Precision quantification method of lactobacillus helveticus cicc 6064, primer, probe, kit and application thereof

Abstract

The present application relates to the technical field of microorganism detection, and in particular to a precision quantification method of lactobacillus helveticus CICC 6064 and application thereof.The present application proposes the application of SEQ ID NO:1 in the precision quantification of lactobacillus helveticus CICC 6064 strain, extracts the genomic DNA of the strain to be detected, designs a primer pair (SEQ ID NO:2 and SEQ ID NO:3) and a TaqMan probe (SEQ ID NO:8) for the strain-specific SNP site on the nucleotide sequence shown in SEQ ID NO:1, and combines with real-time fluorescent quantification PCR technology to realize rapid and precise quantification detection of the strain at the strain level.The method of the present application is suitable for quality control of food, daily chemical, composite microbial agent and other samples added with lactobacillus helveticus CICC 6064, and realizes multidimensional application of real-time monitoring technology.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a precise quantitative method for Lactobacillus helveticus CICC 6064, as well as specific primers, probes, reagent kits for this method and their applications in food, cosmetics, feed and other fields. Background Technology

[0002] Lactobacillus helveticus, a thermophilic lactic acid bacterium that undergoes homofermentation, possesses high protein hydrolysis activity and is a dominant microbial community in natural whey production. It is also widely used in the preparation of traditional fermented milk and commercial fermented beverages. In the field of fermented dairy products, this bacterium not only accelerates cheese maturation and improves flavor through protein hydrolysis but also releases bioactive peptides that exert physiological functions such as immune regulation and antihypertensive effects. In recent years, its probiotic properties, including antibacterial, anticancer, mood-regulating, and cognitive-regulating properties, have also gradually become a research hotspot. However, potential functional differences exist between different strains, and how to achieve accurate detection of functional strains at the strain level remains a common challenge in the industry.

[0003] Lactobacillus helveticus CICC 6064, as a high-quality probiotic strain, is not only used in the field of antibacterial cosmetics due to its excellent antibacterial properties, but also exhibits significant advantages in yogurt starter cultures prepared in combination with Streptococcus thermophilus CICC 6063, including rapid fermentation rate, stable performance, and excellent taste and texture, demonstrating extremely high commercial development value. Therefore, precise strain-level identification and quantitative counting techniques for this strain have become essential key technologies for quality control in its commercial production.

[0004] Existing microbial detection technologies have significant limitations: traditional methods such as 16S rRNA sequencing can only achieve species-level identification and cannot meet the needs of strain-specific analysis; while technologies such as whole-genome sequencing (WGS) can identify strains, they have drawbacks such as long cycle time and high cost, which cannot meet the needs of rapid quality control in the production process; although real-time quantitative PCR (qPCR) technology has the advantages of speed and economy, its specificity depends on high-fidelity primer and probe design, and no specific detection system for CICC 6064 has been reported. There is an urgent need to develop strain-level detection methods that combine high efficiency and specificity to overcome the technical barriers to accurate detection in the process of strain industrialization. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to provide a precise quantitative method for Lactobacillus helveticus CICC 6064. By developing strain-specific primers and probes, combined with qPCR technology, this method enables rapid and accurate detection of this strain in complex samples. It solves the problems of long detection time, high cost, and insufficient specificity in existing strain-level detection methods, and provides efficient intellectual property protection and quality control means for the commercial application of this strain.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention proposes the application of the nucleotide sequence shown in SEQ ID NO:1 in the precise quantification of Lactobacillus helveticus CICC 6064 at the "strain" level.

[0008] Optionally, the nucleotide sequence shown in SEQ ID NO:1 has specific polymorphic sites at positions 234 and 454, and the specific bases at these two sites in Lactobacillus helveticus CICC 6064 are A.

[0009] This invention proposes a precise quantitative method for Lactobacillus helveticus CICC 6064, characterized by comprising at least the following steps:

[0010] S1. Extract genomic DNA from the sample to be tested;

[0011] S2. Design 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 position 234 or 454 of SEQ ID NO:1, and the probes contain the nucleotide sequence corresponding to the polymorphic site.

