Identification method of Streptococcus thermophilus CICC 6063 strain and its primers, kit and application
By designing specific primers to target the conserved gene sequence of Streptococcus thermophilus CICC 6063 strain, and using PCR and gene sequencing technology to detect SNP sites, the problem of rapid identification of Streptococcus thermophilus CICC 6063 strain was solved, and production efficiency and market competitiveness were improved.
Patent Information
- Application Number
- CN202510133080.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-06
- Publication Date
- 2025-08-26
- Estimated Expiration
- 2045-02-06
AI Technical Summary
The prior art is difficult to quickly and accurately identify the CICC 6063 strain of Streptococcus thermophilus in microbiology, resulting in low production efficiency, high cost and insufficient market competitiveness.
Design specific primers to target the unique conserved gene sequence of Streptococcus thermophilus CICC 6063 strain, use PCR technology and gene sequencing technology to detect SNP sites on the nucleotide sequence, and identify the strains by amplification and sequencing.
The rapid and accurate identification of the CICC 6063 strain of Streptococcus thermophilus has been achieved, which has improved production efficiency, reduced costs, and improved market competitiveness.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial identification, and in particular to an identification method, detection primers, a detection kit and related applications of Streptococcus thermophilus CICC 6063 strain. Background Art
[0002] Streptococcus thermophilus is not only a core bacterial species in the dairy fermentation field, but also, thanks to its efficient growth and fermentation capabilities in high-temperature environments, it can rapidly acidify curds, optimize product texture, and significantly shorten the curdling cycle, thereby greatly promoting the efficiency of industrialized dairy production. Furthermore, this strain exhibits a range of beneficial probiotic properties for human health, including bile salt decomposition, hydrophobicity, and β-galactosidase activity. These properties not only help regulate intestinal microbial balance, but also effectively lower blood cholesterol levels and enhance immunity, making a significant contribution to maintaining and improving human health.
[0003] Streptococcus thermophilus CICC 6063 is often used in combination with Lactobacillus helveticus CICC 6064. There have been reports that these strains have been used to produce naked oat fermented beverages. Recent studies have shown that these strains can produce fermented milk, imparting a distinctive floral and fruity aroma to yogurt. This high-quality starter strain has great development potential.
[0004] In the field of microbiology, genetic, physiological, and metabolic characteristics can vary significantly between strains. Strain-level characterization can reveal these differences, providing insights into the diversity and complexity of microorganisms. This allows selection of strains best suited to specific production conditions, thereby improving production efficiency, reducing costs, and enhancing market competitiveness. Summary of the Invention
[0005] To solve the above technical problems, the present invention provides a simple and rapid method for identifying the thermophilic Streptococcus CICC 6063 strain and its detection primers, kit and related applications.
[0006] The present invention proposes the use of the nucleotide sequence shown in SEQ ID NO: 1 in identifying the thermophilic Streptococcus CICC 6063 strain.
[0007] Optionally, the 528th base in the nucleotide sequence shown in SEQ ID NO: 1 is a SNP site.
[0008] The present invention provides a method for identifying the thermophilic Streptococcus CICC 6063 strain, which comprises at least the following steps:
[0009] S1. Extract genomic DNA of the strain to be tested;
[0010] S2. Design primers for the nucleotide sequence shown in SEQ ID NO: 1, wherein the target amplification product of the primers covers the SNP site of SEQ ID NO: 1;
[0011] S3, using primers to amplify the genomic DNA to obtain actual amplification products;
[0012] S4. If the length of the actual amplified product is inconsistent with that of the target amplified product, the strain to be tested is identified as non-thermophilic Streptococcus CICC 6063;
[0013] S5. If the actual amplification product is consistent with the target amplification product in length, sequence the actual amplification product and compare it with the nucleotide sequence shown in SEQ ID NO: 1:
[0014] If the base of the SNP site of the actual amplified product is the same as SEQ ID NO: 1, the strain to be tested is identified as Streptococcus thermophilus CICC 6063;
[0015] If the base of the SNP site of the actual amplified product is different from that of SEQ ID NO: 1, the strain to be tested is identified as non-thermophilic Streptococcus CICC 6063.
