CN111363835A - Method for identifying bifidobacteria in sample at species level and special primer thereof - Google Patents
Method for identifying bifidobacteria in sample at species level and special primer thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying bifidobacteria in a sample on a seed level and a special primer thereof. The invention protects a specific primer pair, which consists of a primer F1 shown in a sequence 1 and a primer R1 shown in a sequence 2. The invention also protects a specific primer group, which consists of a primer pair A and a primer pair B; the primer pair A consists of a primer F2 shown in a sequence 8 and a primer R2 shown in a sequence 9; the primer pair B consists of a primer F3 shown in a sequence 10 and a primer R3 shown in a sequence 11. The invention also protects the application of the specific primer pair or the specific primer group, and aims to identify the bifidobacteria in a sample on a species level. The invention provides a new idea for the supervision of related dairy products in the market and also provides a new method for identifying specific bifidobacteria in intestinal tract samples or other samples.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for identifying bifidobacteria in a sample on a seed level and a special primer thereof.
Background
Nowadays, functional dairy products emerge endlessly in the global food market, and the demand of people for novel probiotic dairy products is increasing day by day. The bifidobacterium is a representative probiotic which is extremely popular in domestic and overseas research at present because of having a plurality of important physiological functions of biological barrier function, nutrition function, anti-tumor function, immunoregulation function, gastrointestinal tract function improvement, anti-aging function and the like on human health.
Bifidobacteria were first isolated from the intestinal flora of breast-fed infants from the paris pasteur institute, france, 1899, and a total of 57 species (subspecies) of bifidobacteria have now been found. However, along with the mass production of dairy products, the random phenomenon that the bifidobacterium adding proportion and the mark of the approved bifidobacterium or the bifidobacterium in the dairy products are inconsistent is not changed by using similar bifidobacterium. Therefore, in order to maintain the legitimate interests of consumers, it is important to identify bifidobacteria quickly and accurately. Meanwhile, the method has important significance for identifying the species and abundance of the bifidobacteria in the complex intestinal flora.
The traditional method for identifying the bifidobacteria is based on the phenotypic characteristics of bacteria in Bergey handbook, but some species with similar phenotypic characteristics and consistent physicochemical properties are difficult to distinguish. With the development of molecular biology, the full-length sequence of the 16S rDNA gene of bacteria has become the gold standard for species identification, but the second-generation high-throughput sequencing cannot be performed due to the overlong fragment (about 1.5 Kb). If the full-length 16S bacteria are used to identify bifidobacteria in the environment, the full-length PCR products of the 16S rDNA of all bacteria in the environment are cloned on a plasmid vector and then sequenced, and the method needs a great deal of manpower and material resources. If the abundance of bifidobacteria in the environment is low, the probability of detecting bifidobacteria using this method is exponentially reduced. In addition, although the 16S hypervariable regions (such as the V3 region, the V3V4 region and the like) are suitable for second-generation high-throughput sequencing, the sequences are relatively conservative, the fragments are too short, the strains can be distinguished only on the genus level, and the resolution is not high for strains with relatively close relativity.
Disclosure of Invention
The invention aims to provide a method for identifying bifidobacteria in a sample on a species level and a special primer thereof.
The invention protects a specific primer pair, which consists of a primer F1 and a primer R1; the primer F1 is shown as a sequence 1 in a sequence table; the primer R1 is shown as a sequence 2 in the sequence table. The specific molar ratio of primer F1 to primer R1 was 1: 1.
The invention also protects a specific primer group, which consists of a primer pair A and a primer pair B; the primer pair A consists of a primer F2 and a primer R2, wherein the primer F2 is shown as a sequence 8 in a sequence table, and the primer R2 is shown as a sequence 9 in the sequence table; the primer pair B consists of a primer F3 and a primer R3, wherein the primer F3 is shown as a sequence 10 in a sequence table, and the primer R3 is shown as a sequence 11 in the sequence table. The specific molar ratio of primer F2 to primer R2 was 1: 1.
The invention also protects the application of the specific primer pair or the specific primer group, and aims to identify the bifidobacteria in a sample on a species level.
The invention also protects the application of the specific primer pair or the specific primer group in the preparation of the kit; the function of the kit is to identify bifidobacteria in a sample at the species level.
The invention also protects a PCR preparation containing the specific primer pair or the specific primer group.
The invention also protects a kit which comprises the specific primer pair or the specific primer group or the PCR preparation.
The invention also protects the application of the PCR preparation or the kit, and the application is to identify the bifidobacteria in a sample on a species level.