[0012] S3. Perform real-time quantitative PCR amplification using the primers and probes described in S2;

[0013] S4. Calculate the concentration of CICC 6064 in the sample based on the standard curve.

[0014] Optionally, in S2, the primer nucleotide sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3, and the amplification product is 123 bp in length, with the amplified region covering the specific SNP site at position 454 of SEQ ID NO:1.

[0015] Optionally, in S2, the probe sequence is as shown in SEQ ID NO:8, the probe sequence length is 16 bp, it contains the 454th polymorphic site of SEQ ID NO:1, and the 5' end is labeled with the fluorescent reporter group FAM and the 3' end is labeled with the quencher group MGB-NFQ.

[0016] Optionally, in S3, the qPCR reaction system is as follows: 10 μL of 2×TaqMan qPCR premix, 1.0 μL of upstream primer with a working concentration of 10 μmol / L, 1.0 μL of downstream primer 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 a final volume of 20 μL.

[0017] Optionally, in S3, the qPCR reaction conditions are: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 64℃ annealing for 32 s, 72℃ extension for 25 s, for a total of 40 cycles.

[0018] This invention proposes a kit for the accurate quantification of Lactobacillus helveticus CICC 6064, which contains at least specific primer pairs SEQ ID NO:2 and SEQ ID NO:3 and a TaqMan probe SEQ ID NO:8, for the identification and quantification of Lactobacillus helveticus CICC 6064 at the strain level in fermented dairy products, cosmetics or compound microbial agents.

[0019] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0020] This invention utilizes the screening of specific SNP sites in Lactobacillus helveticus CICC 6064 and the design of high-fidelity primers and probes to construct a precise quantitative method with significant advantages such as high specificity, high sensitivity and accuracy, high efficiency and economy, and wide applicability.

[0021] In the preferred embodiment, the primers and probes of this invention generate amplification signals only at specific sites of CICC 6064. Validation with 47 bacterial strains confirmed good exclusivity, with a limit of quantitation reaching 300 CFU / mL, and effective even at interfering bacterial concentrations as high as 10⁻⁶ CFU / mL. 8 The difference between the measured value and the theoretical value at CFU / mL should be ≤ ±0.5 log. 10 (CFU / mL), the detection cycle is shortened to 1-2 days compared to whole genome sequencing and the cost is significantly reduced. It can effectively detect different forms of samples such as liquid culture, lyophilized powder, and compound bacterial agents, which can meet the precise quality control needs of this strain in the entire process of research and development, production and supervision in commercial applications such as fermented dairy products, cosmetics and compound bacterial agents. Attached Figure Description

[0022] Figure 1This is an electrophoresis diagram verifying the inclusion amplification between strains of *Lactobacillus helveticus* using primer pairs shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:7. In the diagram, lanes are: M for DNA Marker, 1 for CICC 6064, 2 for CICC 6024, 3 for CICC 6032, 4 for CICC 6102, 5 for CICC 6272, 6 for CICC 20243, 7 for CICC 22171, 8 for CICC 22536, 9 for CICC 22814, 10 for CICC 24208T, 11 for CICC 24071, and CK for blank.

[0023] Figure 2 This is an electrophoresis diagram verifying the exclusive amplification of SEQ ID NO:2 and SEQ ID NO:3 on other species other than *Lactobacillus helveticus*. In the diagram, lane M represents the DNA marker, positive control is CICC 6064, and lane 1 represents CICC 6081. T 2 is CICC25191 T 3 is CICC 6103 T 4 is CICC 25164 T 5 represents CICC 24878 T 6 is CICC 6252 T 7 is CICC25167 T 8 represents CICC 6117 T 9 represents CICC 6263 T 10 is CICC 6224 T 11 is CICC 24209 T 12 is CICC6132 T 13 is CICC 6240 T 14 is CICC 25161 T 15 is CICC 25190 T 16 is CICC 6245 T 17 is CICC6070 T 18 is CICC 6250 T 19 is CICC 24210 T 20 is CICC 6071 T 21 is CICC 6079 T 22 is CICC6186 T 23 is CICC 6069 T 24 is CICC 6222 T 25 is CICC 6246T 26 is CICC 24337 T 27 is CICC25163 T 28 is CICC 24923 T 29 is CICC 25070 T 30 is CICC 25245 T 31 is CICC 25166 T 32 is CICC25165 T 33 is CICC 25162 T 34 is CCUG 33636 T 35 is CICC 25192 T 36 is CCUG 15605 T CK is blank.

