CN116334256B - Identification method of streptococcus thermophilus CICC 6038 strain, and primer, kit and application thereof - Google Patents
Identification method of streptococcus thermophilus CICC 6038 strain, and primer, kit and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganism identification, in particular to an identification method of streptococcus thermophilus CICC 6038 strain, and a primer, a kit and application thereof. The invention provides application of a nucleotide sequence shown as SEQ ID NO. 1 in identifying streptococcus thermophilus CICC 6038 strain. The invention provides an identification method of streptococcus thermophilus CICC 6038 strain, which is characterized in that genome DNA of a strain to be detected is extracted, primers are designed aiming at a nucleotide sequence shown in SEQ ID NO. 1, the genome DNA is amplified, and the streptococcus thermophilus CICC 6038 strain is judged by comparing the lengths of an actual amplified product and a target amplified product and the condition of polymorphic site bases. The invention provides a simple and rapid method for identifying streptococcus thermophilus CICC 6038 strain by utilizing a specific conserved gene sequence of streptococcus.
Description
Technical Field
The invention relates to the technical field of microorganism identification, in particular to an identification method of streptococcus thermophilus CICC 6038 strain, and a primer, a kit and application thereof.
Background
The streptococcus thermophilus (Streptococcus thermophilus) cells are in a spherical or egg-spherical shape, are paired or chain-shaped, have no spores, no flagella and no motility, and are gram-positive bacteria, wherein the diameter of the monomers is generally less than 1 mu m. Streptococcus thermophilus is one of the most commonly used natural fermentation lactobacillus strains, can accelerate the lactic acid of raw materials, promote milk protein curd, has the anti-oxidation activity, adjusts intestinal flora, inhibits pathogenic microorganisms and other probiotics, and improves the texture and flavor of the fermented dairy product, thereby improving the application value of the fermented dairy product.
Research shows that streptococcus thermophilus is not pathogenic and has good fermentation characteristics compared with other streptococcus species. The metabolic characteristics of streptococcus thermophilus such as acidogenesis, glycometabolism, proteolysis, active substance production, post acidification and the like have specificity at the strain level, and directly influence the cost and the texture of the fermented dairy product. The streptococcus thermophilus CICC 6038 has stable hereditary character, strong lactic acid metabolism capability and weak post acidification, and is a high-quality starter strain with great development potential. Therefore, the rapid and accurate 'strain' level identification method is an important technical guarantee for the research, development and commercial application of the streptococcus thermophilus CICC 6038 strain, and has important significance for industrial production.
Genomic data of over 200 strains of Streptococcus thermophilus have been recorded in the NCBI GenBank database. However, most common means of microbial sequencing can only identify microorganisms to species (species) levels, but there are potential functional differences between different isolates of streptococcus thermophilus. Therefore, a specific identification method of each target strain needs to be developed in a targeted manner, so that rapid and accurate identification of the strain level is realized.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for simply and rapidly identifying streptococcus thermophilus CICC 6038 strain, and a primer, a kit and application thereof.
The invention provides application of a nucleotide sequence shown as SEQ ID NO. 1 in identifying streptococcus thermophilus CICC 6038 strain.
Alternatively, the 208 th and 537 th bases on the nucleotide sequence shown in SEQ ID NO. 1 are polymorphic sites.
The invention provides an identification method of streptococcus thermophilus CICC 6038 strain, which at least comprises the following steps:
s1, extracting genome DNA of a strain to be detected;
s2, designing a primer aiming at a nucleotide sequence shown in SEQ ID NO. 1, wherein a target amplification product of the primer covers at least one polymorphic site of the SEQ ID NO. 1;
s3, amplifying the genome DNA by using a primer to obtain an actual amplification product;
s4, if the lengths of the actual amplification product and the target amplification product are inconsistent, identifying the strain to be detected as non-streptococcus thermophilus CICC 6038;
s5, sequencing the actual amplification product according to the length of the actual amplification product and the target amplification product, and comparing the actual amplification product with the nucleotide sequence shown in SEQ ID NO. 1:
if the base of the polymorphism site of the actual amplified product is the same as SEQ ID NO. 1, the strain to be detected is identified as streptococcus thermophilus CICC 6038;
if the actual amplified product polymorphism site has different base from SEQ ID NO. 1, the strain to be detected is identified as non-streptococcus thermophilus CICC 6038.
