CN108103216B - DNA bar code primer, DNA bar code, kit, method and application for rapidly identifying alternaria adefovea strain - Google Patents
DNA bar code primer, DNA bar code, kit, method and application for rapidly identifying alternaria adefovea strain Download PDFInfo
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Abstract
The invention belongs to the field of species and strain identification, and particularly relates to a DNA barcode primer composition, a DNA barcode composition, a kit, a method and application for identifying a desmospora adephagi strain. The DNA barcode composition comprises a first DNA barcode shown as SEQ ID No.1, a second DNA barcode shown as SEQ ID No.2, a third DNA barcode shown as SEQ ID No.3 and a fourth DNA barcode shown as SEQ ID No.4, wherein the first, second, third and fourth DNA barcodes are derived from the genome of the strain Pityrosporum adenini TMCC 70007. The DNA bar code can quickly identify the Pu' er tea fermentation strain of the adenine arthrobotrys yeast TMCC70007, and can quickly and accurately identify the strain from confusable strains or other strains in the same species.
Description
Technical Field
The invention belongs to the field of species and strain identification, and particularly relates to a DNA barcode primer composition, a DNA barcode composition, a kit, a method and application for identifying a desmospora adephagi strain.
Background
Pu' er tea is post-fermented tea with geographical identification of Yunnan, which is prepared by a series of processes by adopting big-leaf sun-dried raw tea as a raw material. Pu' er tea is also concerned by people because of having health care effects of losing weight, reducing blood sugar and blood fat, preventing and improving cardiovascular diseases, resisting aging, resisting cancer, diminishing inflammation, aiding digestion, nourishing stomach and the like. In recent years, Pu 'er tea is more and more popular, and the development of the Pu' er tea industry is pulled by the increasing market demand, so that the economic growth of Yunnan areas is promoted.
Pu ' er tea is fermented tea of Yunnan large-leaf species, and the fermentation production of the Pu ' er tea needs the action of various microorganisms including a Pu ' er tea fermentation production strain, namely, adenine-node yeast (Blastobotrys adeninivorans) TMCC70007 (hereinafter also referred to as TMCC 70007), wherein the TMCC70007 is a key bacterium in the fermentation process. At present, Pu' er tea fermentation is still an empirical fermentation of semi-natural artificial pile fermentation, and although communities mainly comprising few dominant microorganisms are relatively stable in the pile fermentation process, the stability of the product still has a larger space for improvement. Certain potential safety hazards inevitably exist in the production process, and in order to further obtain the favor of consumers and the acceptance of the market, break through the foreign trade barrier and improve the market competitiveness of Pu 'er tea enterprises, the manual control, cleaning and high efficiency of the Pu' er tea production process must be realized, new products are not developed, and the industrial chain is extended. In order to achieve the purpose, technology must be innovated, a series of safe, clean, efficient, artificial, controllable and automatic Pu 'er tea new processes are invented, the healthy development of the Pu' er tea industry can be ensured, and long-term benefits are brought to the nation and people.
With the further development of Pu ' er tea scientific research, more and more people begin to explore the artificial inoculation and fermentation of Pu ' er tea, and at present, a plurality of bacterial strains are applied to the artificial controllable fermentation of Pu ' er tea. In recent years, due to the increase of market demands, a plurality of small-scale manufacturers lack systematic and deep research on Pu 'er tea, and greatly abuse a plurality of other people's patents to profit, for example, according to a plurality of patent methods for inoculating and fermenting Pu 'er tea, a plurality of strains are adopted to ferment Pu' er tea, and a series of methods such as Pu 'er tea processing and production are carried out, so that the economic benefits of Pu' er tea production enterprises with related patents are greatly damaged.
In order to ensure that the germplasm resources of Pu ' er tea fermentation microorganisms are effectively protected, prevent the infringement behavior of abusing the Pu ' er tea fermentation microorganisms and solve the difficult problem of difficult demonstration in the process of artificially controllable fermentation of Pu ' er tea during the infringement of strains, the development of a method for quickly and accurately identifying the Pu ' er tea fermentation strains is imperative, a molecular identification method of the Pu ' er tea fermentation strains needs to be urgently established, a method for quickly identifying and distinguishing similar strains by a DNA bar code technology is developed, so that the problems of identification and identification of industrial production strains are solved by a method combining morphological characteristics and molecular data analysis, a theoretical basis is provided for the development of the controlled industrial fermentation process of Pu ' er tea and resource protection, and the healthy and prosperous development of the Pu ' er tea industry is promoted.