[0016] Optionally, in S2, the length of the target amplification product is 536 bp.
[0017] Optionally, in S2, the target amplification product of the primers covers at least bases 384 to 919 of SEQ ID NO:1.
[0018] Optionally, the nucleotide sequences of the primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3.
[0019] The present invention provides a kit for identifying thermophilic Streptococcus CICC 6063, which uses the nucleotide sequence shown in SEQ ID NO: 1 for strain identification, including using PCR technology and gene sequencing technology to detect SNP sites on the nucleotide sequence shown in SEQ ID NO: 1.
[0020] Optionally, the nucleic acid amplification reagent contains at least a primer pair with nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3.
[0021] The present invention also provides a primer for identifying the thermophilic Streptococcus CICC 6063 strain, wherein the target amplification product of the primer covers position 528 of the nucleotide sequence shown in SEQ ID NO:1.
[0022] The present invention also provides a detection kit for identifying Streptococcus thermophilus CICC 6063, wherein the kit comprises the specific primers described in the present invention.
[0023] The technical solution provided by the embodiment of the present invention has the following advantages compared with the existing technology:
[0024] The present invention utilizes the unique conserved gene sequence of Streptococcus to propose a simple and rapid method for identifying the thermophilic Streptococcus CICC 6063 strain.
[0025] In a preferred technical solution, the primers of the present invention have good specificity for the genus Streptococcus and can accurately and quickly identify the CICC 6063 strain at the "strain" level. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the phylogenetic tree constructed from the conserved gene 6063Scaffolds_6_91175_24 and its 58 closely related sequences in the NCBI database; Figure 2 、 Figure 3 、 Figure 4The electropherograms are respectively the exclusive amplification verification electrophoresis 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 on species other than the genus Streptococcus. Lanes in the figure: M is DNA Marker, positive control is CICC 6063, 1 is CICC 24208, 2 is CICC 6081, 3 is CICC 6103, 4 is CGMCC 1.2625, 5 is CICC 24878, 6 is CICC 6252, 7 is CGMCC 1.3402, 8 is CICC 6117, 9 is CICC 6263, 10 is CICC 6224, 11 is CICC 24209, 12 is CICC 6132, 13 is CICC6240, 14 is CGMCC 1.1881, 15 is JCM 1096, 16 is CICC 6245, 17 is CGMCC 1.1936, 18 is CGMCC1.3992, 19 is CICC 6069, 20 is CICC 6186, 21 is CICC 6070, 22 is CICC 6250, 23 is CICC24210, 24 is CICC 6071, 25 is CICC 6079, 26 is CGMCC 1.2231, 27 is CGMCC 1.2230, 28 is CGMCC1.2138, 29 is CGMCC 1.2503, 30 is CGMCC 1.2696, 31 is CGMCC 1.2695, 32 is CGMCC 1.2009, 33 is JCM 2418, 34 is CICC 6198, 35 is JCM 1185, 36 is CCUG 33636, 37 is CCUG 15605, and CK is blank.
[0027] Figure 5 The figure is an electrophoresis diagram verifying the inclusive amplification between strains within the species of Streptococcus thermophilus using the primer pair shown in SEQ ID NO: 2 and SEQ ID NO: 3; lanes in the figure: M is a DNA marker, 1 is CICC 6063, 2 is CICC 6222, 3 is CICC 6038, 4 is CICC 6035, 5 is CICC 6059, 6 is CICC 6218, 7 is CICC 6219, 8 is CICC 6220, 9 is CICC 22563, 10 is CICC 25079, 11 is CICC 20375, and CK is a blank.
[0028] Figure 6This is an electrophoresis diagram verifying the exclusive amplification of the primer pair represented by SEQ ID NO: 2 and SEQ ID NO: 3 within the genus Streptococcus; in the figure, lanes: M is DNA Marker, 1 is CICC 6063, 2 is CICC 10387, 3 is CICC 10465, 4 is CICC 25094, 5 is CICC 25139, and CK is blank.