Optionally, the identification method of streptococcus thermophilus CICC 6038 strain at least comprises the following steps:
s1, extracting genome DNA of a strain to be detected;
s2, designing a primer aiming at a nucleotide sequence shown in SEQ ID NO. 1, wherein a target amplification product of the primer at least covers bases from 208 th to 537 th positions of the SEQ ID NO. 1; the length of the target amplification product is 330 bp-762 bp;
s3, amplifying the genome DNA by using a primer to obtain an actual amplification product;
s4, if the lengths of the actual amplification product and the target amplification product are inconsistent, identifying the strain to be detected as non-streptococcus thermophilus CICC 6038;
s5, sequencing the actual amplification product according to the length of the actual amplification product and the target amplification product, and comparing the actual amplification product with the nucleotide sequence shown in SEQ ID NO. 1:
if the actual amplification product is T at the 208 th base and C at the 537 th base on the corresponding positions of the nucleotide sequence shown in SEQ ID NO. 1, the strain to be detected is identified as streptococcus thermophilus CICC 6038;
if the actual amplification product is not T at the 208 th base or C at the 537 th base at the corresponding position of the nucleotide sequence shown in SEQ ID NO. 1, the strain to be tested is identified as non-streptococcus thermophilus CICC 6038.
Optionally, in S2, the length of the target amplification product is 400 bp-600 bp.
Alternatively, in S2, the target amplification product of the primer covers at least the 119 th to 625 th bases of SEQ ID NO. 1.
Alternatively, the nucleotide sequences of the primers are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
The invention provides a kit for identifying streptococcus thermophilus CICC 6038 strain, which uses a nucleotide sequence shown in SEQ ID NO. 1 to identify the strain, and comprises the step of detecting polymorphic sites on the nucleotide sequence shown in SEQ ID NO. 1 by adopting a PCR technology and a gene sequencing technology.
Optionally, the kit comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent at least comprises a primer pair with nucleotide sequences shown as SEQ ID NO. 2 and SEQ ID NO. 3.
The invention provides a primer for identifying streptococcus thermophilus CICC 6038 strain, and the nucleotide sequence of the primer is shown as SEQ ID NO. 2 and SEQ ID NO. 3.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the invention provides a simple and rapid method for identifying streptococcus thermophilus CICC 6038 strain by utilizing a specific conserved gene sequence of streptococcus.
In a preferred technical scheme, the primer provided by the invention has good specificity on streptococcus and can accurately and rapidly identify CICC 6038 strain on a strain level.
Drawings
FIG. 1 is a evolutionary tree constructed from the conserved gene 6038GL000641 and the closely related sequences in the 44 NCBI libraries;
FIG. 2 is a diagram of electrophoresis of specific amplification verification of 3 pairs of primers; 1 is CICC 6038,2, CICC 6222,3 is CICC 10135R,4 is CICC 10387,5, CICC 10465,6 is CICC 25094,7 is CICC 25139;
FIG. 3 is a diagram of the specific amplification verification electrophoresis of the primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3; 1 is CICC 6038-1,2 is CICC 6038-2,3 is CICC 6038-3,4 is CICC 6058,5 is CICC 6063,6 is CICC 6222,7 is CICC 10135R,8 is CICC 10138R,9 is CICC 10141R,10 is CICC 10387, 11 is CICC 10465, 12 is CICC 25139, 13 is CICC 25094, 14 is CICC 6246, 15 is CICC 24337, 16 is CICC 6081, 17 is CICC 6117, 18 is CICC 6132, and 19 is blank;
FIG. 4 is a diagram showing the alignment information of 26 homologous representative sequences of single nucleotide polymorphism sites of the sequence shown in SEQ ID NO. 1. Using CICC 6038 (first column, CICC 6038) as the template, the same site is labeled ". Cndot." as the template, and the difference base is labeled differently.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.
