CN111662995A - DNA barcoding, primers, kits, methods and applications - Google Patents

Abstract

The present invention provides DNA barcodes, primers, kits, methods and applications. The DNA barcode is derived from the genome of the Aspergillus niger TMCC 70012 strain, and comprises a sequence of at least 500 bp selected from the DNA sequences shown in SEQ ID No. 4, and the DNA barcode has a length of 500bp-4000bp. The DNA barcode sequence of the present invention can realize the rapid identification and differentiation of the bacterial species within the Aspergillus niger species. Thus, the present invention establishes a standard gene sequence and a sample identification method for Pu'er tea industrial fermentation production strain Aspergillus niger TMCC 70012, which significantly improves the identification efficiency compared with the traditional morphological identification method.

Description

DNA barcoding, primers, kits, methods and applications

technical field

The invention belongs to the field of species and strain identification, and in particular relates to a DNA barcode primer composition, DNA barcode, kit, method and application for identifying Pu'er tea fermentation strains. Specifically, the Pu-erh tea fermentation production strain is Aspergillus niger TMCC 70012.

Background technique

Pu'er tea is a post-fermented tea produced within the scope of Yunnan's geographical indication. The traditional production process of Pu-erh tea is as follows: the picked fresh tea leaves are rolled and dried to make raw green tea. In the production of Pu-erh tea, the process of stacking fermentation is the main factor for the formation of the quality of Pu-erh tea. The special flavor, taste, quality and various health benefits of tea.

In traditional Pu-erh tea production, the humid and hot environment activates the enzymes contained in the tea itself, so that some of the components contained in the tea are converted into substances that can be used by microorganisms; microorganisms also grow in large numbers during the fermentation process of Pu-erh tea, producing abundant Intracellular enzymes and extracellular enzymes catalyze a series of changes in the components contained in tea, thus forming the unique quality of Pu-erh tea. Different origins, microbial species and differences in community structure make Pu-erh tea unique in flavor and quality.

In addition to its unique flavor and culture, Pu-erh tea has also attracted much attention for its health benefits such as weight loss, lowering blood sugar and blood lipids, preventing and improving cardiovascular disease, anti-aging, anti-cancer, anti-inflammatory, aiding digestion, and nourishing the stomach. increasingly popular with consumers. The increasing market demand has driven the development of the Pu'er tea industry and promoted the economic growth of Yunnan.

At present, the production of Pu-erh tea by most manufacturers is still semi-natural artificial piling by empirical fermentation. Although the community dominated by dominant common microorganisms including Aspergillus niger is relatively stable during the piling process, the product is stable. There is still a lot of room for improvement. In order to further gain the favor of consumers and market recognition, break through foreign trade barriers, and enhance the market competitiveness of Pu'er tea enterprises, it is necessary to realize the artificial controllability, cleanliness and efficiency of Pu'er tea production, and constantly develop new products and extend the industrial chain. . To do this, it is necessary to innovate technology and invent a series of new automated Pu-erh tea processes that are safe, clean, efficient, and artificially controllable, so as to ensure the healthy development of the Pu-erh tea industry and bring long-term benefits to the country and the people.

Artificial inoculation and pure-bred fermentation of Pu-erh tea are the new development direction of controllable fermentation of Pu-erh tea. In order to protect Pu-erh tea fermentation process and high-quality microbial germplasm resources, it is imperative to develop a rapid and accurate identification method for Pu-erh tea fermentation strains.

DNA barcoding technology can quickly and easily identify and distinguish similar strains, which can provide theoretical basis and technical means for the development of artificially controllable purebred fermentation process and resource protection of Pu'er tea, and promote the healthy development of Pu'er tea industry.

SUMMARY OF THE INVENTION

In order to overcome the defects of morphology in identifying Pu'er tea fermentation production bacteria, the present invention provides DNA barcodes, primers, test kits, methods and applications for identifying Pu'er tea fermentation strain Aspergillus niger TMCC 70012, thereby It can accurately identify the Aspergillus niger TMCC 70012 strain from confusing species or complex species, and realize rapid identification and differentiation, which can provide rapid identification and evaluation for the new fermentation process of Pu'er tea, and prevent the interference of other miscellaneous bacteria in the fermentation process. , and provide evidence methods and basis for the artificial controllable fermentation process of Pu'er tea and the abuse of strains.

In order to achieve the above object, the present invention adopts the following technical solutions:

(1) a DNA barcode for identifying a strain of Aspergillus niger, the DNA barcode is derived from the genome of Aspergillus niger TMCC70012 strain, and comprises a sequence of at least 500 bp selected from the DNA sequences shown in SEQ ID No. 4, and the The length of the DNA barcode is 500bp-4000bp, preferably 500bp-900bp.

