CN106319081B - The primer and its method of identification leuconostoc mesenteroides subsp mesenteroides and application - Google Patents
The primer and its method of identification leuconostoc mesenteroides subsp mesenteroides and application Download PDFInfo
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- CN106319081B CN106319081B CN201610980765.5A CN201610980765A CN106319081B CN 106319081 B CN106319081 B CN 106319081B CN 201610980765 A CN201610980765 A CN 201610980765A CN 106319081 B CN106319081 B CN 106319081B
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Abstract
The present invention provides a kind of primer for identifying leuconostoc mesenteroides subsp mesenteroides and its method and applications.The present invention identifies that the primer of leuconostoc mesenteroides subsp mesenteroides is designed to using the domain GH70 highly conserved in GH70 glycosyl transferase encoding gene, the region of its height of primer pair variation is expanded, and realizes identification of the Leuconostoc mesenteroides on goldbeater's skin sub-species by carrying out sequencing and subsequent sequence alignment analysis to the resulting segment of amplification.Primer disclosed by the invention can realize the identification on sub-species to Leuconostoc mesenteroides, it is more efficient compared to traditional biochemical identification method using the identification method of the primer, it can be used for carrying out leavening bacterial strain potential in food service industry identification quickly, easy, to be new bacterial strain in food using foundation is provided, accelerate new microorganism resource in the application of field of food.
Description
Technical field
The invention belongs to the technical fields of biological food, and in particular to a kind of identification leuconostoc mesenteroides subsp mesenteroides draw
Object and its method and application.
Background technique
Currently, the Leuconostoc mesenteroides (Leuconostoc mesensteroides) obtained by the method for separation screening
It is a kind of epiphytic bacterium, is common in traditional zymotic dairy product and pickles.Lactic acid and biacetyl class object are generated because it can ferment
Matter is considered having significant contribution to the flavor of these traditional foods.The Ministry of Public Health in 2012 is by leuconostoc mesenteroides subsp mesenteroides
(Leuconostoc mesensteroides subsp.mesenteroides) is included in the strain catalogue (health that can be used for food
Portion announce 2012 No. 8), the active research of fermentation character and its special physiological about Leuconostoc mesenteroides is gradually deeply.
Existing research confirms that Leuconostoc mesenteroides energy fermenting carbohydrate generates lactic acid, and there is high acid ability, oxidation resistance and antagonism to cause
The abilities such as germ.Leuconostoc mesenteroides subsp mesenteroides have been widely used in flavouring agent in field of food at present；And Yin Qite
Different physiological activity, future are expected to become novel probiotics.
The Leuconostoc mesenteroides bacterial strain of characteristic is an important directions of future feature food development.Have many scholars
The bacterial strain with good fermentation character and physiological activity is separated to from traditional food.However, at present for Leuconostoc mesenteroides
The identification of goldbeater's skin subspecies still lacks very efficient technological means.Traditional biochemical identification method is cumbersome, time-consuming；Classical molecule
Biological method is based on 16S rDNA sequence and is identified, but this method can only realize kind of a horizontal identification, for subspecies water
Flat identification is helpless.There is researcher with certain house-keeping genes, if the sequence of gyrB is as appraisal basis, but same is only applicable in
In kind of a horizontal identification.
Summary of the invention
It the problems such as cumbersome, time-consuming the purpose of the present invention is the identification for current leuconostoc mesenteroides subsp mesenteroides, provides
A kind of new primer for identifying leuconostoc mesenteroides subsp mesenteroides and its method and application.
The present invention identifies that the primer of leuconostoc mesenteroides subsp mesenteroides is using high in GH70 glycosyl transferase encoding gene
The conservative domain GH70 of degree is designed to that the region of its height of primer pair variation is expanded, by amplification
Resulting segment carries out sequencing and subsequent sequence alignment analysis to realize mirror of the Leuconostoc mesenteroides on goldbeater's skin sub-species
It is fixed.Identification method of the invention is more simple and effective than traditional biochemical identification, at the same solve current target (such as 16S rDNA and
GyrB etc.) it can not achieve the problem of sub-species identification, one kind is provided efficiently for the identification of leuconostoc mesenteroides subsp mesenteroides
Molecular biology method, promote have good characteristic Leuconostoc mesenteroides bacterial strain screening and its application in food.
The present invention provides a kind of primer for identifying leuconostoc mesenteroides subsp mesenteroides, the upstream primer of the primer and downstream
Primer is as shown in sequence table SEQ ID NO.1, SEQ ID NO.2.