The first aspect of the embodiment of the invention provides application of a specific conserved gene sequence of the streptococcus thermophilus CICC 6038 strain, and the nucleotide sequence is shown as SEQ ID NO. 1.
The genome data of the whole streptococcus thermophilus is collected from a bacterial resource library of China industry microbiological culture collection center (CICC) and a NCBI GenBank database, and a nucleic acid sequence (shown as SEQ ID NO: 1) of a conserved encoding protein on a CICC 6038 genome and specific SNP locus information thereof are obtained through comparative genomics analysis. The nucleic acid sequence was designated 6038GL000641 as a coding gene and was deduced as a tyrosine type site-specific recombinase (site-specific tyrosine recombinase XerD) by alignment with Swissprot and NCBI databases. The homologous sequences were searched by Blast in NCBI NT database, with no results outside the Streptococcus genus and 112 homologous results on the Streptococcus genus. 86 results are streptococcus thermophilus, the sequence coverage rate of the streptococcus thermophilus is 100% compared with the sequence coverage rate of the gene 6038GL000641, and the sequence similarity is more than 98%; the comparison coverage rate of 4 result sequences is only 7%, and the result sequences are directly discarded; the other 22 sequences are distributed on streptococcus salivarius (21) and streptococcus vestibuli (1) which are closely related to streptococcus thermophilus, the coverage rate of the aligned sequences is 100%, and the sequence similarity is between 81 and 84%. The evolution analysis was constructed by combining the gene 6038GL000641 with 22 representative sequences, which are redundant from 86 homologous sequences of Streptococcus thermophilus, and 45 nucleic acid sequences, which are not homologous sequences of Streptococcus thermophilus, and the results are shown in FIG. 1. The results show that the homologous sequence of the nucleotide sequence shown in SEQ ID No. 1 is only present in Streptococcus, is conserved and ubiquitous on Streptococcus thermophilus, and is absent or relatively low in homology on other Streptococcus, and is a conserved genetic marker specific for Streptococcus thermophilus.
Experiments prove that the streptococcus thermophilus CICC 6038 strain and other strains can be identified by utilizing the nucleotide sequence shown in SEQ ID NO. 1 and a specific SNP locus. Wherein, the 208 th and 537 th bases on the nucleotide sequence shown in SEQ ID NO. 1 are polymorphic sites.
In a second aspect of the embodiments of the invention, a method is provided for identifying a streptococcus thermophilus cic 6038 strain, wherein a primer can be used for specifically amplifying a target fragment in streptococcus; on the basis, whether the target strain is CICC 6038 or not is judged, and the specific SNP locus discrimination criteria of the primer amplified fragment must be met. The method specifically comprises the following steps:
s1, extracting genome DNA of a strain to be detected;
s2, designing a primer aiming at a nucleotide sequence shown in SEQ ID NO. 1, wherein a target amplification product of the primer covers at least one polymorphic site of the SEQ ID NO. 1;
s3, amplifying genome DNA of the strain to be detected by using the primer to obtain an actual amplification product;
s4, if the lengths of the actual amplification product and the target amplification product are inconsistent, identifying the strain to be detected as non-streptococcus thermophilus CICC 6038;
s5, sequencing the actual amplification product according to the length of the actual amplification product and the target amplification product, and comparing the actual amplification product with the nucleotide sequence shown in SEQ ID NO. 1:
if the base of the polymorphism site of the actual amplified product is the same as SEQ ID NO. 1, judging that the strain to be detected is identified as streptococcus thermophilus CICC 6038;
if the actual amplified product polymorphism site has different base from SEQ ID NO. 1, judging that the strain to be detected is identified as non-streptococcus thermophilus CICC 6038.