Disclosure of Invention
In order to overcome the defects of morphological identification of the adenine nodularia cerealis strain for fermentation production of Pu 'er tea and the defect that different strains in the strain are difficult to distinguish based on an ITS sequence and an 18S rRNA sequence, the invention provides a group of DNA bar codes of the adenine nodularia cerealis, wherein the DNA bar codes can be used for quickly identifying the Pu' er tea fermentation strain adenine nodularia cerealis TMCC70007, and the strain can be quickly and accurately identified from confusable strains or other strains in the same species.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided a DNA barcode composition for identifying a strain of arthrospora adephaga yeast comprising a first DNA barcode as shown in SEQ ID No.1, a second DNA barcode as shown in SEQ ID No.2, a third DNA barcode as shown in SEQ ID No.3, and a fourth DNA barcode as shown in SEQ ID No.4, wherein the first, second, third and fourth DNA barcodes are derived from the genome of the strain of arthrospora adenosylensis TMCC 70007.
According to a second aspect of the present invention, there is provided a DNA barcode primer composition for identifying a strain of arthrospora adephaga, comprising the following four primer pairs:
a first primer pair:
forward primer LYR _ 1-F: 5'-CAAATCCTTGCCCTGGGGAT-3' the flow of the air in the air conditioner,
reverse primer LYR _ 1-R: 5'-GTTGCAAACGAGCCCAACAT-3', respectively;
a second primer pair:
forward primer ABCB-F: 5'-GGGGATATGGGGACCGAAAC-3' the flow of the air in the air conditioner,
reverse primer ABCB-R: 5'-TTTGGCCCCCTGTACTTGTG-3', respectively; and
a third primer pair:
forward primer DUF 1279-F: 5'-TCTCGGTTGAGGCTTGTTCC-3' the flow of the air in the air conditioner,
reverse primer DUF 1279-R: 5'-GGGCTCATTGCGCTATTTGG-3', respectively;
a fourth primer pair:
forward primer TAE 1-F: 5'-GGAGAAGATCCACGCTCTCG-3' the flow of the air in the air conditioner,
reverse primer TAE 1-R: 5'-GGTACATGCCCCAAGAGGTT-3' the flow of the air in the air conditioner,
wherein the first primer pair is a primer of a first DNA bar code shown as SEQ ID No. 1; the second primer pair is a primer of a second DNA bar code shown as SEQ ID No. 2; the third primer pair is a primer of a third DNA bar code shown as SEQ ID No. 3; the fourth primer pair is a primer of a fourth DNA bar code shown as SEQ ID No. 4.
According to a third aspect of the invention, there is provided a kit for identifying a strain of nodospora adevorans comprising a DNA barcode primer composition according to the invention. In a preferred embodiment, the kit further comprises a DNA barcode composition according to the invention.
According to a fourth aspect of the present invention, there is provided a method for identifying a strain of nodospora adenine dinucleotide, comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) taking the genomic DNA in the step a) as a template, performing PCR amplification by using the first, second, third and fourth primer pairs according to the invention, and determining that the strain to be detected is not the node adenine arthrobotrys strain TMCC70007 if PCR products obtained by respectively amplifying the four primer pairs are not obtained; if a first, a second, a third and a fourth PCR product respectively amplified by the first, the second, the third and the fourth primer pairs are obtained, the following steps are carried out;
c) sequencing the obtained PCR products to obtain respective DNA sequences of the PCR products, wherein the DNA sequence of the first PCR product is a first DNA sequence, the DNA sequence of the second PCR product is a second DNA sequence, the DNA sequence of the third PCR product is a third DNA sequence, and the DNA sequence of the fourth PCR product is a fourth DNA sequence;
d) sequentially splicing the first, second, third and fourth DNA sequences obtained in the step c) to obtain a sequence to be detected; sequentially splicing the first, second, third and fourth DNA barcodes to obtain a standard sequence; comparing the sequence to be detected with the standard sequence for sequence homology, and if the sequence homology is less than 99%, determining that the strain to be detected is not the adenine node B spore-eating yeast strain TMCC 70007; and if the sequence homology is more than or equal to 99 percent, determining that the strain to be tested is the adenine-eating node spore yeast strain TMCC 70007.
In the method of the present invention, preferably, the method further comprises e) performing cluster analysis on the sequence to be tested and the standard sequence, and if the sequence to be tested and the standard sequence are clustered together, determining that the strain to be tested is an adenine-node B.adenosylea strain TMCC 70007.