Further, which designed according to GH70 glycosyl transferase encoding gene.The primer is to utilize
The highly conserved domain GH70 is designed in GH70 glycosyl transferase encoding gene, the variation of its height of the primer pair
Region expanded,
The present invention also provides a kind of identification methods of leuconostoc mesenteroides subsp mesenteroides, method includes the following steps:
(1) genomic DNA of strain to be tested sample is extracted；
(2) using the genomic DNA of bacterial strain sample as template, using primer shown in SEQ ID NO.1, SEQ ID NO.2,
PCR amplification is carried out, pcr amplification product is obtained；
(3) bidirectional sequencing is carried out to pcr amplification product, obtains the sequence of entire amplified fragments；
(4) Blastn analysis is carried out to the sequence of amplified fragments, is obtained corresponding to the highest sequence of similarity by comparing
Species, to judge strain to be tested for which kind of leuconostoc mesenteroides subsp mesenteroides.
The present invention is by being sequenced the resulting segment of PCR amplification and then carrying out Blastn sequence alignment analysis come real
Existing identification of the Leuconostoc mesenteroides on goldbeater's skin sub-species.BLAST(Basic LocalAlignment Search Tool)
It is a set of analysis tool that similarity system design is carried out in Protein Data Bank or DNA database.Blast program can rapidly with public affairs
Database progress similitude sequence is opened to compare.Score in BLAST result is the statistical description to a kind of pair of similitude.
Further, in step (2), the reaction system of PCR amplification are as follows: 2 μ l of 2ng/ μ l DNA profiling, 0.5 μm of ol/L draw
The MgSO of 1 μ l of object, 2 0.2mmol/LdNTP μ l, 1.5mmol/L41 μ l, 10 × PCR reaction buffer, 2 μ l, 5U/ μ l rTaq
0.5 μ l of archaeal dna polymerase, adds water to 20 μ l.
Further, in step (2), the response procedures of PCR amplification are as follows: 95 DEG C of initial denaturation 10min；95 DEG C of denaturation 30s, 48
DEG C annealing 30s, 72 DEG C of extensions 120s, 35 recycle；72 DEG C of extension 10min.
Further, in step (2), pcr amplification product is subjected to agarose gel electrophoresis, whether to determine strain to be tested
For Leuconostoc mesenteroides.
Further, in step (2), take 5 μ L of pcr amplification product in 1% Ago-Gel with the voltage of 5V/cm, electricity
Swim 20-30min, is dyed using EB, electrophoresis result is taken pictures on ultraviolet gel imaging system.
Further, in step (3), such as SEQ ID NO.1 of primer sequence used in bidirectional sequencing, SEQ ID NO.2 institute
Show.
Further, in step (3), after two primer sequencing results are spliced, the sequence of entire amplified fragments is obtained
Column.
The application of above-mentioned primer of the invention in identification leuconostoc mesenteroides subsp mesenteroides.
The invention has the benefit that primer disclosed by the invention can realize the mirror on sub-species to Leuconostoc mesenteroides
Fixed, the region highly to make a variation in primer pair GH70 glycosyl transferase encoding gene is expanded, by resulting of amplification
Duan Jinhang sequencing and subsequent sequence alignment analysis realize identification of the Leuconostoc mesenteroides on goldbeater's skin sub-species.Utilize this
The identification method of primer is more efficient compared to traditional biochemical identification method, can be used for starter bacteria potential in food service industry
Strain carries out identification quickly, easy, thus be new bacterial strain in food using foundation is provided, accelerate new microorganism resource to exist
The application of field of food.
Detailed description of the invention
Fig. 1 is schematic diagram of the present invention for the gene gtf1 in leuconostoc mesenteroides subsp mesenteroides identification method；
Fig. 2 is the digital pcr amplification figure of leuconostoc mesenteroides subsp mesenteroides known to present invention identification primer pair；
Fig. 3 is that the present invention is based on the chadograms of segment building obtained by identification primer digital amplification；
Fig. 4 is that the present invention is based on the chadograms that genome sequence constructs；
Fig. 5 is the electrophoresis result figure of strain to be tested BD1710 subspecies of the present invention identification.
Specific embodiment
Embodiment of the present invention is described in detail below with reference to embodiment and Figure of description, but this field skill
Art personnel will be understood that the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.
