitative PCR amplification using the primers and probes described in S2; S4. Calculate the concentration of CICC 6069 in the sample based on the standard curve; The real-time fluorescence quantitative PCR reaction system was as follows: 10 μL of 2× TaqMan qPCR premix, 0.4 μL of upstream primer at a working concentration of 10 μmol / L, 0.4 μL of downstream primer at a working concentration of 10 μmol / L, 0.4 μL of probe at a working concentration of 10 μmol / L, 2 μL of DNA template, and sterile water to 20 μL. The qPCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 63°C for 32 s, and extension at 72°C for 25 s.

4. The method according to claim 3, characterized in that The limit of quantification of the method was 500 CFU / mL.

5. The method according to claim 3, characterized in that In S2, the nucleotide sequences of the primers are shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the amplified product thereof is 190 bp in length, which is suitable for real-time fluorescence quantitative PCR detection.

6. The method according to claim 3, characterized in that In S2, the sequence of the probe is shown in SEQ ID NO: 14, the probe sequence is 20 bp in length, and contains the 627th polymorphic site of SEQ ID NO:

1.

7. A specific primer, characterized in that The nucleotide sequences of the primers are shown in SEQ ID NO: 4 and SEQ ID NO: 5, and are used for the identification and detection of Bifidobacterium longum subspecies infantis CICC 6069 strain.

8. The primer according to claim 7, characterized in that The amplified product of the primer pair is 190 bp in length, covers the 627th specific SNP site of SEQ ID NO: 1, is suitable for real-time fluorescence quantitative PCR detection, and has an annealing temperature of 63°C.

9. An LNA-modified TaqMan probe, characterized in that: The nucleotide sequence of the probe is shown in SEQ ID NO: 14, and is used for the identification and detection of Bifidobacterium longum subspecies infantis CICC 6069 strain.

10. The probe according to claim 9, characterized in that The probe is designed based on the 627th SNP site of SEQ ID NO: 1, with the 5' end labeled with a fluorescent reporter group FAM, the 3' end labeled with a quencher group BHQ1, and the T base polymorphism site that specifically recognizes CICC 6069 is modified with LNA.

11. A kit for accurate detection of Bifidobacterium longum subspecies infantis CICC 6069, characterized in that: Comprising the specific primer pairs according to claim 7 and claim 8, the LNA probes according to claim 9 and claim 10, a qPCR premix and a standard.

12. Use of the specific primer pairs according to claims 7 and 8, the LNA probes according to claims 9 and 10, and the kit according to claim 11 for horizontal identification and quantitative detection of Bifidobacterium longum subsp. infantis CICC 6069 strain, wherein such applications include quality control, R&D testing, and colonization research of probiotic products, fermented dairy products, cosmetics, feed, and intestinal microbial samples.

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