[0024] Figure 3 This is an electrophoresis diagram verifying the exclusive amplification of SEQ ID NO:6 and SEQ ID NO:7 on other species besides *Lactobacillus helveticus*. In the diagram, lane M represents the DNA marker, positive control is CICC 6064, and lane 1 represents CICC 6081. T 2 is CICC25191 T 3 is CICC 6103 T 4 is CICC 25164 T 5 represents CICC 24878 T 6 is CICC 6252 T 7 is CICC25167 T 8 represents CICC 6117 T 9 represents CICC 6263 T 10 is CICC 6224 T 11 is CICC 24209 T 12 is CICC6132 T 13 is CICC 6240 T 14 is CICC 25161 T 15 is CICC 25190 T 16 is CICC 6245 T 17 is CICC6070 T 18 is CICC 6250 T 19 is CICC 24210 T 20 is CICC 6071 T 21 is CICC 6079 T 22 is CICC6186 T23 is CICC 6069 T 24 is CICC 6222 T 25 is CICC 6246 T 26 is CICC 24337 T 27 is CICC25163 T 28 is CICC 24923 T 29 is CICC 25070 T 30 is CICC 25245 T 31 is CICC 25166 T 32 is CICC25165 T 33 is CICC 25162 T 34 is CCUG 33636 T 35 is CICC 25192 T 36 is CCUG 15605 T CK is blank.

[0025] Figure 4 The figures show the qPCR results of *Lactobacillus helveticus* under different annealing temperatures using the probes shown in SEQ ID NO:8 and SEQ ID NO:9. The red curve in the figure represents CICC 6064, and the other colored curves represent CICC 6024, CICC 6272, CICC22171, CICC 22536, and CICC 24208. T Five strains of Lactobacillus helveticus were found.

[0026] Figure 5 This is the result of genetic stability verification of specific loci in strain CICC 6064. Figure A shows the primer amplification product bands at different generations. In each lane of the figure: M is the DNA Marker, 1 to 10 are the number of generations of CICC 6064, and CK is the blank. Figure B is the sequence alignment diagram of the specific locus and surrounding bases in CICC 6064 at 10 generations.

[0027] Figure 6 This is the result of the exclusion verification of the CICC 6064 strain-level qPCR detection method established in this invention. The red curve in the figure represents CICC 6064, and the other colored curves represent other Lactobacillus species. Detailed Implementation

[0028] To better understand the above-mentioned objectives, features, and advantages of this invention, the following description further details the solutions proposed in this invention. All strains involved in this invention are existing strains, deposited at the China Industrial Microbial Culture Collection Center (CICC). The public can obtain them through formal channels by applying to CICC after fulfilling research ethics and legal procedures. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments. Example 1

[0030] This example illustrates the primer and probe design and specificity verification process.

[0031] This invention provides the application of a unique conserved gene sequence of Lactobacillus helveticus in the detection of Lactobacillus helveticus strain CICC 6064 at the strain level. This nucleotide sequence is shown in SEQ ID NO:1.

