CN108315402B - Amplification primer pair for sequencing bifidobacterium in intestinal tract and sequencing method - Google Patents
Amplification primer pair for sequencing bifidobacterium in intestinal tract and sequencing method Download PDFInfo
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Abstract
The invention provides an amplification primer pair for sequencing bifidobacterium in intestinal tracts, which comprises an upstream primer Bif-11F and a downstream primer Bif-11R, wherein the upstream primer Bif-11F and the downstream primer Bif-11R are used as the amplification primer pair for PCR amplification to obtain a PCR amplification product, after SMRT high-throughput sequencing, species annotation is carried out on an amplification sequence by using a QIIME (V1.7) platform for all known bifidobacterium sequences to obtain an accurate identification result. The amplification primer for sequencing the bifidobacteria in the intestinal tract is designed based on the consensus core gene sequence of the bifidobacteria, can amplify the bifidobacteria at the species level, has better specificity and practicability, and can better reflect the diversity of the bifidobacteria in the intestinal tract. Meanwhile, the method is suitable for analyzing the bifidobacteria in samples such as fermented dairy products, feces, soil and the like.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to an amplification primer for sequencing bifidobacterium in intestinal tracts and a quantitative method.
Background
Bifidobacteria are a gram-positive anaerobic bacterium present in the gastrointestinal tract of humans and animals. It was originally isolated from the faeces of breast-fed infants and currently contains 69 defined species. Generally considered to have health promoting effects and are therefore widely used in food and probiotic products. Also, bifidobacteria were considered to be a key symbiont in human-microbial interactions at the beginning of the last century and to play a key role in maintaining a healthy gastrointestinal tract. With the development of biotechnology, more and more non-culturable microorganisms are found. Bifidobacteria, one of the common bacteria in the intestinal tract, are generally considered to have a probiotic effect, and can be one of the key indicators for assessing health. However, it is not currently possible to achieve which bifidobacteria play a role in the gut, and what associations they exist with other bacteria in the gut.
The present bacterial universal primers for bacterial diversity analysis in intestinal tract can simultaneously amplify most of the bacteria, especially the dominant flora, but cannot amplify bifidobacteria with relatively low content at species level, and especially lack an amplification primer pair capable of realizing high-throughput sequencing on most or all species of bifidobacteria in intestinal tract.
Disclosure of Invention
In view of the above, the present invention provides an amplification primer pair for sequencing bifidobacterium in intestinal tract, wherein the upstream sequence of the primer pair is Bif-11F shown in SEQ ID No.1 and Bif-11R shown in SEQ ID No. 2:
Bif-11F: 5 '-AAGAAGAAGGCCACCAAGTAYT-3', wherein, Y is degenerate base, representing C or T;
Bif-11R：5’-GGTAAGAGTCGGACGCTGTGCAATAA-3’。
preferably, in the amplification primer pair for sequencing bifidobacterium in intestinal tract, the PCR amplification fragment size of the primer pair is about 625 bp.
Preferably, in the amplification primer pair for sequencing bifidobacterium in intestinal tract, the optimal annealing temperature for PCR amplification of the primer pair is 60 ℃.
Another object of the present invention is to provide the above method for identifying bifidobacterium by using the amplification primer pair for bifidobacterium sequencing in intestinal tract, comprising the following steps:
s1: extracting genome DNA of a sample to be detected containing the bifidobacterium, and carrying out PCR amplification by taking the obtained genome DNA as a template and the Bif-11F and Bif-11R as amplification primer pairs to obtain a PCR amplification product;
s2: sequence determination and alignment: purifying the PCR amplification product to obtain a PCR amplification fragment, and performing SMRT high-throughput sequencing on the PCR amplification fragment;
s3: all known sequences of bifidobacteria are used as an alignment database, and species annotation is carried out on the amplified sequences by using a QIIME (V1.7) platform, so as to obtain an accurate identification result.
Preferably, in the method for identifying bifidobacterium by using the amplification primer pair for sequencing bifidobacterium in intestinal tract according to the invention, the PCR amplification system in step S1 is: the amplification system consisted of 1ul of 20ng/ul DNA template, 2 XKAPA HiFi HotStartStread Mix25ul, 10. mu.M Bif-11F and Bif-11R each at 1.2ul, with the balance being distilled or sterile deionized water, based on a 50ul total system.
Preferably, in the method for identifying bifidobacterium by using the amplification primer pair for sequencing bifidobacterium in intestinal tract according to the invention, the PCR amplification reaction program in step S1 is: pre-denaturation at 95 ℃ for 5min for 1 cycle, denaturation at 95 ℃ for 1min for 30 cycles, annealing at 60 ℃ for 1min, extension at 72 ℃ for 2min, and final extension at 72 ℃ for 7min for 1 cycle.