In the method of the present invention, preferably, the length of the first PCR product amplified by the first primer pair is 758 bp; the length of a second PCR product obtained by amplification of the second primer pair is 441 bp; the length of a third PCR product obtained by amplification of the third primer pair is 775 bp; the length of a fourth PCR product obtained by amplification of the fourth primer pair is 580 bp.
In the method of the present invention, preferably, the procedure of PCR amplification is: 1) pre-denaturation at 94-96 deg.C for 8 min; 2) denaturation at 94-96 ℃ for 45 seconds, annealing at 55-57 ℃ for 45 seconds, and extension at 72 ℃ for 1min 15 seconds, wherein the procedure 2) is performed for 30-35 cycles; 3) extension at 72 ℃ for 10 min. In a further preferred embodiment the procedure for PCR amplification is: 1) pre-denaturation at 95 ℃ for 8 min; 2) denaturation at 94 ℃ for 45 seconds, annealing at 56 ℃ for 45 seconds, and extension at 72 ℃ for 1min 15 seconds, wherein the procedure 2) is performed for 32-35 cycles; 3) extension at 72 ℃ for 10 min.
In the method of the present invention, preferably, the clustering analysis is construction of a phylogenetic tree.
A fifth aspect according to the present invention relates to the use of a DNA barcode composition according to the present invention for identifying strains of Arthrospora adefovea.
According to a sixth aspect the invention relates to the use of a DNA barcode primer composition according to the invention for identifying strains of Arthrospora adenantha.
Compared with the prior art, the invention has the following advantages and positive effects:
1. 4 DNA barcode fragments most suitable for identifying the industrial fermentation production strain of the puer tea, namely, the adenine arthrospora cerealis TMCC70007 are found and selected as DNA barcode combinations, and compared with other DNA sequences, the 4 DNA sequences have the characteristics of universality, easiness in amplification and easiness in comparison.
2. Based on the findings, the sample identification method of the Puer tea industrial fermentation production strain Arthrosporium adenini TMCC70007 is established, compared with the traditional morphological identification method and ITS and 18S rRNA and other sequence identification methods, the identification accuracy is greatly improved, and the defect that different strains in the strain are difficult to distinguish is overcome. The method has low requirements on the strain, can quantify the identification indexes, provides an accurate and effective method for judging the adenine-saving spore yeast TMCC70007 in time, increases the specificity and the accuracy of identification, provides a rapid identification method for the Pu 'er tea fermentation strain, namely the adenine-saving spore yeast TMCC70007, solves the problems of difficult demonstration of strains in the manual controllable fermentation process of the Pu' er tea, difficult evidence demonstration in the process patent infringement case and the like, ensures the benefits of the special rights of the Pu 'er tea fermentation process, and ensures the healthy and prosperous development of the Pu' er tea industry.
Drawings
FIG. 1 shows TMCC70007, CBS 8244 of the species Alternaria adephagaTCBS 8335, CBS 7350, CBS 7370 and CBS 6800 of the Blastotrys raffinositifera speciesTThe combined sequences of (a) were based on the genetic distance of the Kimura-2-parameter model to construct an NJ tree.
Detailed Description
The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.
One embodiment of the invention relates to a DNA barcode composition for identifying a strain of Arthrospora adenantha, comprising a first DNA barcode shown in SEQ ID No.1, a second DNA barcode shown in SEQ ID No.2, a third DNA barcode shown in SEQ ID No.3, and a fourth DNA barcode shown in SEQ ID No.4, wherein the first, second, third, and fourth DNA barcodes are derived from the genome of the strain of Arthrospora adenantha TMCC 70007.
The DNA barcode technology (DNA barcoding) is a new molecular identification technology for identifying species by using a standard and short DNA fragment in a genome, and can quickly and accurately identify the species.