The invention also provides a method for identifying bifidobacteria in a sample at the species level, comprising the following steps in sequence:
(1) extracting the genome DNA of the sample;
(2) taking genome DNA as a template, and carrying out PCR amplification by adopting the specific primer pair;
(3) recovering PCR amplification products and sequencing;
(4) and performing BLAST comparison on the sequencing result in a database, and judging whether the sample contains the bifidobacteria and which bifidobacteria according to the most similar strains in the database.
In the PCR amplification reaction system in the step (2), the specific molar ratio of the primer F1 to the primer R1 is 1: 1. In the PCR amplification reaction system in the step (2) of the method, the concentration of the primer F1 and the concentration of the primer R1 are both 10 mu M. In the PCR amplification reaction system in the step (2) of the method, the content of the template DNA is 50-100 ng. The annealing temperature for the PCR amplification of step (2) of the method may specifically be 54 ℃. The reaction conditions of the PCR amplification of step (2) of the method are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.
The most similar strain is the strain which has the highest sequence identity with a certain sequencing result in a database and the sequence of which the identity is more than 90 percent.
The database may specifically be the NCBI.
The invention also provides a method for identifying bifidobacteria in a sample at the species level, comprising the following steps in sequence:
(1) extracting the genome DNA of the sample;
(2) taking the genomic DNA obtained in the step (1) as a template, performing PCR amplification on the A by using the primer, and recovering a PCR amplification product;
(3) taking the PCR amplification product obtained in the step (2) as a template, performing PCR amplification on the B by adopting the primer, and recovering the PCR amplification product;
(4) and (3) mixing PCR amplification products obtained in the step (3) of a plurality of samples, performing high-throughput sequencing to obtain sequencing results of all samples, performing BLAST comparison on the sequencing results of all samples in a database, and judging whether the samples contain bifidobacteria and which bifidobacteria according to the most similar strains in the database.
In the PCR amplification reaction system in the step (2), the specific molar ratio of the primer F2 to the primer R2 is 1: 1. In the PCR amplification reaction system in the step (2) of the method, the concentration of the primer F2 and the concentration of the primer R2 are both 10 mu M. In the PCR amplification reaction system in the step (2) of the method, the content of the template DNA is 50-100 ng. The annealing temperature for the PCR amplification of step (2) of the method may specifically be 54 ℃. The reaction conditions of the PCR amplification of step (2) of the method are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.
The high-throughput sequencing specifically adopts an Illumina HiSeq2500 sequencing platform.
The most similar strain is the strain which has the highest sequence identity with a certain sequencing result in a database and the sequence of which the identity is more than 90 percent.
The database may specifically be the NCBI.
Any one of the above samples is a biological sample, a biological metabolite, a bioprocessed product.
Any of the samples described above is a stool sample, a dairy product. The dairy product may be a yoghurt.
Any one of the above-mentioned bifidobacteria is Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium animalis subsp.
The invention provides a method for rapidly and accurately identifying bifidobacteria in an environmental sample at a seed level. The method provided by the invention can identify the types of the probiotics in the dairy products and human intestinal tracts on the market at present, and is quick and accurate, wide in application range and less in limitation. On one hand, the invention can effectively monitor the disorder of the dairy product market, so that consumers can buy real effective products. On the other hand, the invention can supplement the needed probiotics in time by identifying the species of the bifidobacteria in the intestinal tract of the human. The invention provides a new idea for the supervision of related dairy products in the market and also provides a new method for identifying specific bifidobacteria in intestinal tract samples or other samples.
Drawings
FIG. 1 is an electrophoretogram of example 2.
FIG. 2 is an electrophoretogram of example 3.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 obtaining of specific primer pairs
Design and synthesis of special primers for identifying bifidobacterium strains
A specific primer pair is obtained by comparing a large number of sequences, obtaining the sequences, designing primers and comparing the performances of the primers.
The specific primer pair consists of a primer F1 and a primer R1.
F1 (SEQ ID NO: 1 of the sequence Listing): 5 '-GTSACCGTYGARGACAACAAC-3';
r1 (SEQ ID NO: 2 of the sequence Listing): 5 '-ACGGCGCAGGASTTGAAVGTG-3';
in the primer, S represents C or G, Y represents C or T, R represents A or G, and V represents A or G or C.
The specific primer pair can realize the classification and identification of bifidobacterium species on the nucleic acid level.
Example 2 verification of Effect of specific primer pairs
Test strains: strain 1.5043, strain 1.2226, strain 1.2190, strain 1.2202 and strain 1.5014. Each strain was obtained from CGMCC (China General microbiological culture Collection Center, CGMCC), and the numbers are the numbers in CGMCC.