[0032] The sequence information for SEQ ID NO:1 is as follows:

[0033] 185 Lactobacillus helveticus genome sequences were retrieved from the China Industrial Microbial Culture Collection (CICC) and the NCBI GenBank database. Comparative genomics analysis identified a conserved protein-coding nucleotide sequence (as shown in SEQ ID NO:1) in the CICC 6064 genome, presumed to be a peptidase metallopeptidase domain-containing protein. Homology searching in the NCBI database revealed no homologous sequences outside the Lactobacillus helveticus species, but 32 homologous sequences were found within the species, all with 100% coverage and a similarity greater than 98%, confirming that the sequence shown in SEQ ID NO:1 is a conserved and ubiquitous characteristic sequence within the Lactobacillus helveticus species. Further comparative analysis of 185 Lactobacillus helveticus genomes revealed that the nucleotide sequence shown in SEQ ID NO:1 contains polymorphic sites at positions 234 and 454 specific to CICC 6064: CICC 6064 has an A at position 234 (C for other Lactobacillus helveticus) and an A at position 454 (G for other Lactobacillus helveticus). This specific SNP site allows for precise detection of CICC 6064 at the strain level. Primers were designed using Primer Premier 6 software for the nucleotide sequence shown in SEQ ID NO:1 and its polymorphic site at position 454. Oligo 7 software was used to comprehensively evaluate parameters such as Tm value, GC content, and dimer formation of the primers. Finally, three pairs of specific primers and two TaqMan probes were selected. Specific sequence information is shown in Table 1.

[0034] Table 1: Specific primers and probes for CICC 6064

[0035]

[0036] Note: Bases marked with * are CICC 6064-specific SNP sites on the probe sequence.

[0037] Using the NCBI primer tool Primer-BLAST, bacteria (Bacteria) <taxid:2>The specificity of the primer pairs represented by SEQ ID NO: 2 and SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and SEQ ID NO: 6 and SEQ ID NO: 7 was verified genome-wide, allowing up to 4 base mismatches per primer, with an amplification fragment length of no more than 2000 bp, to screen primers with good specificity on Lactobacillus helveticus. The results showed that SEQ ID NO: 4 and SEQ ID NO: 5 might produce non-specific amplification on multiple species of Actinomyces; the primer specificity of SEQ ID NO: 2 and SEQ ID NO: 3, and SEQ ID NO: 6 and SEQ ID NO: 7 were both good, and could be used for physical verification.

[0038] To systematically evaluate the intra-species inclusivity and inter-species exclusivity of the two primer pairs, 11 strains of Lactobacillus helveticus (including CICC 6064) and 36 strains of non-Lactobacillus helveticus model strains in the "List of Strains for Use in Food" (a total of 47 strains) were selected for experimental verification. The strain information is shown in Table 2. The PCR amplification results showed that the primers represented by SEQ ID NO: 2 and SEQ ID NO: 3, and SEQ ID NO: 6 and SEQ ID NO: 7 had good inclusivity on Lactobacillus helveticus (as shown in Figure 1 ), and could effectively amplify all Lactobacillus helveticus. The PCR product was consistent with the length of the target fragment (123 bp), and no non-specific bands were detected. The exclusivity results of the 36 non-Lactobacillus helveticus strains are shown in Figure 2 and Figure 3 The primers represented by SEQ ID NO: 2 and SEQ ID NO: 3 did not appear to have non-specific amplification in the model strains of non-Lactobacillus helveticus, and the primers represented by SEQ ID NO: 6 and SEQ ID NO: 7 appeared to have non-target fragment amplification products in the model strains of non-Lactobacillus helveticus, indicating that the former had better specificity. Therefore, the primers represented by SEQ ID NO: 2 and SEQ ID NO: 3 were preferred for the development of the subsequent quantitative detection system.

[0039] Table 2: Strain information for verification experiments

[0040]

[0041] For specificity verification of the probe at the strain level, 6 strains of Lactobacillus helveticus CICC 6064, CICC 6024, CICC 6272, CICC 22171, CICC 22536 and CICC 24208 T were selected for qPCR experiments, and the results are shown in Figure 4 As shown. At annealing temperature 55℃, both probes SEQ ID NO: 8 and SEQ ID NO: 9 can detect the amplification signal of L. helveticus except CICC 6064, but probe SEQ ID NO: 8 can significantly distinguish CICC 6064 (red line) from other 5 strains of L. helveticus, which can be used as a candidate probe. Further optimization of reaction conditions, with the continuous increase of annealing temperature, the specificity of probe SEQ ID NO: 8 for CICC 6064 detection is continuously improved, which can effectively distinguish CICC 6064 and other L. helveticus at 63℃ ~ 64℃.