Another embodiment of the invention relates to a DNA barcode primer composition for identifying a strain of arthrospora adephaga, comprising the following four primer pairs:
a first primer pair:
forward primer LYR _ 1-F: 5'-CAAATCCTTGCCCTGGGGAT-3' (SEQ ID No.5),
reverse primer LYR _ 1-R: 5'-GTTGCAAACGAGCCCAACAT-3' (SEQ ID No. 6);
a second primer pair:
forward primer ABCB-F: 5'-GGGGATATGGGGACCGAAAC-3' (SEQ ID No.7),
reverse primer ABCB-R: 5'-TTTGGCCCCCTGTACTTGTG-3' (SEQ ID No. 8); and
a third primer pair:
forward primer DUF 1279-F: 5'-TCTCGGTTGAGGCTTGTTCC-3' (SEQ ID No.9),
reverse primer DUF 1279-R: 5'-GGGCTCATTGCGCTATTTGG-3' (SEQ ID No. 10);
a fourth primer pair:
forward primer TAE 1-F: 5'-GGAGAAGATCCACGCTCTCG-3' (SEQ ID No.11),
reverse primer TAE 1-R: 5'-GGTACATGCCCCAAGAGGTT-3' (SEQ ID No.12),
wherein the first primer pair is a primer of a first DNA bar code shown as SEQ ID No. 1; the second primer pair is a primer of a second DNA bar code shown as SEQ ID No. 2; the third primer pair is a primer of a third DNA bar code shown as SEQ ID No. 3; the fourth primer pair is a primer of a fourth DNA bar code shown as SEQ ID No. 4.
The primer of the invention can realize the specific amplification of the DNA bar code.
Yet another embodiment of the invention relates to a kit for identifying a strain of arthrospora adephaga comprising a DNA barcode primer composition according to the invention.
In the kit according to the present invention, four pairs of DNA barcode primers are preferably placed in four separate packages.
In a further preferred embodiment, the kit further comprises a DNA barcode composition according to the invention. The DNA barcode composition is preferably present on a recording medium. The recording medium is, for example, an optical disc.
Yet another embodiment of the present invention relates to a method for identifying a strain of nodospora adephagi comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) taking the genomic DNA in the step a) as a template, performing PCR amplification by using the first, second, third and fourth primer pairs according to the invention, and determining that the strain to be detected is not the node adenine arthrobotrys strain TMCC70007 if PCR products obtained by respectively amplifying the four primer pairs are not obtained; if a first, a second, a third and a fourth PCR product respectively amplified by the first, the second, the third and the fourth primer pairs are obtained, the following steps are carried out;
c) sequencing the obtained PCR products to obtain respective DNA sequences of the PCR products, wherein the DNA sequence of the first PCR product is a first DNA sequence, the DNA sequence of the second PCR product is a second DNA sequence, the DNA sequence of the third PCR product is a third DNA sequence, and the DNA sequence of the fourth PCR product is a fourth DNA sequence;
d) sequentially splicing the first, second, third and fourth DNA sequences obtained in the step c) to obtain a sequence to be detected; sequentially splicing the first, second, third and fourth DNA barcodes to obtain a standard sequence; comparing the sequence to be detected with the standard sequence for sequence homology, and if the sequence homology is less than 99%, determining that the strain to be detected is not the adenine node B spore-eating yeast strain TMCC 70007; and if the sequence homology is more than or equal to 99 percent, determining that the strain to be tested is the adenine-eating node spore yeast strain TMCC 70007.
According to an embodiment of the present invention, in the polymerase chain reaction, the amplification conditions are preferably pre-denaturation at 94 ℃ for 8min, followed by denaturation at 94 ℃ for 45 sec, annealing at 56 ℃ for 45 sec, extension at 72 ℃ for 1min 15 sec for a total of 32-35 cycles, and final extension at 72 ℃ for 10 min.
In a further preferred embodiment, the method further comprises e) performing cluster analysis on the test sequence and the standard sequence, and if the test sequence and the standard sequence are clustered together, determining that the test strain is the alternaria adefovea strain TMCC 70007. The clustering analysis may be to construct a phylogenetic tree.
In a specific embodiment of the invention, the four primer pairs of the invention are used to perform PCR amplification of genomic DNA extracted from the strain to be identified, followed by detection by agarose gel electrophoresis. Identifying the strains based on detecting the presence or absence of PCR products: if the strain to be identified does not amplify a corresponding target band, the strain is not TMCC 70007; if the corresponding target band is amplified, the strain is proved to be possible TMCC 70007. Then, for further identification, sequencing the PCR product, performing series combination on 4 DNA sequences obtained by sequencing, and performing homologous comparison on the combined sequences (standard sequences) of 4 DNA barcode sequences of the TMCC70007 strain to obtain the similarity (namely homology) between the sequences, and if the homology is more than or equal to 99%, judging that the strain to be detected is the adenine-noderum TMCC 70007.
In a more preferred embodiment, the sequence to be tested can be clustered with a standard sequence, such as a phylogenetic tree, and a phylogenetic tree method based on the clustering analysis is used to establish an identification rule to identify the species.