The identification method of the embodiment of the invention overcomes the defect that the homology of the 16S rDNA sequence is required to be analyzed in the existing method, fully utilizes the specific conserved nucleic acid sequence of streptococcus thermophilus, establishes a simple and rapid identification method, does not need complex instruments, and is suitable for large-scale popularization and application. In order to further ensure the accuracy of the identification, it is preferable that the target amplification product covers the polymorphic sites of two SEQ ID NO. 1, and that the bases of the two polymorphic sites in the actual amplification product are identical to those of SEQ ID NO. 1, and that the streptococcus thermophilus CICC 6038 strain is determined.
As a further preferable technical scheme of the embodiment of the invention, the identification method of the streptococcus thermophilus CICC 6038 strain at least comprises the following steps:
s1, extracting genome DNA of a strain to be detected;
s2, designing a primer aiming at a nucleotide sequence shown in SEQ ID NO. 1, wherein a target amplification product of the primer at least covers bases from 208 th to 537 th positions of the SEQ ID NO. 1; the length of the target amplification product is 330 bp-762 bp;
s3, amplifying the genome DNA by using a primer to obtain an actual amplification product;
s4, if the lengths of the actual amplification product and the target amplification product are inconsistent, identifying the strain to be detected as non-streptococcus thermophilus CICC 6038;
s5, sequencing the actual amplification product according to the length of the actual amplification product and the target amplification product, and comparing the actual amplification product with the nucleotide sequence shown in SEQ ID NO. 1:
if the actual amplification product is T at the 208 th base and C at the 537 th base on the corresponding positions of the nucleotide sequence shown in SEQ ID NO. 1, the strain to be detected is identified as streptococcus thermophilus CICC 6038;
if the actual amplification product is not T at the 208 th base or C at the 537 th base at the corresponding position of the nucleotide sequence shown in SEQ ID NO. 1, the strain to be tested is identified as non-streptococcus thermophilus CICC 6038.
In the preferable technical scheme, when the target amplification product of the primer covers the 208 th to 537 th bases of SEQ ID NO. 1, the length of the target amplification product is 330bp; i.e., the target amplification product covers both polymorphic sites at position 208 and 537. In order to ensure the accuracy of sequencing, the length of the amplification product of interest may be suitably extended, for example, the upstream primer may be designed at a position 10 to 100 bp upstream of the 208 th base of SEQ ID NO. 1, and the downstream primer may be designed at a position 10 to 100 bp downstream of the 537 th base of SEQ ID NO. 1. When the target amplification product of the primer covers all the bases of SEQ ID NO. 1, the length of the target amplification product is 762bp.
As a further preferable technical scheme of the embodiment of the invention, in S2, the length of the target amplification product can be 400 bp-600 bp, more preferably 450 bp-550 bp; the target amplified product within the length range can not only have good coverage on the target fragment, but also can amplify and sequence the nucleotide fragment with the length more quickly, efficiently and accurately.
As a further preferred embodiment of the present invention, in S2, the target amplification product of the primer covers at least the 119 th to 625 th bases of SEQ ID NO. 1. Aiming at 119 th to 625 th bases of SEQ ID NO. 1, the embodiment of the invention obtains a pair of primer pairs with optimal amplification effect through primer screening, and the nucleotide sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 3 (see Table 1).
Table 1: specific primer information for CICC 6038
The length of the PCR product obtained by amplification of the primer pair is 507bp, the base of the 90 # locus of the product is T (the SNP locus is positioned on a 15bp nucleic acid sequence ACGTAAGTGCTCGGC and underlined) and the base of the 419 # locus of the product is C (the SNP locus is positioned on a 15bp nucleic acid sequence TTAAAAACGACAATA and underlined).