Preferably, in the method for identifying bifidobacterium by using the amplification primer pair for sequencing bifidobacterium in intestinal tract, the PCR amplification fragment in the step S2 has a size of 625 bp.
The invention also provides application of the amplification primer pair for sequencing the bifidobacterium in the intestinal tract in preparing a kit for detecting the bifidobacterium in the intestinal tract; and the application of the amplification primer pair for sequencing the bifidobacterium in the intestinal tract in the species analysis of the bifidobacterium in the fermented dairy products, the feces or the soil samples.
Compared with the prior art, the invention has the following advantages:
the invention screens out the core shared by bifidobacteria by comparing 48 strains of complete gene sequencing bifidobacteria core genes, performs blast in NCBI, and finally designs a specific primer of the bifidobacteria by taking the core gene rpsK as a target sequence. And a pair of bifidobacterium specific primers which can be used for high-throughput sequencing is screened out through agarose gel primary screening and Pac bio SMRT sequencing. The pair of primers can amplify the bifidobacteria in the intestinal tract at species level, and provides possibility for researching the diversity of the bifidobacteria in the intestinal tract and the connection of the bifidobacteria with other bacteria.
The amplification primer for sequencing the bifidobacteria in the intestinal tract is designed based on the consensus core gene sequence of the bifidobacteria, can amplify the bifidobacteria at the species level, has better specificity and practicability, and can better reflect the diversity of the bifidobacteria in the intestinal tract. Meanwhile, the method is suitable for analyzing the bifidobacteria in samples such as fermented dairy products, feces, soil and the like.
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FIG. 1 shows the result of specific amplification of Bifidobacterium primers Bif-11 from 22 test materials in one embodiment of the present invention;
FIG. 2 is a graph of the sequencing results of enhanced high throughput sequencing by amplification of 2 different fecal samples using the amplification primer pairs described above in one embodiment of the present invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The invention provides an amplification primer pair for sequencing lactobacillus in intestinal tracts, which has the following nucleotide sequences:
Bif-11F: 5 '-AAGAAGAAGGCCACCAAGTAYT-3', wherein, Y is degenerate base, representing C or T;
Bif-11R：5’-GGTAAGAGTCGGACGCTGTGCAATAA-3’。
the following provides an example of achieving accurate quantification of bifidobacteria using the amplification primer pair described above for the sequencing of bifidobacteria in the gut.
The method for obtaining the amplification primer pair comprises the following steps:
downloading the whole genome sequence of all bifidobacteria from NCBI, searching the common core gene sequence fragments of all bifidobacteria by comparison, designing a Bifidobacterium Primer with the PCR amplification product of about 700 and 1000bp by means of Primer design software Primer 5.0, searching and aligning the designed Bifidobacterium Primer by using MEGE6.0 software, and designing degenerate bases if necessary. The designed lactobacillus primers are subjected to specificity evaluation through agarose gel for preliminary screening, the preliminarily screened bifidobacterium primers with different Barcodes are utilized to amplify different sample DNA diluents containing bifidobacterium, and finally, the amplification products are subjected to high-throughput sequencing by virtue of the SMRT (Single-molecule Real-Time) technology of Pacific Bioscience company for further screening to obtain the amplification primer pair suitable for sequencing lactobacillus in intestinal tracts.
The following provides an example for achieving accurate identification of bifidobacterium species using the amplification primer pair for bifidobacterium sequencing in the gut described above.
Example 1 method for amplification of Bifidobacterium and non-Bifidobacterium strains Using pairs of amplification primers
S1: randomly selecting DNA of 2 bifidobacteria as a positive template, 14 bifidobacteria related lactic acid bacteria and 6 common pathogenic bacteria as negative templates, and carrying out PCR amplification by using Bif-11F and Bif-11R as primers to obtain a PCR amplification product;
the nucleotide sequences of the primer pairs are respectively as follows:
Bif-11F: 5 '-AAGAAGAAGGCCACCAAGTAYT-3', wherein, Y is degenerate base, representing C or T;
Bif-11R：5’-GGTAAGAGTCGGACGCTGTGCAATAA-3’。
wherein, the information of the 22 strains is shown in the following table:
the PCR amplification system is:
the PCR amplification reaction program is as follows:
detecting PCR amplification product with 1% agarose gel, performing electrophoresis for 20-30min, and ultraviolet photographing to detect amplification effect, as shown in FIG. 1. S2: sequence determination and alignment
Purifying PCR amplification products, mixing samples, repairing DNA fragments, connecting joints and controlling library quality to obtain an amplification fragment with the size of about 625bp, and sequencing the amplification fragment by means of a Single molecule Real-Time Sequencer (SMRT) (Pacific Biosciences company); a DNA sequencing library was constructed using the kit SMRT bell TM template kit 1.0, a Pacific Biotechnology company, and the detailed procedures were carried out according to the kit instructions.