Specifically, an N J phylogenetic tree is constructed by applying MEGA 5.1 or PAUP software to the standard sequence of the Arthrospora adenine-eating yeast TMCC70007 and the sequence to be tested of each strain to be identified, if the sequence to be tested of the strain to be identified is clustered with the standard sequence of the Arthrospora adenine-eating yeast TMCC70007, the strain to be tested is identified as the Arthrospora adenine-eating yeast TMCC70007, and the term "clustering" used herein means that the strain to be tested and the strain TMCC70007 are in the same branch and have the same evolutionary distance after being analyzed by the phylogenetic tree.
In the above scheme, in step b), the length of the first PCR product amplified by the first primer pair is 758 bp; the length of a second PCR product obtained by amplification of the second primer pair is 441 bp; the length of a third PCR product obtained by amplification of the third primer pair is 775bp, and the length of a fourth PCR product obtained by amplification of the fourth primer pair is 580 bp.
Other embodiments of the invention relate to the use of the DNA barcode primer composition according to the invention for identifying a strain of arthrospora adensis, and the use of the DNA barcode composition according to the invention for identifying a strain of arthrospora adensis.
Examples
The present invention is further illustrated by the following specific examples. The methods used in the examples are conventional, unless otherwise specified.
Example 1: based on the homology comparison of the sequences obtained by amplifying the first primer pair, the second primer pair, the third primer pair and the fourth primer pair, whether the strain to be detected is the adenine-node-B-eating yeast TMCC70007 or not is judged
1. The strain source is as follows: the information of the selected related strains is shown in table 1 below:
TABLE 1
In the above table 1, the strain TMCC70007 is deposited at China General Microbiological Culture Collection Center (CGMCC) with the Collection number of CGMCC No. 8683; the CBS strains were purchased from the CBS-KNAW fungal biodiversity center, the Netherlands.
2. Respectively extracting strain DNA: OMEGA e.z.n.a was used.TMYeast DNA Kit D3370-01 Kit (manufactured by OMEGA) extracts Yeast genomic DNA and dilutes the DNA concentration of the sample to 0.5. mu.g/. mu.L with sterilized deionized water.
3. Amplifying DNA fragments, and respectively carrying out Polymerase Chain Reaction (PCR) on the extracted DNA of each strain by adopting the following 4 pairs of primer pairs:
a first primer pair:
forward primer LYR _ 1-F: 5'-CAAATCCTTGCCCTGGGGAT-3' (SEQ ID No.5),
reverse primer LYR _ 1-R: 5'-GTTGCAAACGAGCCCAACAT-3' (SEQ ID No. 6);
a second primer pair:
forward primer ABCB-F: 5'-GGGGATATGGGGACCGAAAC-3' (SEQ ID No.7),
reverse primer ABCB-R: 5'-TTTGGCCCCCTGTACTTGTG-3' (SEQ ID No. 8); and
a third primer pair:
forward primer DUF 1279-F: 5'-TCTCGGTTGAGGCTTGTTCC-3' (SEQ ID No.9),
reverse primer DUF 1279-R: 5'-GGGCTCATTGCGCTATTTGG-3' (SEQ ID No. 10);
a fourth primer pair:
forward primer TAE 1-F: 5'-GGAGAAGATCCACGCTCTCG-3' (SEQ ID No.11),
reverse primer TAE 1-R: 5'-GGTACATGCCCCAAGAGGTT-3' (SEQ ID No.12),
four primer pairs are used for carrying out PCR respectively, and the PCR reaction system is 50 mu L: ddH2O 37.7μL、MgCl25 μ L, 4 μ L dNTPs, 1 μ L forward primer, 1 μ L reverse primer, 0.3 μ L Taq DNA polymerase, 1 μ L DNA template, and no dye. And (3) amplification procedure: pre-denaturation at 94 deg.C for 8min, subsequent denaturation at 94 deg.C for 45s, annealing at 56 deg.C for 45s, and extension at 72 deg.C for 1min and 15s, for 32-35 cycles in total, and final extension for 10 min.
4. And detecting an amplification product, performing electrophoresis detection by using 1.0% agarose gel and 1 × TBE electrophoresis solution, and detecting the size of a PCR fragment by using DNASMarker. If the strain to be detected does not amplify a corresponding target band, the strain is not TMCC 70007; if obvious clear bands appear and no miscellaneous bands exist, the DNA fragments are sequenced, sequencing selection adopts sequencing, splicing results are provided by sequencing company (Huada gene), and the DNA fragments can be spliced by self.