The specific primer for identifying CICC 6038 developed based on the gene sequence has specificity on the streptococcus thermophilus species level, 507bp target products can be obtained, and the strain with target strips amplified by PCR can be identified as streptococcus thermophilus. Experiments prove that the primer pair has good detection accuracy and 100% differentiation on the near-edge strains. The specificity of the Primer pairs shown in SEQ ID NO. 2 and SEQ ID NO. 3 was verified in bacterial (Bacteria < maxid: 2 >) genome using NCBI Primer tool Primer-BLAST, allowing NO more than 6 base mismatches per Primer and NO more than 2000bp amplified fragment length. The primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3 only exist on streptococcus thermophilus in the NCBI database, and the primer pair has good specificity on the streptococcus thermophilus.
A third aspect of the embodiments of the present invention provides a kit for identifying a Streptococcus thermophilus CICC 6038 strain, wherein the identification of the strain is performed by using the nucleotide sequence shown in SEQ ID NO. 1, and the kit comprises PCR technology and gene sequencing technology for detecting the polymorphic site on the nucleotide sequence shown in SEQ ID NO. 1. Specifically, the 208 th and 537 th bases on the nucleotide sequence shown in SEQ ID NO. 1 are polymorphic sites.
As an improvement of the embodiment of the invention, the kit comprises a nucleic acid amplification reagent, and the nucleic acid amplification reagent at least comprises a primer pair with nucleotide sequences shown as SEQ ID NO. 2 and SEQ ID NO. 3. Besides primers, dNTPs, buffers, DNA polymerase, and the like are included. The method comprises the following steps: primer (10. Mu. Mol/L) 2. Mu.L.times.2, 2 XPCR Taqmix 25. Mu.L, ddH 2 O21μL。
In a third aspect, the embodiment of the invention provides a primer for identifying streptococcus thermophilus CICC 6038 strain, the nucleotide sequence of which is shown as SEQ ID NO. 2 and SEQ ID NO. 3.
The following examples are further illustrative of the present invention, wherein all experimental reagents used in the following examples are commercially available, and all strains used in the following examples are existing strains and are deposited in China center for type culture collection (CICC).
Example 1
This example is used to illustrate the primer design and specificity verification process.
1) Primer design and selection:
primers containing SNP sites were designed using Primer Premier 6 against the nucleic acid sequence of gene 6038GL000641, and comprehensive evaluation was performed on the primers by Oligo 7, and finally 3 pairs of specific primers were selected as shown in Table 1. Based on the above, 7 strains of streptococcus genus strains such as streptococcus thermophilus CICC 6038, streptococcus thermophilus CICC 6222, streptococcus thermophilus CICC 10135R, streptococcus mutans CICC 10387, streptococcus agalactiae CICC 10465, streptococcus pyogenes CICC 25094, streptococcus agalactiae Ma Yang subspecies CICC 25139 and the like in the CICC strain resource physical library are selected for carrying out experiments of verifying the primer amplification efficiency, and the optimal primer scheme is optimized.
Table 2 validates the experimental strain information
The PCR reaction system was 50. Mu.L total volume: DNA template 2. Mu.L, primer (10. Mu. Mol/L) 2. Mu.L.times.2, 2 XPCR Taqmix 25. Mu.L, ddH 2 O21. Mu.L; PCR amplification conditions were 95℃for 5min;95 ℃ 30s,55 ℃ 30s,72 ℃ 35s,35 cycles; and at 72℃for 10min. The amplified products were subjected to gel electrophoresis, and the results are shown in FIG. 2.
As shown in the experimental results shown in FIG. 2, the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3 have high amplification efficiency and better specificity. The other 2 pairs of primers present amplification products of non-target fragments in the control strain. And carrying out a specific amplification verification experiment on the strain.