[0029] Figure 7 This is an electrophoresis diagram showing the amplification of CICC 6063 strains at different passages using primers shown in SEQ ID NO: 2 and SEQ ID NO: 3. Lanes M represents a DNA marker, 1 to 10 represent passage numbers of CICC 6063, and CK represents a blank.
[0030] Figure 8 This is a comparison of single nucleotide polymorphisms (SNPs) between the sequence represented by 6063Scaffolds_6_91175_24 and 39 representative homologous sequences from Streptococcus thermophilus. CICC 6063 (first column, CICC 6063) was used as the template. Identical sites are marked with a "·"; sites that differ from the template are marked with a differential base. DETAILED DESCRIPTION
[0031] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme 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.
[0032] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all the embodiments.
[0033] The genome sequence of Streptococcus thermophilus was retrieved from the China Industrial Culture Collection (CICC) and the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ). Comparative genomic analysis revealed a conserved protein-encoding nucleotide sequence (SEQ ID NO:1) and its SNP location within the CICC 6063 genome. This nucleotide sequence is hypothesized to be the acetylornithine aminotransferase (argD) gene. A search of the NCBI NT database for homologous sequences revealed no sequences outside the genus Streptococcus, while 166 homologous sequences within the genus Streptococcus were found. Among them, 140 sequences were from the genome of Streptococcus thermophilus, and their sequence coverage with gene CICC 6063Scaffolds_6_91175_24 was 100%, and the sequence similarity was greater than 98%; the other 26 sequences were from the genomes of Streptococcus salivarius (21 sequences), Streptococcus vestibulus (1 sequence), and Streptococcus (4 sequences), all of which had 100% sequence coverage and 87% to 90% sequence similarity. After de-redundancy processing of the 140 sequences from the genome of Streptococcus thermophilus, 33 representative sequences were obtained. This gene was subjected to phylogenetic tree development analysis with the above 33 representative sequences homologous to Streptococcus thermophilus and the remaining 26 homologous sequences from non-Streptococci thermophilus, a total of 60 nucleotide sequences. The results are detailed in [ 140 ] Figure 1 The results showed that the homologous sequence of the nucleotide sequence shown in SEQ ID NO: 1 only exists in the genus Streptococcus, is conserved and ubiquitous in Streptococcus thermophilus, and does not exist in other Streptococci or has relatively low homology, indicating that it is a conserved genetic marker unique to Streptococcus thermophilus.
[0034] Experimental verification has shown that the nucleotide sequence shown in SEQ ID NO: 1 and its specific SNP site can be used to horizontally identify Streptococcus thermophilus CICC 6063. The 528th base in the nucleotide sequence shown in SEQ ID NO: 1 is a polymorphic site.
[0035] A second aspect of the present invention provides a method for identifying the thermophilic Streptococcus CICC 6063 strain. Primers are used to specifically amplify a target fragment in Streptococcus. Based on this, determining whether the target strain is CICC 6063 requires that the primer-amplified fragment meets the specific SNP site discrimination criteria. Specifically, the method includes the following steps:
[0036] S1. Extract genomic DNA of the strain to be tested;
[0037] S2. Design primers for the nucleotide sequence shown in SEQ ID NO: 1, wherein the target amplification product of the primers covers the SNP site at position 528 of SEQ ID NO: 1;
[0038] S3, using primers to amplify the genomic DNA of the strain to be tested to obtain the actual amplification product;
[0039] S4. If the length of the actual amplified product is inconsistent with that of the target amplified product, the strain to be tested is identified as non-thermophilic Streptococcus CICC 6063;
[0040] S5. If the actual amplification product is consistent with the target amplification product in length, sequence the actual amplification product and compare it with the nucleotide sequence shown in SEQ ID NO: 1:
[0041] If the base of the SNP site of the actual amplified product is the same as SEQ ID NO: 1, the strain to be tested is determined to be Streptococcus thermophilus CICC 6063;
[0042] If the base of the SNP site of the actual amplified product is different from that of SEQ ID NO: 1, the strain to be tested is determined to be non-thermophilic Streptococcus CICC 6063.