[0042] In summary, through strict screening of primer probe specificity and condition optimization, the primer group SEQ ID NO: 2 and SEQ ID NO: 3 and the probe SEQ ID NO: 8 with higher specificity are finally selected to construct the CICC 6064 strain level detection system. This combination shows excellent performance in species identification and strain specific recognition, which can ensure the accuracy and reliability of the detection results. Example 2

[0043] This example is used to illustrate the universality of the polymorphic site in the identification of CICC 6064 strain within L. helveticus species.

[0044] To fully verify the universality of the polymorphic site for L. helveticus CICC 6064 strain level identification standard within the species, 185 L. helveticus genomes were collected from NCBI database and CICC resource library as reference genomes for supplementary verification at the genome level. The homologous sequences of SEQ ID NO: 1 in 185 genomes were retrieved, and 100% sequence similarity was used as the threshold for clustering and de-duplication, and finally 39 homologous groups were obtained. One representative nucleotide sequence was selected from each group for difference alignment analysis with SEQ ID NO: 1.

[0045] The results show that the full-length nucleotide sequence of SEQ ID NO: 1 is 687 bp, and 51 SNP sites are detected by alignment with homologous genes (see Table 3). Among them, CICC 6064 has specific differences at positions 234 (A / C) and 454 (A / G) with other L. helveticus homologous sequences, which can be used as specific genetic markers for strain level identification, and the discrimination standard has universality within the L. helveticus species.

[0046] Table 3: Difference sites of SEQ ID NO: 1 between CICC 6064 strain and other L. helveticus

[0047]

[0048] Note: "." indicates that the sequence is identical to the reference sequence of CICC 6064. Example 3

[0049] This example illustrates the genetic stability of polymorphic sites across different generations of CICC 6064 strains.

[0050] Strain CICC 6064 was obtained from the CICC strain resource bank, cultured, and passaged to obtain strains from generations 1 to 10. Genomic DNA was extracted from the strain and used as a template for PCR amplification using primers SEQ ID NO:2 and SEQ ID NO:3. The PCR reaction system consisted of a total volume of 50 μL: 2 μL DNA template, 1 μL × 2 primers at a working concentration of 10 μmol / L, 25 μL of 2×PCR Taqmix, and 21 μL of ddH2O. The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 1 min, 72℃ for 35 s, for 35 cycles; and 72℃ for 10 min. The PCR products were subjected to 1% agarose gel electrophoresis and sequencing.

[0051] The results showed that the lengths of the amplified fragments all conformed to the theoretically designed length (approximately 123 bp). See the attached results. Figure 5 A. The PCR amplification product, after sequencing, yielded a nucleotide sequence of 123 bp. The nucleotide sequence at position 454 of SEQ ID NO:1 was consistently A, showing a 100% nucleotide match and being completely consistent with the PCR product sequence predicted by the conserved sequence SEQ ID NO:1 on the CICC 6064 genome. This indicates that the SNP site upon which the discrimination criterion proposed in this invention is genetically stable within the CICC 6064 strain and can serve as a valid reference for the identification of CICC 6064 strains.

[0052] Lactobacillus helveticus strain CICC 6064 was obtained from the CICC strain resource bank and passaged under anaerobic conditions at 37℃ using MRS culture to obtain strains from generation 1 to generation 10. Genomic DNA was extracted from each generation of strains as PCR templates, and primers covering the SNP site at position 454 of SEQ ID NO:1 were used to amplify SEQ ID NO:2 and SEQ ID NO:3. The PCR reaction system (50 μL) was prepared as follows: 2 μL DNA template, 1 μL each of primers at a working concentration of 10 μmol / L, 25 μL 2×PCR Taqmix, and 21 μL ddH2O; the reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 60℃ annealing for 1 min, 72℃ extension for 35 s (35 cycles), and a final extension at 72℃ for 10 min. The amplified products were verified by 1% agarose gel electrophoresis and analyzed by Sanger sequencing.