2) Primer specificity verification:
in order to comprehensively evaluate the specificity of the primers shown in SEQ ID No. 2 and SEQ ID No. 3 on streptococcus thermophilus, 18 strains are selected as experimental verification strains, and the experimental verification strains comprise 3 strains of different generations of the same strain of CICC 6038, 6 strains of streptococcus thermophilus which are not CICC 6038, 4 strains of other species of streptococcus, 2 strains of other genera of the family Streptococcaceae and 3 strains of other families. The specific information of the verified strain is shown in table 2, and the taxonomic conditions of different generations (in plants), different strains (between plants), different species (between species) of the same genus, different genera (between genus) of the same family and different families are covered. After the strains are activated, transferred and cultured and the purity is confirmed, the genome DNA is extracted, and a primer PCR amplification verification experiment is carried out.
The PCR reaction system was 50. Mu.L total volume: DNA template 2. Mu.L, primer (10. Mu. Mol/L) 2. Mu.L.times.2, 2 XPCR Taqmix 25. Mu.L, ddH 2 O21. Mu.L; PCR amplification conditions were 95℃for 5min;95 ℃ 30s,55 ℃ 30s,72 ℃ 35s,35 cycles; and at 72℃for 10min. The amplified products were subjected to gel electrophoresis, and the results are shown in FIG. 3.
As shown in FIG. 3, 9 Streptococcus thermophilus had bright bands at the corresponding positions, and 9 non-Streptococcus thermophilus strains including 4 Streptococcus strains were not banded. The results show that the primers SEQ ID NO. 2 and SEQ ID NO. 3 have high amplification efficiency in the streptococcus thermophilus and good specificity, and other species than streptococcus thermophilus cannot amplify the products.
Example 2:
this example illustrates the process of identifying CICC 6038 strain in the genome of Streptococcus thermophilus species by specific primers.
To examine whether the criteria for identification of CICC 6038 "strain" level in Streptococcus thermophilus are universally applicable, 176 genomes from the NCBI RefSeq database and 6 genomes from the CICC library (other than the Streptococcus thermophilus strain of CICC 6038 in Table 2) were collected, together with 182 Streptococcus thermophilus genomes as reference genomes, and supplemental verification was performed at the genome level.
The homologous sequences of the SEQ ID NO. 1 on 182 genomes are searched by using software Blast, and the homologous sequences are clustered and de-redundant according to 100% similarity by using software cd-hit, so that 26 homologous groups are finally obtained. The thermophilic chain ball nucleic acid sequences in each homologous group are completely identical, so that each group selects a representative sequence, and the representative sequence is compared with SEQ ID NO. 1 to obtain gene difference site information, and the detail is shown in FIG. 4.
The result shows that the base at 208 th and 537 th positions of SEQ ID NO. 1 of CICC 6038 are different from the base at the same position of the homologous sequence of other streptococcus thermophilus, can be used as a special genetic marker for identifying the strain level thereof, and the identification standard has universality on streptococcus thermophilus species. Thus, primers SEQ ID NO. 2 and SEQ ID NO. 3, whose amplification products cover these two SNP sites, are able to identify the CICC 6038 strain unambiguously at the strain level.
Example 3:
this example is presented to demonstrate the genetic stability of two polymorphic sites in different strains of CICC 6038 strain.
1) Sample information and DNA template extraction
Bacterial strain CICC 6038 is obtained from CICC strain resource library, and is cultured and passaged in an aerobic environment at 37 ℃ by using an M17 culture medium; three strains of generation 1 (6038-1), generation 3 (6038-2) and generation 5 (6038-3) were obtained, and genomic DNA of the strains was extracted using a bacterial genomic DNA extraction kit (Cat.#DP302-02, TIANGEN, beijing).