[0043] The identification method of the embodiment of the present invention overcomes the disadvantage of the existing method that the 16S rDNA sequence homology needs to be analyzed, fully utilizes the unique conserved nucleotide sequence of Streptococcus thermophilus, and establishes a simple and rapid identification method that does not require complex instruments and is suitable for large-scale promotion and application.
[0044] As a further preferred technical solution of the present invention, in S2, targeting the polymorphic site 528 of SEQ ID NO:1, the present invention obtained a pair of primers with the best amplification effect through primer screening, the nucleotide sequences of which are shown in SEQ ID NO:2 and SEQ ID NO:3 (see Table 1). The PCR product amplified using this primer pair is 536 bp in length, and the base at position 125 of the product (excluding the primer sequence) is G (the SNP site is located in a 15 bp nucleotide sequence TTCAAGGGGAATCAG, underlined).
[0045] Table 1: Specific primer information for CICC 6063
[0046]
[0047]
[0048] The specific primers developed by the present invention based on the gene sequence for identifying CICC 6063 are specific at the species level of Streptococcus thermophilus and can obtain a target product of 536bp. The strains that amplify the target band using PCR can be identified as Streptococcus thermophilus. Experiments have confirmed that the primer pair has good detection accuracy and can distinguish closely related strains with 100%. Using the NCBI primer tool Primer-BLAST in bacteria (Bacteria <taxid:2>The specificity of the primer pair shown in SEQ ID NO: 2 and SEQ ID NO: 3 was verified genome-wide, allowing for no more than four base mismatches per primer and amplified fragments no longer than 2000 bp. The primer pair shown in SEQ ID NO: 2 and SEQ ID NO: 3 was found only in Streptococcus thermophilus in the NCBI database, demonstrating good specificity for this bacteria.
[0049] As an improvement to an embodiment of the present invention, the kit includes a nucleic acid amplification reagent, which contains at least a primer pair with the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. In addition to the primers, the kit also includes dNTPs, buffer, and DNA polymerase. Specifically, the kit includes: 2 primers (10 μmol / L) (1 μL), 2×PCR Taqmix (25 μL), and ddH2O (21 μL).
[0050] The present invention is further illustrated by specific examples. The experimental reagents used in the following examples are all commercially available. The strains involved in the present invention are all existing strains deposited in the China Industrial Culture Collection (CICC), among which Streptococcus thermophilus CICC 6063 is deposited with CICC 6063 in October 1989.
[0051] Example 1
[0052] This example is used to illustrate the primer design and specificity verification process.
[0053] 1) Primer design and simulation verification:
[0054] Primers were designed using Primer Premier 6 for the nucleotide sequence of gene SEQ ID NO: 1, and the primers were comprehensively evaluated by Oligo 7, and finally three pairs of specific primers were screened out, as shown in Table 1. <taxid:2>) Genome-wide specificity verification 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 conducted. Four base mismatches per primer were allowed, and the amplified fragment length did not exceed 2000 bp. Primers with good specificity for S. thermophilus were screened. The results showed that all three primer pairs had good specificity and were suitable for subsequent field validation.
[0055] 2) Physical verification:
[0056] To comprehensively evaluate the specificity of the three primer pairs for Streptococcus thermophilus, 37 non-Streptococcus type strains from the "List of Bacteria Acceptable for Food Use" were first selected as experimental validation strains for primer exclusivity verification and selection of the optimal primer scheme. Eleven strains of Streptococcus thermophilus, including CICC 6063, were then selected as experimental validation strains for primer inclusion within the species. Four strains of Streptococcus from other species within the genus Streptococcus were also selected as experimental validation strains for primer exclusivity within the genus and between species. Genomic DNA was extracted from all 62 experimental validation strains and PCR amplification was performed using these primer sets. Strain information is shown in Table 2.