The strain 1.5043 belongs to Bifidobacterium bifidum (Bifidobacterium bifidum). Strain 1.2226 belongs to the species Bifidobacterium animalis subsp. Strain 1.2190 belongs to Bifidobacterium adolescentis (Bifidobacterium adolescentis). Strain 1.2202 belongs to Bifidobacterium longum subsp. Strain 1.5014 belongs to Bifidobacterium longum subsp.
1. Extracting the genome DNA of the test strain.
2. The genomic DNA obtained in step 1 was used as a template, and PCR amplification was carried out using the specific primer pair designed in example 1.
The reaction system (25. mu.L) was KAPA HiFi GC Buffer (5 ×) 5. mu.L, KAPA HiFi HotStart DNApolymerase 0.5. mu.L, KAPA dNTP Mix 0.5. mu.L, primer F10.5. mu.L, primer R10.5. mu.L, and genomic DNA 10. mu.L, and in sterile water 8. mu.L, the concentration of primer F1 was 10. mu.M, the concentration of primer R1 was 10. mu.M, and the content of template DNA was 50-100 ng.
KAPA HiFi GC Buffer (5 ×), KAPA HiFi HotStart DNA Polymerase, and KAPA dNTPmix are all products of KAPA Biosystems, USA.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.
3. After completion of step 2, the PCR amplification product was subjected to 2% agarose gel electrophoresis (using 100bp DNA Marker; 100V, 60 min). The electrophoresis results of the PCR products are shown in FIG. 1.
4. After step 2 is completed, the amplified fragment is recovered by magnetic bead purification and then sequenced.
The amplified fragments were all in the range of 305. + -. 25 bp.
The sequencing result of the strain 1.5043 is shown as a sequence 3 in the sequence table. BLAST alignment was performed at NCBI with CP022723.1 being the closest sequence and 99% identity.
The sequencing result of the strain 1.2226 is shown as a sequence 4 in the sequence table. BLAST alignment was performed at NCBI with CP031154.1 being the closest sequence and 99% identity.
The sequencing result of the strain 1.2190 is shown as a sequence 5 in the sequence table. BLAST alignment was performed at NCBI with CP010437.1 being the closest sequence and 99% identity.
The sequencing result of the strain 1.2202 is shown as a sequence 6 in the sequence table. BLAST alignment was performed at NCBI with CP010411.1 being the closest sequence and 100% identity.
The sequencing result of the strain 1.5014 is shown as a sequence 7 in the sequence table. BLAST alignment was performed at NCBI with the closest sequence KX922701.1 and 100% identity.
The results show that the comparison results of 5 test strains are in accordance with the actual strain information, and the sequence identity reaches more than 99 percent, which indicates that the designed specific primers can be used for identifying the bifidobacterium species level.
Example 3 identification of which bifidobacteria and bifidobacteria classes contained in a biological sample by means of specific primer pairs
The population A is a healthy newborn population (consisting of 10 newborns, which are named as sample 1-1 to sample 1-10 in sequence, wherein the newborn refers to an infant within 6 months of birth and all the newborns have informed consent of guardians).
Population B was a healthy group of women who were 4 women (from sample 2-1 to sample 2-4 in that order; here, women within 6 months of birth; all volunteers with informed consent).
Population C is a general adult population (consisting of 6 normal persons, named samples 3-1 to 3-6 in sequence, all non-pregnant and non-parturient women, all volunteers with informed consent).
Population D was a general adult population (consisting of 8 normal persons, designated in sequence as samples 4-1 to 4-8; all non-pregnant and non-parturient women; all volunteers with informed consent).
1. Feces of each individual was taken as a sample, and genomic DNA was extracted using a feces genomic DNA extraction kit from Tiangen corporation.
2. And (3) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and adopting a primer pair consisting of F2 and R2. F2 (sequence 8):
r2 (sequence 9):
in the primer, S represents C or G, Y represents C or T, R represents A or G, and V represents A or G or C.
The reaction system (25. mu.L) was KAPA HiFi GC Buffer (5 ×) 5. mu.L, KAPA HiFi HotStart DNApolymerase 0.5. mu.L, KAPA dNTP Mix 0.5. mu.L, primer F20.5. mu.L, primer R20.5. mu.L, and genomic DNA 10. mu.L, and in sterile water 8. mu.L, the concentration of primer F2 was 10. mu.M, the concentration of primer R2 was 10. mu.M, and the content of template DNA was 50-100 ng.
KAPA HiFi GC Buffer (5 ×), KAPA HiFi HotStart DNA Polymerase, and KAPA dNTPmix are all products of KAPA Biosystems, USA.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.