2) Specific primers amplify fragments of interest
The PCR reaction system was 50. Mu.L total volume: DNA template 2. Mu.L, primer (10. Mu. Mol/L) 2. Mu.L.times.2, 2 XPCR Taqmix 25. Mu.L, ddH 2 O21. Mu.L; PCR amplification conditions were 95℃for 5min;95 ℃ 30s,55 ℃ 30s,72 ℃ 35s,35 cycles; and at 72℃for 10min. The PCR products were subjected to 1% agarose gel electrophoresis, and the amplified fragment lengths were all consistent with the theoretical design length (507 bp), and the results were as shown in FIG. 3, wherein 1 was CICC 6038-1,2 was CICC 6038-2, and 3 was CICC 6038-3.
3) Sequencing and identification results
The PCR amplified product is sequenced to obtain a nucleic acid sequence with the length of 507bp, and the 90 th base and the 419 th base of the product correspond to the 208 th base and the 537 th base of SEQ ID NO. 1 respectively. The result shows that the amplified fragment length of the primer in three samples 6038-1, 6038-2 and 6038-3 is consistent, the nucleic acid sequence is 100% matched, and the primer is completely consistent with the PCR product sequence predicted by the conserved sequence SEQ ID NO. 1 on the CICC 6038 genome. The SNP locus based on the discriminant criterion provided by the invention is genetically stable in CICC 6038 strain, and can be used as an effective reference for CICC 6038 strain identification.
Example 4:
this example is used to demonstrate the verification process of rapid and accurate identification of bacterial strain CICC 6038 between different strains within the species of Streptococcus thermophilus.
1) Sample information and DNA template extraction
Strains CICC 6058, CICC 6063, CICC 6222 and CICC 10135R, CICC 10138R, CICC 10141R are obtained from a CICC strain resource library and serve as experimental strains for inter-strain specific primer verification. All strains have been subjected to high throughput sequencing to obtain their genome data and identified as belonging to Streptococcus thermophilus by bioinformatic analysis. Among them, whole genome SNP analysis can determine that they belong to different strains, and SNP information is detailed in Table 3. The strain culture conditions and the genomic DNA extraction method were the same as in example 3.
Table 3: SNP distance matrix of whole genome of experimental strain
2) Specific primers amplify fragments of interest
The PCR amplification system and the amplification conditions are the same as in example 3, the PCR product is subjected to agarose gel electrophoresis with concentration of 1%, the length of the amplified fragment accords with the theoretical design length (507 bp), and the result is shown in FIG. 3 in detail.
3) Sequencing and identification results
Table 4: amplified fragment differences of CICC 6038 from other Streptococcus thermophilus strains
In addition to CICC 6038 strain, the PCR products of other 6 strains of streptococcus thermophilus are sequenced to obtain 507bp nucleic acid sequences respectively. The sequences obtained by these sequencing were compared with the amplified product sequences of CICC 6038 strain to obtain the gene difference site information, as shown in Table 4. The 90 th and 419 th bases of the amplified product of the 6 non-CICC 6038 strain are C and T respectively, and are different from the CICC 6038 strain, and neither meets the discriminant criterion. This shows that SNP locus differences according to the discriminant criteria provided by the invention are universal on streptococcus thermophilus and can be used for CICC 6038 strain identification.
Example 5:
this example is used to illustrate the process of specificity verification of primers on non-Streptococcus thermophilus.
1) Sample information and DNA template extraction
Obtaining 9 non-thermophilic streptococcus strains from a CICC strain resource library, wherein CICC 10387 (streptococcus mutans), CICC 10465 (streptococcus agalactiae), CICC 25139 (streptococcus agalactiae Ma Yang subspecies) and CICC 25094 (streptococcus pyogenes) are used as experimental strains verified by different species specific primers in streptococcus; CICC 6246 (lactococcus lactis subspecies lactis) and CICC 24337 (lactococcus lactis subspecies milk fat) are used as experimental strains verified by specific primers in different genera in the streptococcaceae; CICC 6081 (Lactobacillus acidophilus), CICC 6117 (Lactobacillus casei) and CICC 6132 (Lactobacillus reuteri) as experimental strains verified by other family specific primers. The strain culture conditions and the genomic DNA extraction method were the same as in example 3.