Finally, use
PorRS _ ReadsOfinsert.1 protocol in tal (V2.7) for sequencing quality control; wherein, the shortest insertion segment length and the maximum insertion segment length in the quality control parameters have parameter setting values of 525 and 725; carrying out subsequent analysis on the sequence qualified by quality control according to the difference between the barcode and the primer sequence;
s3: in the figure, M represents Marker, and from left to right 1-22 represents Bifodobacterium gallicum, Bifodobacterium lognum subsp. infirans, Enterococcus asini, Enterococcus faecalis, Enterococcus italicus, Lactobacillus casei, Lactobacillus salivarivar, Lactobacillus helveticus, Weissella belteri, Penicoccus acidicifuga, Pedicoccus saccharolyticus, Leucospora leucotrichia, Leucospora, Streptococcus acidilactici, Penicoccus pectococcus, Leucospora serovar, Streptococcus mutans, Streptococcus thermophilus, Streptococcus mutans, Streptococcus thermophilus, Streptococcus lactis, Streptococcus mutans, Streptococcus lactis, Streptococcus lacticola, Streptococcus mutans, Streptococcus mutans, Streptococcus.
As can be seen from fig. 1, the results of amplifying 2 randomly extracted test bifidobacteria were all positive and negative for 20 non-bifidobacteria tested using the bifidobacterium primer set provided by the present invention, which further indicates that the bifidobacterium primer set provided by the present invention has good specificity.
Example 2: high throughput sequencing of fecal samples Using the amplification primer pairs of the invention
S1: 2 stool DNA dilutions (labeled 1 and 2 respectively) determined to contain bifidobacteria were amplified with bacterial universal primers (27f/1492r: SEQ ID NO.35 '-AGAGAGTTTGATCCTGGCTCCAG-3'/5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID NO.4)) with different Barcodes and the amplification primer pair provided in S1 (the nucleotide sequence of the primer pair is the same as in example 1), and the PCR amplification system and PCR amplification reaction procedure were the same as in example 1 to obtain PCR amplification products;
s2: sequence determination and alignment
And (3) purifying the PCR amplification product, mixing samples, repairing DNA fragments, connecting joints and controlling library quality to obtain an amplification fragment with the size of about 625bp, and sequencing the obtained amplification fragment by Pacio SMRT, wherein the specific operation process is the same as that in the example 1.
S3: and (3) taking all the bifidobacteria with known sequences as an alignment database, and performing species annotation on the amplified sequences by using a QIIME (V1.7) platform to obtain an accurate identification result. The results of the amplification with the bacterial universal primer (27f/1492r) and the amplification with the Bifidobacterium primer (Bif-11) were analyzed in comparison, and are shown in Table 1. The heat map was drawn using the R software according to the presence or absence of the bacterial species, wherein black represents the presence of amplified species and white represents the absence of amplified species, and the results are shown in FIG. 2.
TABLE 1 relative content (‰) of target bacteria in feces amplified by different primers
As can be seen from Table 1, the Bifidobacterium primers provided by the present invention amplify more Bifidobacterium than the bacterial universal primers (27f/1492) amplified in fecal sample 1 and fecal sample 2, and have higher content of Bifidobacterium than the bacterial universal primers. FIG. 2 is a graph of the results of sequencing by amplification of 2 different fecal samples using the amplification primer pairs described above to enhance high throughput sequencing. Wherein 27f represents a universal bacterial primer (27f/492r), Bif-11 represents a primer of the present invention, black represents stool sample No.1, and gray represents stool sample No. 2; in the figure, black boxes indicate presence and white boxes indicate absence.