[0053] The results showed that the PCR amplification products of each generation of the strain all exhibited a 123 bp target band (Figure 5A). Sequencing results showed that the product sequence matched the predicted sequence of SEQ ID NO:1 by 100%, and the 454th base was a characteristic A base, with no base mutations or polymorphisms (Figure 5B). This result confirms that the SNP site at position 454 of SEQ ID NO:1 has high genetic stability in CICC 6064 strain and can be used as a specific molecular marker for strain-level identification. Example 4

[0054] This example illustrates the inclusiveness and exclusivity of the precise quantification method for Lactobacillus helveticus CICC 6064.

[0055] To verify the inclusiveness of the established CICC 6064 strain-level qPCR detection method, ten CICC 6064 samples from different sources and forms (including two liquid cultures, two solid cultures, three lyophilized powders, and three compound bacterial agent samples) were selected, and the concentration of the target strain was adjusted to 1.75 × 10⁻⁶. 7 The samples were tested at approximately CFU / mL, and the results are shown in Table 4. Experimental data show that the absolute deviation of the detected values ​​from the theoretical values ​​for all 10 samples was <0.5 Log. 10 (CFU / mL) showed no significant differences between different batches, morphological samples, and single / mixed bacterial systems, indicating that the method can efficiently detect the target strain and confirms its good adaptability to samples from different sources, with different morphologies and complex matrices.

[0056] To assess the exclusivity of the method, 47 physical strains (including 11 *Lactobacillus helveticus* strains and 36 non-*Lactobacillus helveticus* model strains) listed in Table 2 were tested, with a focus on validating the specific recognition ability of the 11 *Lactobacillus helveticus* strains. The test results are shown in Figure 6. The validation results showed that no amplification signal was detected in any of the 36 non-*Lactobacillus helveticus* model strains. Among the 11 *Lactobacillus helveticus* strains, only CICC 6064 showed a specific Ct value, while the other strains of the same species showed no amplification response. This result fully confirms that the established qPCR method has strict species and strain-level specificity, effectively eliminating interference from non-target strains, significantly reducing the false positive rate, and ensuring the accuracy and reliability of the test results.

[0057] Table 4: Method Inclusivity Validation

[0058] Example 5

[0059] This embodiment is used to verify the practical applicability of the accurate quantification method for Lactobacillus helveticus CICC 6064 in compound microbial agents, complex foods, and daily chemical products.

[0060] By simulating the complex microbial community background of real-world application scenarios, a concentration of 10 8 Fourteen interfering bacteria at CFU / mL (including nine heterologous strains and five different strains of the same species of Lactobacillus helveticus) and gradient concentrations (10... 6 -10 8 The CICC 6064 bacterial suspension (CFU / mL) was mixed and the repeatability was detected using the established qPCR method. The accuracy of the method was evaluated by the difference between the measured value and the theoretical value of plate count. The results are shown in Table 5.

[0061] Experimental data showed that when 9 non-Lactobacillus helveticus strains and 5 different strains of the same species of Lactobacillus helveticus were used as high concentrations (10... 8 When interfering with bacteria (CFU / mL), the target strain CICC 6064 was at 10 6 -10 8 The difference between the measured and theoretical values ​​within the CFU / mL concentration range is ≤|±0.5 log 10 | (CFU / mL), within the acceptable error range of ISO 16140-2 standard, with good consistency in test results. Especially when the concentration of interfering bacteria is extremely high while the concentration of target bacteria is as low as 10. 6 Even under extreme conditions of CFU / mL, this qPCR method can still achieve accurate identification thanks to its highly specific primer-probe combination, demonstrating its efficient detection capability for CICC 6064 in complex microbial matrices and providing reliable technical support for the practical detection of complex samples such as fermented dairy products and cosmetics.

[0062] Table 5: Applicability of CICC 6064 Detection under Mixed Bacterial Conditions

[0063] Example 6

[0064] This example illustrates the limit of quantitation (LOQ) of the precise quantification method for Lactobacillus helveticus CICC 6064.