Embodiment 1
Design of primers and its verifying for the identification of leuconostoc mesenteroides subsp mesenteroides level:
Existing Leuconostoc mesenteroides genome sequence (each bacterial strain and its genome sequence extraction code see attached list 1) is carried out
Comparative analysis identifies a generally existing GH70 glycosyl transferase encoding gene, is named as gtf1.On gtf1 gene
The highly conserved base sequence for encoding GH70 structural domain comprising two sections has one section between this two sections of highly conserved sequences
The sequence of height variation, this section of sequence show high polymorphism (polymorphism) in different strains.
1 Leuconostoc mesenteroides full-length genome information of table
The schematic diagram of gtf1 gene and its coded product is as shown in Fig. 1, we pass through in highly conserved GH70 coding
Region design primer realizes that the region to make a variation to center height expands, and is realized by the sequencing of amplified production to bacterial strain
Identification.
The code area GH70 DNA sequence dna in each bacterial strain is compared point in each Genomic sequence information according to shown in table 1
Analysis, design has the universal primer of certain degeneracy on this basis.
Upstream primer: 5'-AAAATYAMASAATGGTC-3'(SEQ ID No.1)
Downstream primer: 5'-AAGTAACGYAAHTKRCC-3'(SEQ ID No.2)
Using above-mentioned primer, in the method for digital pcr (in silico PCR) amplification to the goldbeater's skin of gene order-checking
Leukonid carries out digital amplification, and as a result as shown in Fig. 2, strain DSM 20484 does not obtain specific band, is negative right
According to group；Bacterial strain KM20 is the reference culture with strain lemon leukonid similar in Leuconostoc mesenteroides, and bacterial strain KM20 is studied
Comparative maturity, used herein as control.The resulting segment of digital amplification is compared, constructs chadogram (such as 3 institute of attached drawing
Show), acquired results are similar to the evolutionary relationship based on genome sequence, and the chadogram based on genome sequence is as shown in Fig. 4.
The above results show that four plants of leuconostoc mesenteroides subsp mesenteroides can be distinguished with close bacterial strain.Therefore, the identification primer is utilized
Theoretically being feasible is identified to leuconostoc mesenteroides subsp mesenteroides.
Embodiment 2
For the Leuconostoc mesenteroides being separated in traditional food in the identification method of goldbeater's skin sub-species, this method includes following
Step:
(1) the Leuconostoc mesenteroides BD1710 being separated to from traditional food with this laboratory is bacterium to be measured, to be tested
Bacterial strain carries out genome DNA extraction, and gained DNA carries out PCR amplification as template.
(2) using the testing gene group DNA extracted from above-mentioned bacterial strains as template, pcr amplification reaction system is as follows: 2ng/ μ l
The MgSO of 2 μ l of DNA profiling, 0.5 μm of 1 μ l of ol/L primer, 2 μ l of 0.2mmol/L dNTP, 1.5mmol/L41 μ l, 10 × PCR are anti-
2 μ l of buffer, 0.5 μ l of 5U/ μ l rTaq archaeal dna polymerase are answered, 20 μ l are added to.
(3) response parameter of PCR amplification: 95 DEG C of initial denaturation 10min；95 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C extend
2min is recycled 35 times；72 DEG C of extension 15min, 4 DEG C of preservations.
(4) agarose gel electrophoresis: by obtained 5 μ l of pcr amplification product with the electricity of 5V/cm in 1% Ago-Gel
Pressure, electrophoresis 20-30min, EB dyeing, electrophoresis result are taken pictures on ultraviolet gel imaging system.Acquired results are shown in attached drawing 5.From Fig. 5
As can be seen that identification primer provided by the present invention can amplify specific band, Leuconostoc mesenteroides can be amplified.
(5) DNA sequencing is carried out to above-mentioned amplified production, respectively using SEQ ID No.1 and SEQ ID No.2 as upstream and downstream
Primer carries out bidirectional sequencing, and acquired results are spliced, and splices the sequence of completion as shown in SEQ ID No.3.What splicing was completed
Sequence carries out BLAST analysis, the results show that matching degree highest two are respectively Leuconostoc mesenteroides
Subsp.mesenteroides ATCC8293 (100% coverage rate, 100% similarity) and Leuconostoc
Mesenteroides subsp.mesenteroides J18 (100% coverage rate, 99% similarity), therefore, it was confirmed that
BD1710 is leuconostoc mesenteroides subsp mesenteroides ATCC8293.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement and simple modifications etc., should all be included in the protection scope of the present invention in content.