3. After completion of step 2, the amplified fragment was recovered by magnetic bead purification.
4. And 3, performing PCR amplification by using the amplified fragment recovered in the step 3 as a template and adopting a primer pair consisting of F3 and R3. F3 (sequence 10): 5' -AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAG-3'; r3 (sequence 11): 5' -CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3’。
In the primer, N represents A or T or C or G.
In the primer, several N underlined constitute a sample tag, and each sample has a unique sample tag.
5. After completion of step 4, the PCR amplification product was subjected to 2% agarose gel electrophoresis (using 100bp DNA Marker; 100V, 60 min).
The electrophoresis results of the PCR products are shown in FIG. 2. In fig. 2, A, B, C, D, F, I, J represents a portion of individuals in population a and E, G, H represents a portion of individuals in population B. It can be seen that the designed specific primers are capable of amplifying bifidobacteria in fecal samples.
6. And (3) purifying and recovering PCR amplification products obtained in the step (4) of all samples (carrying out quantification and quality inspection), mixing, and carrying out high-throughput sequencing (using an Illumina HiSeq2500 sequencing platform, carrying out double-end 250bp sequencing, wherein amplified fragments are in the range of 441bp +/-25 bp.
7. The original sequencing result is assigned to each sample according to the sample label, then BLAST comparison is carried out on the sequencing result in NCBI (length threshold is set to be 250), whether the most similar strain in NCBI (the most similar strain is the bacterium belonging to the sequence with the highest sequence identity and more than 90% identity with a certain sequencing result in NCBI) is bifidobacterium or not is further bifidobacterium is judged, and the relative abundance of the bifidobacterium is determined according to the number of the sequence of the bifidobacterium and the total number of the sequence of the bifidobacterium in the sequencing result.
The results of the most similar strains of bifidobacteria in NCBI and the relative abundance of this bifidobacteria in the faeces of individual individuals in population a are shown in tables 1 and 2.
TABLE 1
TABLE 2
The results for the most similar strains of bifidobacteria in NCBI and the relative abundance of this bifidobacteria in the faeces of individual individuals in population B are shown in table 3.
TABLE 3
The results for the most similar strains of bifidobacteria in NCBI and the relative abundance of this bifidobacteria in the faeces of individual individuals in population C are shown in table 4.
TABLE 4
The results of the most similar strains of bifidobacterium in NCBI in the faeces of individual individuals in population D and the relative abundance of this bifidobacterium are shown in tables 5 and 6.
TABLE 5
TABLE 6
Experimental results show that the method established by the invention not only can rapidly and accurately identify the bifidobacteria on the species level, but also has wide application range, can be applied to the detection of the bifidobacteria in intestinal tracts and probiotic products, and has good application prospect.
SEQUENCE LISTING
<110> institute of microbiology of Chinese academy of sciences
<120> method for identifying bifidobacteria in sample at species level and special primer thereof
<130>GNCYX182073
<160>11
<170>PatentIn version 3.5
<210>1
<211>21
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<213>Artificial sequence
<400>1
gtsaccgtyg argacaacaa c 21
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acggcgcagg asttgaavgt g 21
<210>3
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<213>Bifidobacterium bifidum
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ccaagcggat cttgttcagg atgagggtcg gcagcgcctc gccgtcgacg tcctcggcga 60
tgatcagcag cggcttgccg gacttcatga cgagctcggc gatgtggacg acgtcctgct 120
ggctggaaac cttgccggag gtcaggagga tgtacggatc ctcaagaacc gcggtctggt 180
cgtccgcgtt ggtcacgaag tacggggcga tgtagccctt gtcgaaacgc atgccctcgg 240
tgaactcaag gtcaaggccg aagcggttgt tgtcttcaac ggtcacctgt ctcttataca 300
catctgacgc tgccgacgag aca 323
<210>4
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<213>Bifidobacterium animalis subsp. lactis
<400>4
cctacggatg ttgttgagaa tcagcgtggg cagtgcctcg ccatcgacat cctcggccac 60
gatcagcaac ggcttgccgg tcttcatcac cagttcggcg atgtggacca catcctgctg 120
cgaggagacc ttgctcgagg tgagcagaat gtacgggtcg tcgagcacgg ccgtctggtc 180
ttccgcattg gtgacgaagt agggggaaat ataacccttg tcgaagcgca tgccctcggt 240
gaaatcgaga tcgaggccga agcggttgtt gtcttcaacg gtcacctgtc tcttatacac 300