[0057] Table 2 Verification experiment strain information
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064] 2) Specific primers amplify the target fragment
[0065] The PCR reaction system was 50 μL in total volume: 2 μL DNA template, 1 μL primer (10 μmol / L) × 2, 2× PCR Taqmix 25 μL, 21 μL ddH2O; PCR amplification conditions were 95°C for 5 min, 30 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 40 s, and 72°C for 10 min. The amplified products were subjected to gel electrophoresis. The results are detailed in the table. Figure 2-7 .
[0066] The exclusive results of non-thermophilic streptococci in the "List of bacteria that can be used in food" are as follows Figure 2 、 Figure 3 、 Figure 4 As shown, the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 did not show non-specific amplification in non-Streptococcus model strains, indicating that the primers have high amplification efficiency and better specificity; the other two primer pairs showed amplification products of non-target fragments in non-Streptococcus model strains, indicating poor specificity. Therefore, the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 were selected to conduct specific amplification verification experiments between species within the genus, and the results are shown in FIG. Figure 5 and Figure 6 The PCR products of 11 strains of Streptococcus thermophilus were consistent with the target fragment length ( Figure 5 ), and 4 strains of other Streptococcus species had no bands ( Figure 6 The above results indicate that primers SEQ ID NO: 2 and SEQ ID NO: 3 have high amplification efficiency and good specificity in Streptococcus thermophilus, and other species other than Streptococcus thermophilus cannot amplify products.
[0067] 3) Sequencing and identification results
[0068] Eleven PCR amplification products were sequenced to yield a 536-bp nucleotide sequence. Comparative analysis revealed differential site information for the CICC 6063 strain (Table 3). The 125th base (excluding primer sequences) of the amplification product sequences of non-CICC 6063 strains was always "A," unlike the "G" of the CICC 6063 strain. The results demonstrate that the SNP site differences underlying the proposed discrimination criteria are universally applicable for identifying the CICC 6063 strain in S. thermophilus and can be used for identification of S. thermophilus CICC 6063 strains at the "strain" level.
[0069] Table 3: Differential sites in the amplified fragments of CICC 6063 strain and other thermophilic Streptococci
[0070]
[0071] Example 2
[0072] This example is used to illustrate the universal applicability of polymorphic sites in identifying the CICC 6063 strain within the species Streptococcus thermophilus.
[0073] To fully test whether the identification criteria of CICC 6063 "strain" within the species S. thermophilus are universally applicable, a total of 182 S. thermophilus genomes from the NCBI database and the CICC resource library were collected as reference genomes for supplementary verification at the genome level.
[0074] Using SEQ ID NO: 1 as a template, we searched for homologous sequences across 182 genomes. Clustering and redundancy removal were performed based on 100% similarity, ultimately yielding 39 homology groups. The nucleotide sequences of S. thermophilus within each homology group were identical, so a representative sequence from each group was selected and aligned with SEQ ID NO: 1 to obtain information on differentially expressed sites (see Table 4 for details).
[0075] The results showed that the base at position 528 of SEQ ID NO: 1 differs from the base at the same position in other homologous sequences of Streptococcus thermophilus, making it a unique genetic marker for strain-level identification. This criterion is universal across all Streptococcus thermophilus species. Therefore, primers SEQ ID NO: 2 and SEQ ID NO: 3, whose amplification products encompass these two SNPs, can clearly identify CICC 6063 at the strain level.
[0076] Example 3
[0077] This example is used to illustrate the genetic stability of polymorphic sites in different generations of CICC 6063 strains.
[0078] 1) Sample information and DNA template extraction
[0079] The strain CICC 6063 was obtained from the CICC strain resource bank and cultured and passaged using M17 medium at 37°C under an aerobic environment; the 1st to 10th generation strains were obtained, and the genomic DNA of the strain was extracted using a DNA crude extraction reagent (Cat.#9182, TARAKA, Beijing).