5. Firstly, checking the quality of a sequence peak image obtained after sequencing in software Chromas, and splicing forward and reverse sequences by using SeqMan in a DNASTAR software package after determining that the quality of the peak image meets the requirement of data analysis. Sequencing finds that the length of a first PCR product obtained by amplification of the first primer pair is 758 bp; the length of a second PCR product obtained by amplification of the second primer pair is 441 bp; the length of a third PCR product obtained by amplifying the third primer pair is 775bp, the length of a fourth PCR product obtained by amplifying the fourth primer pair is 580bp, and the first, second, third and fourth DNA sequences of the first, second, third and fourth PCR products are manually proofread and sequentially spliced according to the sequence to obtain a sequence to be detected; the first, second, third and fourth DNA barcodes of TMCC70007 are spliced in sequence according to the sequence. Specifically, SeqMan in DNAStar software was used, see software description for splicing, to obtain a standard sequence. Comparing the sequence to be tested with the standard sequence, and if the homology of the sequence to be tested and the standard sequence of TMCC70007 is lower than 99 percent, judging that the strain to be tested is not the TMCC70007 strain; if the homology is more than 99 percent, the strain to be detected can be preliminarily determined to be the TMCC70007 strain. For example, as can be seen from table 2, the sequence similarity of other strains combinations is less than 99%, indicating that these strains are not TMCC70007 strains.
Table 2 comparison of the similarity of the combined sample sequences with the standard sequence Blastn is shown in the table below.
Example 2: identification rule established by phylogenetic tree method for identifying species
Obtaining a sequence to be tested of the strain to be tested by adopting the steps in the embodiment 1, and carrying out TMCC70007 and CBS 8244 treatment on the strain of the Alternaria adephagaTCBS 8335, CBS 7350 Strain and CBS 7370 and CBS 6800 of the Blutobotrys raffinoses speciesTMeGA 5.10 is used for combining sequences to be detected of the strains and constructing a Neighbor-join phylogenetic tree based on a Kimura-2-parameter model, the method is a Bootstrap method, and the Bootstrap repeated No. is 1000. If the sequence to be tested of the strain to be tested is clustered with the standard sequence of the TMCC70007 (namely the same branch and the same evolutionary distance), the strain to be tested is the Pu' er tea fermentation strain TMCC 70007.
As shown in FIG. 1, TMCC70007, CBS 8244 of Alternaria adephaga strainTCBS 8335, CBS 7350 Strain and CBS 7370 and CBS 6800 of the Blutobotrys raffinoses speciesTThe strain presents different branches, so that whether the strain to be detected is the Pu' er tea fermentation strain TMCC70007 or not can be further verified.
Industrial applicability
Compared with the traditional morphological identification method and the sequence identification based on ITS, 18S rRNA and the like, the method can distinguish and identify whether the strain to be detected is the Arthrospora adenine-eating yeast TMCC70007 strain in the species.
SEQUENCE LISTING
<110> Menghai tea industry, Limited liability company
<120> DNA barcode primer, DNA barcode, kit, method and application for rapidly identifying alternaria adenantha strain
<130> FI-163426-59:52/C
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 758
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 1
cgggcgagct tacccttcag cagaccgata tggtattcgg tagccttgtt cttttgagtc 60
ctcgcaagct cctcctggat ttgctggatc ttttccaaca ctcccattct ttcactattg 120
aactgaacgt ttaacaaacc tgattttgat caattatcca gtactttata atgataggtg 180
accacggagg ttaactgtct tccaacgctg atgggtgacc agattctcgt tccgacatgg 240
ctcgccatag cggattacaa agagatggta agcagaggat aaagatacgt ctgaaggagg 300
atggacaatg tgctaacgag tactgaacct gtataggagg tgtattcgcg cagtacacaa 360
aaagcccgct gaaaaccagc gggggttctt gctgtttgtt cgccaaagct tccatcagta 420
tgaatctctg cctcgaaagg actttggcac cattgagcat ctgctacgaa agggcaacaa 480
gaagcttgaa ctgtattccg accccaatgt gcgcaagatt tcatgatcaa ggtaaacatt 540
gtaaatagta tgaatagcat gaagcatgaa cagtatggta tcacctgaaa tagtcctcgt 600
taccaagcat gtataagaat tggtcggtat taccaaagat ctgggcatcg aatgacaaag 660