2) Specific primers amplify fragments of interest
The PCR amplification system and amplification conditions were the same as in example 3. The PCR products of 9 strains of non-Streptococcus thermophilus were subjected to 1% agarose gel electrophoresis, and none of them had the target bands, and the results are shown in FIG. 3. The 4 Streptococcus strains were also not amplified efficiently, which is consistent with the sequence verification results during the primer design process.
In summary, the experimental verification results and the sequence verification results show that the effective amplification of the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3 can judge that the strain is streptococcus thermophilus, and the CICC 6038 strain can be effectively identified on the strain level by combining the information of SNP loci on the amplified products.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. The application of the primer in the identification of streptococcus thermophilus CICC 6038 strain is characterized in that the primer is used for detecting the 208 th and 537 th base polymorphism sites on the nucleotide sequence shown in SEQ ID NO. 1, the 208 th base is T and the 537 th base is C on the corresponding position of the nucleotide sequence shown in SEQ ID NO. 1, and the strain to be detected is identified as streptococcus thermophilus CICC 6038; the nucleotide sequences of the primers are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
2. A method for identifying streptococcus thermophilus CICC 6038 strain, which is characterized by at least comprising the following steps:
s1, extracting genome DNA of a strain to be detected;
s2, designing a primer aiming at a nucleotide sequence shown in SEQ ID NO. 1, wherein the nucleotide sequence of the primer is shown in SEQ ID NO. 2 and SEQ ID NO. 3;
s3, amplifying the genome DNA by using the primer to obtain an actual amplification product;
s4, if the lengths of the actual amplification products and the target amplification products are inconsistent, identifying the strain to be detected as non-streptococcus thermophilus CICC 6038;
s5, sequencing the actual amplification product and comparing the actual amplification product with the nucleotide sequence shown in SEQ ID NO. 1 if the lengths of the actual amplification product and the target amplification product are consistent:
if the base of the polymorphism site of the actual amplification product is the same as SEQ ID NO. 1, the strain to be detected is identified as streptococcus thermophilus CICC 6038;
if the base of the polymorphism site of the actual amplification product is different from SEQ ID NO. 1, the strain to be detected is identified as non-streptococcus thermophilus CICC 6038;
wherein, the 208 th and 537 th bases on the nucleotide sequence shown in SEQ ID NO. 1 are polymorphic sites.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002046393A1 (en) * | 2000-12-07 | 2002-06-13 | Toyo Boseki Kabushiki Kaisha | Method of identifying nucleotide polymorphism |
| CN106480185A (en) * | 2016-10-12 | 2017-03-08 | 内蒙古农业大学 | The rapid screening method of the streptococcus thermophilus of rich extracellular polysaccharide and realize the Polymorphism nucleotide sequence of methods described |
| CN110093433A (en) * | 2019-04-22 | 2019-08-06 | 中山大学 | The SNP marker and its application of Streptococcusagalactiae |
| CN114891703A (en) * | 2022-07-05 | 2022-08-12 | 中国食品发酵工业研究院有限公司 | Compound leaven with weak post-acidification function, application thereof and preparation method of yogurt |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002046393A1 (en) * | 2000-12-07 | 2002-06-13 | Toyo Boseki Kabushiki Kaisha | Method of identifying nucleotide polymorphism |
| CN106480185A (en) * | 2016-10-12 | 2017-03-08 | 内蒙古农业大学 | The rapid screening method of the streptococcus thermophilus of rich extracellular polysaccharide and realize the Polymorphism nucleotide sequence of methods described |
| CN110093433A (en) * | 2019-04-22 | 2019-08-06 | 中山大学 | The SNP marker and its application of Streptococcusagalactiae |
| CN114891703A (en) * | 2022-07-05 | 2022-08-12 | 中国食品发酵工业研究院有限公司 | Compound leaven with weak post-acidification function, application thereof and preparation method of yogurt |
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