Sequence table
<110>Shanghai Bright Dairy & Food Co., Ltd.
<120>primer of identification leuconostoc mesenteroides subsp mesenteroides and its method and application
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>artificial sequence
<400> 1
aaaatyamas aatggtc 17
<210> 2
<211> 17
<212> DNA
<213>artificial sequence
<400> 2
aagtaacgya ahtkrcc 17
<210> 3
<211> 1920
<212> DNA
<213>artificial sequence
<400> 3
gtcaacaaat caatacttta atgtttcaag tggtagtgaa tttttaccta agcaactgtt 60
aggcgaaaaa acaagtacag ggtttaccaa cgtggacaat ggcaagactg agttttattc 120
tacgagtggc taccaagcaa agaatacatt tattcaagat aatgacaatt ggtattattt 180
tgataatgat ggctatatgg ttgttggcgg tcaagaaatt aatggtaaaa aatattattt 240
cctaccaaat ggtgtagagt tacaagatgc ttatttgtct gatgggacta gtgagtatta 300
ctacagtagt gatggtcgtc aaatttctaa tcaatattat caaggatcag acaacaactg 360
gcgttatttc tttgcagatg gtcatatggc tgtagggtta gcaacaatta ctacagaaaa 420
tggtacaaca aatcaacaat atttcgatgc aaatggtatg caacttaagg gcgtagctat 480
aaaggatact gatggcaatg tgcactattt tgatggtaag acaggaaaca tggttataaa 540
ttcctggggc aaaataagcg atggttcatg gttatactta aatgatagcg gtgtagcggt 600
cacaggaccg caaaatatta acggtcaaaa tctttacttc aacgaagacg gtattcaagt 660
aaagggtgaa gccattactg ataatagtgg aaacatacat tattatgatc gcagcacagg 720
aaatatggtt gtgaactcat ggggtgaaac gaataatggt tcatggctat acttgaacga 780
caagggtgat gccgttacag gagaacaagt tattgacggt caaaaactat atttcagtag 840
taatggaatc caacttaaaa atacattcaa gaagctatcc gatggttcat ggctatattt 900
gaacgataaa ggtcttccag tgacaggagc acaggtcatt gatggacaaa acttgtattt 960
cgaccaagat gggaagcaag tcaaaggtga cgttgctaca gatggacaag gtaacactca 1020
ttattatgac ggcaacacag gaaatatggt tactaattca tgggcagagt tagcggacgg 1080
ttcatggatg tatctagata atgatggcaa tcctttaaca ggaccgcaaa agattgatgg 1140
ccagtcactc tactttaatg atgctggtaa gcaaatcaaa aacgcattgg ttaaactaga 1200
tgatgggtca acaatttacc tcgatgataa aggtgtttca tcaaccggta ttcaaagaat 1260
tgatgataag atatattatt ttgatcctga tggtaaacaa gtagtatgtc gttttgaaga 1320
attaccagat ggttcatgga tgtatctaga tgatgacggt gttgctgcta cgggcgctca 1380
aaaaattaat ggccaggaat tatatttcga caataacggg aaacaagtca aaaatgacaa 1440
agtaattaat gacgatggaa caataaacta ttacacaggt atgagcggtg aaaaactaaa 1500
aaatgatttt ggtgaattac cagacggttc atggatgtac ttggataatc aaggtaatgc 1560
tgtaataggt gcccaaaaaa ttaatggcca gaatctttac ttcaagacag acggacgaca 1620
ggttaagggt gaagcaaatg ttgattcatc gggtgaaatg cacttctatg atcctgattc 1680
tggcgagcta attacaaata gatttgaaca agttgctagt ggtgtatggg cttactttga 1740
tgccaacggt gttgctgtaa ctggtgagca acgcattggt aagcaaaatt tattttttga 1800
tccaactggt tatcaagtta aaggcgacaa acgaacaatt gacggcgttc tctatacctt 1860
tgataaagaa agtggtgaga gaaagggttt agattctata tcggtattac ccaccaatgg 1920
Claims (9)
1. a kind of primer pair for identifying leuconostoc mesenteroides subsp mesenteroides, which is characterized in that the primer pair such as sequence table SEQ ID
Shown in NO.1 and SEQ ID NO.2.