gtgccatttt atctaggttc acttggtcca ggtcgagctc gcgggtttct cggaagctca 720
caattaggtc cagtagtttt acaattcgct ttccaatg 758
<210> 2
<211> 441
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 2
gaccgaaact aggtggaatg gtgtccactc agaggataat actgataggt atcaggatca 60
ctctgatata gacaggaata cacctcctcc acagtattcc tatgatgact acccttcacc 120
gactgtccct atgctcattg gcggcagcgc agctctagtt atgctagcac tggtgtatac 180
atcggctaag gacttgttaa tgtgcaaaga agaccatagg ggccctgaga cagagcgatg 240
cgggtcgaaa gtttggacgc tagttaacta tcttttggtg tttgtgggat tggtgattat 300
tcttatcgga aagatcctaa aggacctcag aacaattgga gaaataaaca agaaaaaaga 360
agagacatgg cgaattcaag accagaaaaa acatgcattg cgagctgaac aagagggtct 420
agatcaagcc ctcgcacaag t 441
<210> 3
<211> 775
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 3
gttcccaccg tccatcatgt atattactcg ggacgcaacg gccctggtca acctggtgcg 60
cctggtgagc ctgttgaacc tgcgggatcg aggcccagat ctcatttaaa tgaacaaaat 120
aggccaagcg ctggagtaaa tggacagcct gtaaatggta aaccgaatgc ttcggctgca 180
tatccaccaa aaggacctga gagtataaat gagtttaaca ctacagcatc gtcaccgaaa 240
gttgcgcctg gtctagacca accgggacct tccgctaagc ctggattcgt tagaagatat 300
atgaatgcag tggccagtgc tccagtgagc catctcgtct cgtttgtctc catgtacact 360
acggtttatg gattcacgat ggtttcggtg tgggcagtgg tgcatcactt cgggtatgca 420
ccgctaggtg gaatccccaa ttggattctt gttcagggcc tggactttgt tagtgggtta 480
tcagaatggt tcagctggtt gaacgttgcc agccagagcg ctcgatgggt tagccagggt 540
gcagctgcat attccatcgc caaagtgctg ctaccagtaa gactgttctt taccttactt 600
ctaactcgat ggtttgctcg ggtcgcggtc attcccatta ataaccggat cgccagaatt 660
ttcaaatggt ttattccaaa gaggtttaga agccaaaaat agtgtagtgt agaattgtat 720
attttttgtt attattcgta catcattagg agtggatctt cagcgctccg tccgg 775
<210> 4
<211> 580
<212> DNA
<213> Arthrospora adenantha (Blastobotrys adeninivorans)
<400> 4
gctctcgttg gtcaactgca tgtagctggt agcttgacgc tcagcagatg ccaggttctg 60
tcgtcgctcg gctgcggcct ggtcttcgtc tgcaggcgca attgcaggaa cttccttctg 120
gagattatga atctcagcca gtttagcaag agactcgtca aacagatacg ttgcatcgtt 180
caaaagaaga gctacaaatt ggacaaaaaa gtggacatcc tccttgctct ctttctcaag 240
cttgtctcgg taccggttat tggcccagat gactttgatc actctggaaa tgtaatggcg 300
agagttgaac ttgtcataaa actgtgaact agttccagtt tgctcaactt caatgtagaa 360
cttcatgaga gcatgaagca agtgctccaa acttactgga tcggtatcga acaaatggac 420
catgaatccg ggccctccct gcatgttttc gagagctcca aagaatagaa actccaccag 480
tttcgacttg agataaggat tcttgacata ggtcgtgttg gtcaaaaagg tcacacagaa 540
aaccaacacc ttatcaatgg atccgtgcac aacctcttgg 580
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
caaatccttg ccctggggat 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
gttgcaaacg agcccaacat 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
ggggatatgg ggaccgaaac 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
tttggccccc tgtacttgtg 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
tctcggttga ggcttgttcc 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
gggctcattg cgctatttgg 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
ggagaagatc cacgctctcg 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence
<400> 12
ggtacatgcc ccaagaggtt 20
Claims (7)
1. A DNA barcode primer composition for identifying the adenine node B.sp.tmcc 70007 strain comprises the following four primer pairs:
a first primer pair:
forward primer LYR _ 1-F: 5'-CAAATCCTTGCCCTGGGGAT-3' the flow of the air in the air conditioner,
reverse primer LYR _ 1-R: 5'-GTTGCAAACGAGCCCAACAT-3', respectively;
a second primer pair:
forward primer ABCB-F: 5'-GGGGATATGGGGACCGAAAC-3' the flow of the air in the air conditioner,
reverse primer ABCB-R: 5'-TTTGGCCCCCTGTACTTGTG-3', respectively; and
a third primer pair:
forward primer DUF 1279-F: 5'-TCTCGGTTGAGGCTTGTTCC-3' the flow of the air in the air conditioner,
reverse primer DUF 1279-R: 5'-GGGCTCATTGCGCTATTTGG-3', respectively;
a fourth primer pair:
forward primer TAE 1-F: 5'-GGAGAAGATCCACGCTCTCG-3' the flow of the air in the air conditioner,
reverse primer TAE 1-R: 5'-GGTACATGCCCCAAGAGGTT-3' the flow of the air in the air conditioner,
wherein the first primer pair is a primer of a first DNA bar code shown as SEQ ID No. 1; the second primer pair is a primer of a second DNA bar code shown as SEQ ID No. 2; the third primer pair is a primer of a third DNA bar code shown as SEQ ID No. 3; the fourth primer pair is a primer of a fourth DNA bar code shown as SEQ ID No. 4.