(2) The DNA barcode according to (1), the nucleotide sequence of which is shown in SEQ ID No. 1, SEQ ID No. 4 or SEQ ID No. 7.

(3) A primer pair for amplifying the DNA barcode of (1) or (2).

(4) The primer pair according to (3), wherein the nucleotide sequence of the forward primer is the same as the sequence in the genome of the Aspergillus niger TMCC 70012 strain: the sequence is as shown in SEQ ID from the genome of the TMCC 70012 strain A sequence in the region from position 1 of the nucleotide sequence shown in No. 4 to position 3510 of the nucleotide sequence shown in SEQ ID No. 4, and the length of the forward primer is 15-30 bp; Its reverse primer is reverse complementary to the sequence in the genome of the TMCC 70012 strain: the sequence is from the 471st position of the nucleotide sequence shown in SEQ ID No. 4 in the genome of the TMCC 70012 strain to the The sequence in the region of the last bit of the nucleotide sequence shown in SEQ ID No. 4, and the length of the reverse primer is 15-30 bp.

(5) The primer pair according to (4), wherein the nucleotide sequences of the forward primer and the reverse primer are respectively as follows:

Forward primer: 5'-ACGCCTCAGTAGAAGATAGT-3';

Reverse primer: 5'-CTTGCCATGGTGGTGGGATA-3'.

(6) A kit for identifying a strain of Aspergillus niger, comprising the primer pair according to any one of (3)-(5).

(7) a method for identifying Aspergillus niger bacterial strains, comprising the following steps:

a) provide the genomic DNA of the strain to be tested;

b) using the genomic DNA described in step a) as a template, using the primer pair according to any one of (3)-(5) to carry out PCR amplification to obtain a PCR product;

c) Detect the PCR product by agarose gel electrophoresis, if there is no target band, then determine that the strain to be tested is not Aspergillus niger TMCC 70012 strain, if there is a target band, then proceed to step d);

d) Sequencing the obtained PCR product to obtain a nucleotide sequence to be tested; aligning the sequence to be tested with the nucleotide sequence of the DNA barcode described in (1) or (2) for homology, if the same If the origin is above 97%, it is determined that the strain to be tested is Aspergillus niger TMCC 70012 strain.

(8) Application of the DNA barcode according to (1) or (2) in identifying Aspergillus niger strains.

(9) Use of the primer pair according to any one of (3)-(5) in identifying Aspergillus niger strains.

(10) The application of the kit according to (6) in identifying strains of Aspergillus niger.

Compared with the prior art, the present invention has the following advantages and positive effects:

1. The present invention uses proteomics technology to find out the missing annotation peptide segment from the genome of Aspergillus niger TMCC 70012 strain, and the peptide segment is located in the protein GLF A encoded by the UDP-galactopyranosyl mutase GLF A gene. The present invention further develops a DNA bar code based on the GLF A gene through careful research and comparative analysis, and the bar code sequence can realize the rapid identification and differentiation of Aspergillus niger species.

2. The present invention further finds that, compared with other genes, the sequence of the GLF A gene (for example, SEQ ID No. 1) has the characteristics of universality, easy amplification and easy comparison, and it is different between different strains in Aspergillus niger species. obvious.

3. The present invention establishes a standard gene sequence and a sample identification method for Pu'er tea industrial fermentation production strain Aspergillus niger TMCC 70012, which significantly improves the identification efficiency compared with the traditional morphological identification method. The method has low requirements for the integrity of the sample and the identification index can be quantified, which provides an effective basis for the timely determination of Pu'er tea fermentation process and its germplasm resources, and makes up for the strain identification of this kind of Pu'er fermented tea production strain based on DNA barcoding technology. Whitespace. In addition, morphological confounding species were further added and identification rules were established by phylogenetic tree method based on cluster analysis. The reliability and accuracy of identification were also much better than conventional molecular identification methods.

Description of drawings

Figure 1 shows the mass spectrum of the newly identified peptide VPSFQDILQGR.

Figure 2 shows the comparison between the mass spectrum of the synthetic peptide VPSFQDILQGR and the original identified peptide; the original identified peptide is the peptide obtained by mass spectrometry analysis and identification in the present invention; the upper part of the figure is the mass spectrum of the original identified peptide Figure, the bottom is the mass spectrum of the synthesized peptide.

Figure 3 shows the nucleotide sequence of SEQ ID NO. 1, with introns in gray.