atctgacgct gccgacgaaa caa 323
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<213>Bifidobacterium adolescentis
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cctagcggat gttgttcaga atcagggtcg gcagagcctc gccgtcaacg tcctcagcga 60
tgatcagcag cggcttgccg gtcttcataa ccaactcggc gatgtggacg acatcctgct 120
gggagctcag cttgccggag gtcaggagga tgtacggatc ctcgagaacg gcggtctggt 180
cctcggcgtt ggtcacgaag tacggagcga tgtagccctt gtcgaagcgc atgccctcgg 240
tgaagtcaag gtcaagaccg aagcggttgt tgtcttcaac ggtcacctgt ctcttataca 300
catctgacgc tgccgacgaa acaa 324
<210>6
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<213>Bifidobacterium longum subsp. Infantis
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cctacggatg ttgttcagga tcagggtcgg cagtgcttcg ccatcgacgt cctcagcgat 60
gatcagcagc ggcttgccgg tcttcatgac cagctcggcc acgtgcacga tgtcctgctg 120
ggaggagacc ttgccggagg tcagcagaat gtacggatct tcgagcactg cggtctggtc 180
gtcagcgttg gtgacgaagt acggggcgat gtagcccttg tcgaaacgca tgccctcggt 240
gaagtcgagg tccaggccga agcggttgtt gtcttcaacg gtcacctgtc tcttatacac 300
atctgacgct gccgacgaaa caa 323
<210>7
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<213>Bifidobacterium longum subsp. Longum
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cctacggatg ttgttcagga tcaaggtcgg cagtgcttcg ccgtcgacgt cctcagcgat 60
gatcagcagc ggcttgccgg tcttcatgac cagctcggcc acgtgcacga tgtcctgctg 120
ggatgagacc ttgccggagg tcagcagaat gtacggatct tcgagcactg cggtctggtc 180
gtcagcgttg gtgacgaagt acggggcgat gtagcccttg tcaaaacgca tgccctcggt 240
gaagtcgagg tccaggccga agcggttgtt gtcttcaacg gtcacctgtc tcttatacac 300
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Claims (9)
1. The specific primer pair consists of a primer F1 and a primer R1; the primer F1 is shown as a sequence 1 in a sequence table; the primer R1 is shown as a sequence 2 in the sequence table.
2. The specific primer group consists of a primer pair A and a primer pair B;
the primer pair A consists of a primer F2 and a primer R2; the primer F2 is shown as a sequence 8 in the sequence table; the primer R2 is shown as a sequence 9 in the sequence table;
the primer pair B consists of a primer F3 and a primer R3; the primer F3 is shown as a sequence 10 in the sequence table; the primer R3 is shown as a sequence 11 in the sequence table.
3. Use of the specific primer set according to claim 1 or the specific primer set according to claim 2 for identifying bifidobacteria in a sample at the species level.
4. Use of the specific primer pair according to claim 1 or the specific primer set according to claim 2 for the preparation of a kit; the function of the kit is to identify bifidobacteria in a sample at the species level.
5. A PCR preparation comprising the specific primer set according to claim 1 or the specific primer set according to claim 2.
6. A kit comprising a specific primer pair according to claim 1 or a specific primer set according to claim 2 or a PCR preparation according to claim 4.
7. Use of a PCR preparation according to claim 5 or a kit according to claim 6 for identifying bifidobacteria in a sample at the species level.
8. A method of identifying bifidobacteria in a sample at the species level, comprising the sequential steps of:
(1) extracting the genome DNA of the sample;
(2) performing PCR amplification by using the specific primer pair of claim 1 and using the genome DNA as a template;
(3) recovering PCR amplification products and sequencing;
(4) and performing BLAST comparison on the sequencing result in a database, and judging whether the sample contains the bifidobacteria and which bifidobacteria according to the most similar strains in the database.
9. A method of identifying bifidobacteria in a sample at the species level comprising the steps of:
(1) extracting the genome DNA of the sample;
(2) performing PCR amplification on the A by using the genomic DNA obtained in the step (1) as a template and adopting the primer in claim 2, and recovering a PCR amplification product;
(3) taking the PCR amplification product obtained in the step (2) as a template, carrying out PCR amplification on the B by adopting the primer in claim 2, and recovering the PCR amplification product;
(4) and (3) mixing PCR amplification products obtained in the step (3) of a plurality of samples, performing high-throughput sequencing to obtain sequencing results of all samples, performing BLAST comparison on the sequencing results of all samples in a database, and judging whether the samples contain bifidobacteria and which bifidobacteria according to the most similar strains in the database.
Priority Applications (1)
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