[0065] Based on the characteristics of TaqMan-qPCR technology, a Ct value of 35 was set as the upper limit of detection. Using standard curve data from 10 replicate assays, the concentration of CICC 6064 strain at Ct=35 was calculated to be approximately 300 CFU / mL (verified by plate counting method). To evaluate the consistency and accuracy of low-concentration detection, DNA samples were diluted to 300 CFU / mL and 10 replicate assays were performed. The results are shown in Table 6.

[0066] Experimental data showed that the logarithmic difference between the measured values ​​of 10 repeated tests and the theoretical values ​​of plate counts was ≤|±0.5 log| 10 The concentration (CFU / mL) met the acceptable error range of ISO 16140-2, and all repeated experiments consistently detected the target signal. This result confirms that the limit of quantitation for this method is 300 CFU / mL, demonstrating good repeatability and accuracy even at low concentrations, meeting the detection requirements for the low-abundance target strain CICC 6064 in the product.

[0067] Table 6: Validation of the method limit of quantitation

[0068]

[0069] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the 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 invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the nucleotide sequence shown in SEQ ID NO:1 in the precise quantification of *Lactobacillus helveticus* CICC 6064 at the "strain" level, characterized in that... The nucleotide sequence shown has a specific polymorphic site at position 454, and when this site is A, the test strain is identified as Lactobacillus helveticus CICC 6064, where the application is not for diagnostic purposes.

2. A precise quantitative method for *Lactobacillus helveticus* CICC 6064 for non-diagnostic purposes, characterized in that, At least the following steps are included: S1. Extract genomic DNA from the sample to be tested; S2. Real-time quantitative PCR amplification was performed using specific primers and probes, wherein the nucleotide sequences of the primers are shown in SEQ ID NO:2 and SEQ ID NO:3, and the nucleotide sequence of the probe is shown in SEQ ID NO:8; S3. Calculate the concentration of CICC 6064 in the sample based on the standard curve; The combination of primers and probes enables precise quantification of CICC 6064, with a quantification limit of 300 CFU / mL, and a range up to 10 8 It can still maintain specific quantification capability in the presence of non-target Lactobacillus helveticus at CFU / mL.

3. The method according to claim 2, characterized in that, The real-time quantitative PCR reaction system is as follows: 10 μL of 2×TaqManqPCR premix, 0.4 μL of upstream primer with a working concentration of 10 μmol / L, 0.4 μL of downstream primer 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 a final volume of 20 μL.

4. The method according to claim 2, characterized in that, The qPCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 64℃ annealing for 32 s, 72℃ extension for 25 s, for a total of 40 cycles.

5. The application of primer-probe combination in the precise quantification of *Lactobacillus helveticus* CICC 6064 at the strain level, characterized in that the nucleotide sequences of the primers are shown in SEQ ID NO:2 and SEQ ID NO:3, and their amplification product is 123 bp in length, covering the specific SNP site at position 454 of SEQ ID NO:1, suitable for real-time quantitative PCR detection of *Lactobacillus helveticus* CICC 6064; the nucleotide sequence of the probe is shown in SEQ ID NO:8, designed based on the SNP site at position 454 of SEQ ID NO:1, with a fluorescent reporter group labeled FAM at the 5' end and a quencher group labeled BHQ1 at the 3' end, wherein the application is for non-diagnostic purposes.

6. The application of the detection kit in the accurate quantification of Lactobacillus helveticus CICC 6064 at the strain level, characterized in that... The kit comprises the primer and probe combination as described in claim 5, qPCR premix, and standards. The kit enables accurate quantification of Lactobacillus helveticus CICC 6064 with a limit of quantification of 300 CFU / mL, wherein the application is for non-diagnostic purposes.

7. The application of the primer-probe combination as described in claim 5 or the detection kit as described in claim 6 in the preparation of products for quantitative detection of Lactobacillus helveticus CICC 6064 at the "strain" level, wherein the application includes quality control, research and development testing and colonization studies of probiotic products, fermented dairy products, cosmetics, feed and intestinal microbial samples.

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