2. a kind of method of identification leuconostoc mesenteroides subsp mesenteroides in Leuconostoc mesenteroides, which is characterized in that this method packet
Include following steps:
(1) genomic DNA of Leuconostoc mesenteroides bacterial strain sample to be measured is extracted；
(2) using the genomic DNA of bacterial strain sample as template, using primer shown in SEQ ID NO.1 and SEQ ID NO.2, into
Row PCR amplification, obtains pcr amplification product；
(3) bidirectional sequencing is carried out to pcr amplification product, obtains the sequence of entire amplified fragments；
(4) Blastn analysis is carried out to the sequence of amplified fragments, obtains object corresponding to the highest sequence of similarity by comparing
Kind, to judge strain to be tested for which kind of leuconostoc mesenteroides subsp mesenteroides.
3. the identification method of the leuconostoc mesenteroides subsp mesenteroides according to claim 2 in Leuconostoc mesenteroides, special
Sign is, in step (2), the reaction system of PCR amplification are as follows: 2 μ l of 2ng/ μ l DNA profiling, 0.5 μm of 1 μ l of ol/L primer,
The MgSO of 2 μ l of 0.2mmol/L dNTP, 1.5mmol/L4 1 μ l, 10 × PCR reaction buffer, 2 μ l, 5U/ μ l rTaq DNA are poly-
0.5 μ l of synthase, adds water to 20 μ l.
4. the identification method of the leuconostoc mesenteroides subsp mesenteroides according to claim 2 in Leuconostoc mesenteroides, special
Sign is, in step (2), the response procedures of PCR amplification are as follows: 95 DEG C of initial denaturation 10min；95 DEG C of denaturation 30s, 48 DEG C of annealing 30s,
72 DEG C of extension 120s, 35 circulations；72 DEG C of extension 10min.
5. the identification method of the leuconostoc mesenteroides subsp mesenteroides according to claim 2 in Leuconostoc mesenteroides, special
Sign is, in step (2), pcr amplification product is carried out agarose gel electrophoresis to determine whether strain to be tested is the bright string of goldbeater's skin
Pearl bacterium goldbeater's skin subspecies.
6. the identification method of the leuconostoc mesenteroides subsp mesenteroides according to claim 5 in Leuconostoc mesenteroides, special
Sign is, in step (2), takes 5 μ L of pcr amplification product in 1% Ago-Gel with the voltage of 5V/cm, electrophoresis 20-30min,
It is dyed using EB, electrophoresis result is taken pictures on ultraviolet gel imaging system.
7. the identification method of the leuconostoc mesenteroides subsp mesenteroides according to claim 2 in Leuconostoc mesenteroides, special
Sign is, in step (3), such as SEQ of primer sequence used in bidirectional sequencing
Shown in ID NO.1 and SEQ ID NO.2.
8. the identification method of the leuconostoc mesenteroides subsp mesenteroides according to claim 2 in Leuconostoc mesenteroides, special
Sign is, in step (3), after two primer sequencing results are spliced, obtains the sequence of entire amplified fragments.
9. application of the primer described in claim 1 in identification Leuconostoc mesenteroides in leuconostoc mesenteroides subsp mesenteroides.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974450A (en) * | 2010-09-13 | 2011-02-16 | 郑州大学 | Leuconostoc mesenteroides and application thereof |
| CN102433385A (en) * | 2011-12-20 | 2012-05-02 | 北京大北农科技集团股份有限公司 | Primers for identifying leuconostoc mesenteroides and application thereof |
| CN105349477A (en) * | 2015-12-21 | 2016-02-24 | 光明乳业股份有限公司 | Leuconostoc mesenteroides as well as preparation method and application thereof |
-
2016
- 2016-11-08 CN CN201610980765.5A patent/CN106319081B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974450A (en) * | 2010-09-13 | 2011-02-16 | 郑州大学 | Leuconostoc mesenteroides and application thereof |
| CN102433385A (en) * | 2011-12-20 | 2012-05-02 | 北京大北农科技集团股份有限公司 | Primers for identifying leuconostoc mesenteroides and application thereof |
| CN105349477A (en) * | 2015-12-21 | 2016-02-24 | 光明乳业股份有限公司 | Leuconostoc mesenteroides as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 利用特异性引物确定肠膜明串珠菌菌株BD1710的亚种归属;高彩霞等;《乳业科学与技术》;20160101(第01期);5-7 |
| 肠膜明串珠菌胞外多糖合成的蛋白组学分析;鄢明辉等;《食品工业科技》;20160412(第14期);158-160 |
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