[0080] 2) Specific primers amplify the target fragment
[0081] The PCR reaction system was a 50 μL total volume: 2 μL DNA template, 1 μL primer (10 μmol / L) × 2, 2× PCR Taqmix 25 μL, and 21 μL 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 72°C for 10 min. PCR products were subjected to 1% agarose gel electrophoresis. The amplified fragment lengths all met the theoretical design length (536 bp). The results are shown in Figure 2. Figure 7 , where 1-10 are CICC 6063-1 to CICC 6063-10 respectively.
[0082] 3) Sequencing and identification results
[0083] The PCR amplification product was sequenced to obtain a 536-bp nucleotide sequence. Base 125 of the product (excluding the primer sequence) corresponds to base 528 of SEQ ID NO:1. The results showed that the primers amplified fragments of consistent length and 100% nucleotide sequence match across all 10 CICC 6063 samples, completely consistent with the PCR product sequence predicted by SEQ ID NO:1, a conserved sequence within the CICC 6063 genome. This demonstrates that the SNPs underlying the proposed discrimination criteria are genetically stable within the CICC 6063 strain and can serve as an effective reference for identification of the CICC 6063 strain.
[0084] In summary, the experimental verification results and sequence verification results show that the effective amplification of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 can determine that the strain is Streptococcus thermophilus. Combined with the information of the SNP sites on the amplified products, the CICC 6063 strain can be effectively identified at the strain level.
[0085] 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. Use of the nucleotide sequence shown in SEQ ID NO: 1 in identifying Streptococcus thermophilus CICC 6063 strain, characterized in that: The 528th base of the nucleotide sequence shown in SEQ ID NO: 1 is G, and the 528th base G is a specific single nucleotide polymorphism (SNP) marker of Streptococcus thermophilus CICC 6063; the specific SNP marker was confirmed by comparing 182 Streptococcus thermophilus genome sequences and is uniquely present in the strain.
2. A method for identifying the thermophilic Streptococcus CICC 6063 strain, characterized in that: At least the following steps are included: S1. Extract genomic DNA of the strain to be tested; S2. Design primers for the nucleotide sequence shown in SEQ ID NO: 1, wherein the target amplification product of the primers covers the 528th polymorphic site of SEQ ID NO: 1; S3, amplifying the genomic DNA using the primers to obtain an actual amplification product; S4. If the length of the actual amplification product is inconsistent with the length of the target amplification product, the strain to be tested is identified as non-thermophilic Streptococcus CICC 6063; S5. If the actual amplification product is consistent with the target amplification product in length, sequence the actual amplification product and compare it with the nucleotide sequence shown in SEQ ID NO: 1: If the actual amplified product corresponds to the nucleotide sequence shown in SEQ ID NO: 1, the base at position 528 is G, and the strain to be tested is identified as Streptococcus thermophilus CICC 6063; If the actual amplified product corresponds to a nucleotide sequence shown in SEQ ID NO: 1, where the base at position 528 is not G, the strain to be tested is identified as non-thermophilic Streptococcus CICC 6063.
3. The identification method according to claim 2, characterized in that The nucleotide sequences of the primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3; the primers do not amplify non-Streptococcus strains, and only amplify a product containing the 528th G base in the CICC6063 strain of thermophilic Streptococcus, and the length of the target amplification product is 536 bp.
4. Use of primers in identifying Streptococcus thermophilus CICC 6063 strain, characterized in that, The nucleotide sequences of the primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the target amplification product covers bases 384 to 919 of SEQ ID NO: 1, wherein base 528 is G. The specificity of the primers was verified by the following: (a) No amplification was found in 37 non-Streptococcus strains; (b) Among the 11 strains of Streptococcus thermophilus, only CICC 6063 amplified a product containing the 528th G base.
5. Use of the detection kit in identifying Streptococcus thermophilus CICC 6063 strain, characterized in that: Comprising the primer described in claim 4, and the following components: (a) SEQ ID NO: 2 and SEQ ID NO: 3 primers at a concentration of 10 μM; (b) 2× PCR Taq premix; The PCR amplification conditions of the kit are as follows: pre-denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 40 s, and extension at 72°C for 10 min.