From table 1 and fig. 2, it can be seen that 3 species of bifidobacteria can be amplified only by amplifying fecal sample 1 with the bacterial universal primer (27f/1492), and that a total of 11 species of bifidobacteria can be amplified by amplifying fecal sample 1 with the bifidobacterium primer set provided by the present invention, with 8 species of bifidobacteria being amplified more than with the universal primer; the fecal sample 2 is amplified by using the bacterial universal primer (27f/1492) to amplify only 4 bifidobacteria, and the fecal sample 2 is amplified by using the bifidobacterium primer pair provided by the invention to amplify 7 bifidobacteria in total, and 3 bifidobacteria are amplified compared with the universal primer. Further, the diversity of the bifidobacterium amplified by the bifidobacterium primer pair provided by the invention is obviously better than that of the general bacterial primer, which is beneficial to further research on the diversity of the bifidobacterium in intestinal tracts.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> amplification primer pair for sequencing bifidobacterium in intestinal tract and sequencing method
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Claims (7)
1. An amplification primer pair for sequencing bifidobacterium in intestinal tracts, which is characterized in that the upstream sequence of the primer pair is Bif-11F shown in SEQ ID NO.1 and Bif-11R shown in SEQ ID NO. 2:
Bif-11F: 5 '-AAGAAGAAGGCCACCAAGTAYT-3', wherein, Y is degenerate base, representing C or T;
Bif-11R：5’- GGTAAGAGTCGGACGCTGTGCAATAA-3’；
the size of the PCR amplified fragment of the primer pair is 625 bp;
the optimal annealing temperature for PCR amplification of the primer pair is 60 ℃.
2. A method for identifying the genus bifidobacterium using the amplification primer set of claim 1 for the purpose of non-disease diagnosis, comprising the steps of:
s1: extracting genome DNA of a sample to be detected containing the bifidobacterium, and carrying out PCR amplification by taking the obtained genome DNA as a template and the Bif-11F and Bif-11R as amplification primer pairs to obtain a PCR amplification product;
s2: sequence determination and alignment: purifying the PCR amplification product to obtain a PCR amplification fragment, and performing SMRT high-throughput sequencing on the PCR amplification fragment;
s3: and taking the sequences of all known bifidobacteria as an alignment database, and performing species annotation on the amplified sequences by using a QIIME platform to obtain an accurate identification result.
3. The method of claim 2, wherein the PCR amplification system in step S1 is: the amplification system consisted of 1ul of 20ng/ul DNA template, 2 XKAPA HiFi HotStartStread Mix25ul, 10. mu.M of Bif-11F and Bif-11R each at 1.2ul, with the balance being distilled or sterile deionized water, based on a 50ul total system.
4. The method according to claim 2, wherein the PCR amplification reaction procedure in step S1 is as follows: pre-denaturation at 95 ℃ for 5min for 1 cycle, denaturation at 95 ℃ for 1min for 30 cycles, annealing at 60 ℃ for 1min, extension at 72 ℃ for 2min, and final extension at 72 ℃ for 7min for 1 cycle.
5. The method according to claim 2, wherein the PCR amplified fragment of step S2 has a size of 625 bp.
6. Use of the amplification primer pair of claim 1 in the preparation of a kit for detecting bifidobacteria in the intestinal tract.
7. Use of the pair of amplification primers of claim 1 for bifidobacteria species analysis in fermented dairy products, stool or soil samples.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1816196A1 (en) * | 2002-04-09 | 2007-08-08 | Societe Des Produits Nestle S.A. | La1 - The genome of a Lactobacillus strain |
| CN107653306A (en) * | 2017-11-13 | 2018-02-02 | 江南大学 | A kind of Bifidobacterium quick determination method and application based on high-flux sequence |
| CN107937581A (en) * | 2017-12-28 | 2018-04-20 | 内蒙古农业大学 | Amplimer for lactic acid bacteria sequencing is to, lactic acid bacteria kind identification method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1816196A1 (en) * | 2002-04-09 | 2007-08-08 | Societe Des Produits Nestle S.A. | La1 - The genome of a Lactobacillus strain |
| CN107653306A (en) * | 2017-11-13 | 2018-02-02 | 江南大学 | A kind of Bifidobacterium quick determination method and application based on high-flux sequence |
| CN107937581A (en) * | 2017-12-28 | 2018-04-20 | 内蒙古农业大学 | Amplimer for lactic acid bacteria sequencing is to, lactic acid bacteria kind identification method and application |
Non-Patent Citations (3)
| Title |
|---|
| Genomic evidence for antibiotic resistance genes of actinomycetes as origins of antibiotic resistance genes in pathogenic bacteria simply because actinomycetes are more ancestral than pathogenic bacteria;Patrick C.Y. Woo et al.;《Medical Hypotheses》;20061231;第67卷(第6期);摘要部分，表1，表2，图2 * |
| Patrick C.Y. Woo et al..Genomic evidence for antibiotic resistance genes of actinomycetes as origins of antibiotic resistance genes in pathogenic bacteria simply because actinomycetes are more ancestral than pathogenic bacteria.《Medical Hypotheses》.2006,第67卷(第6期),第1297–1304页. * |
| 利用核心基因比较评价细菌域高等级分类阶元的关系;张维维;《中国优秀硕士学位论文全文数据库 基础科学辑》;中国学术期刊（光盘版）电子杂志社;20150716(第8期);第A006-211页 * |
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