2. A kit for identifying a nodospora adenantha TMCC70007 strain comprising the DNA barcode primer composition of claim 1.
3. A method for identifying a strain of the nodospora adephagi TMCC70007, comprising the steps of:
a) providing the genome DNA of a strain to be tested;
b) performing PCR amplification by using the genomic DNA in the step a) as a template and using the first, second, third and fourth primer pairs according to claim 1, and determining that the strain to be detected is not the Arthrospora adenantha strain TMCC70007 if PCR products obtained by respective amplification of the four primer pairs are not obtained; if a first, a second, a third and a fourth PCR product respectively amplified by the first, the second, the third and the fourth primer pairs are obtained, the following steps are carried out;
c) sequencing the obtained PCR products to obtain respective DNA sequences of the PCR products, wherein the DNA sequence of the first PCR product is a first DNA sequence, the DNA sequence of the second PCR product is a second DNA sequence, the DNA sequence of the third PCR product is a third DNA sequence, and the DNA sequence of the fourth PCR product is a fourth DNA sequence;
d) sequentially splicing the first, second, third and fourth DNA sequences obtained in the step c) to obtain a sequence to be detected; sequentially splicing a first DNA bar code, a second DNA bar code, a third DNA bar code and a fourth DNA bar code which are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 to obtain a standard sequence; comparing the sequence to be detected with the standard sequence for sequence homology, and if the sequence homology is less than 99%, determining that the strain to be detected is not the adenine node B spore-eating yeast strain TMCC 70007; and if the sequence homology is more than or equal to 99 percent, determining that the strain to be tested is the adenine-eating node spore yeast strain TMCC 70007.
4. The method according to claim 3, further comprising e) performing cluster analysis on the sequence to be tested and the standard sequence, wherein the cluster analysis is to construct a phylogenetic tree, and if the strain to be tested and the strain TMCC70007 are in the same branch and have the same evolutionary distance after being analyzed by the phylogenetic tree, determining that the strain to be tested is the Alternaria adefovea strain TMCC 70007.
5. The method of claim 3, wherein the first PCR product amplified by the first primer pair is 758bp in length; the length of a second PCR product obtained by amplification of the second primer pair is 441 bp; the length of a third PCR product obtained by amplification of the third primer pair is 775 bp; the length of a fourth PCR product obtained by amplification of the fourth primer pair is 580 bp.
6. The method of claim 3, wherein the procedure for PCR amplification is: 1) pre-denaturation at 94-96 deg.C for 8 min; 2) denaturation at 94-96 ℃ for 45 seconds, annealing at 55-57 ℃ for 45 seconds, and extension at 72 ℃ for 1min 15 seconds, wherein the procedure 2) is performed for 30-35 cycles; 3) extension at 72 ℃ for 10 min.
7. The use of the DNA barcode primer composition of claim 1 for identifying a strain of nodospora adefovea TMCC 70007.
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| CN105779297A (en) * | 2014-12-16 | 2016-07-20 | 勐海茶业有限责任公司 | Strain of Arxula adeninivorans for producing high activity polyphenoloxidase and application thereof to production of Pu'er tea |
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| CN105779297A (en) * | 2014-12-16 | 2016-07-20 | 勐海茶业有限责任公司 | Strain of Arxula adeninivorans for producing high activity polyphenoloxidase and application thereof to production of Pu'er tea |
Non-Patent Citations (1)
| Title |
|---|
| The complete genome of Blastobotrys(Arxula) adeninivorans LS3- a yeast of biotechnological interest;Gotthard Kunze et al.;《Biotechnology for Biofuels》;20140424(第66期);全文 * |
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