Figure 4 shows the correspondence between SEQ ID NO. 1 and the amino acid sequence of the encoded protein, and the gray part is the newly identified peptide segment VPSFQDILQGR.

Figure 5A shows the SDS-PAGE separation map of the whole cell protein of TMCC 70012, and the band shown in bold is the location of GLF A protein; Figure 5B shows the molecular weight verification map of GLF A protein. In Figure 5B, the abscissa represents the logarithm of molecular weight MW, and the ordinate represents the ratio of the migration amount of each molecular weight protein marked by protein molecular weight in SDS-PAGE electrophoresis separation to the total migration amount of SDS-PAGE.

Fig. 6 shows the sequence alignment of barcode sequence SEQ ID NO. 1 obtained by NCBI-BLASTN with homology thereto, and the gray line segment below Fig. 6 shows the homology sequence.

Figure 7 shows the nucleotide sequence of SEQ ID NO.4, the light gray part is SEQ ID NO.1, the dark gray part is the intron in SEQ ID NO.1, the bold font is the primer sequence, and the underlined part is The sequence of the product amplified with this primer is SEQ ID NO.7.

Figure 8 shows a sequence alignment of the barcode sequence SEQ ID NO.4 obtained by NCBI-BLASTN with homology to it, and the grey line segment below Figure 8 shows the homology sequence.

9 shows the results of agarose gel electrophoresis of products obtained by PCR amplification using primers designed for the Aspergillus niger TMCC 70012GLF A gene of the present invention.

Detailed ways

The present invention will be further described below through the description of specific embodiments and with reference to the accompanying drawings, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention The basic idea of the present invention is within the scope of the present invention.

In the present invention, a species-specific gene-encoded protein that is difficult to be found by traditional gene prediction software is found from the Aspergillus niger TMCC 70012 strain by using the systematic proteogenomics technology. Since the protein encoded by this gene has high homology with UDP-galactopyranosyl mutase, it was named GLF A. The present invention finds that the open reading frame (SEQ ID No. 1) of the gene GLFA encoding the protein can identify the strain of Aspergillus niger TMCC 70012 from confusing species, so it can be used to develop and identify the strain of Aspergillus niger produced by industrial fermentation of Pu'er tea DNA barcoding of TMCC 70012. Compared with the prior art, the specificity of the DNA barcode obtained by the above method of the present invention is higher.

Considering that the DNA barcode should have an appropriate length and sufficient specificity between strains, the present invention further obtains a DNA barcode that can accurately and effectively identify the Aspergillus niger TMCC70012 strain based on the above-mentioned unique DNA sequence through careful research and comparative analysis.

Thus, one aspect of the present invention provides a DNA barcode for identifying A. adeninotropha strains, the DNA barcode is derived from the genome of the Aspergillus niger TMCC 70012 strain, and comprising selected from the group consisting of as shown in SEQ ID No. 4 A sequence of at least 500bp in the DNA sequence, and the length of the DNA barcode is 500bp-4000bp, preferably 500bp-900bp.

The present invention discovers a protein coding sequence that is difficult to be found by traditional gene prediction software through systematic protein genomics research, and supports the existence of the peptide segment of the gene coding sequence product and the related proteomics mass spectrometry data is accurate and reliable; based on the protein coding sequence in the The position in the genome determined the possible gene sequence (SEQ ID No. 1) and protein sequence (SEQ ID No. 3) encoding the protein. The length of SEQ ID No. 1 is 1964 bp. According to the comparison analysis, SEQ ID No. 1 is unique to the genome of Aspergillus niger TMCC 70012 strain, so the sequence itself can be used as a DNA barcode for identifying Aspergillus niger TMCC 70012 strain. In addition, through theoretical analysis and experimental verification, the present invention proves that the specificity of DNA barcodes containing this sequence and longer in length is more guaranteed. When the length of the DNA barcode is too long (eg, greater than 4000 bp), it is not ideal for the amplification operation. Therefore, based on the gene sequence, the present invention finds a longer and more specific DNA barcode sequence in the genome of Aspergillus niger TMCC 70012 strain. On the other hand, generally, when the length of the DNA barcode is not less than 500 bp, the operation requirements of easy amplification and easy comparison can also be satisfied. Therefore, the present invention proves that a sequence of at least 500 bp selected from the DNA sequences shown in SEQ ID No. 4 can also achieve rapid and accurate identification and differentiation of Aspergillus niger strains through theoretical analysis and experimental verification.

The barcode sequence of the present invention can realize the rapid and accurate identification and differentiation of strains within the species of Aspergillus niger.

In particular, the present invention found that in the genome of Aspergillus niger TMCC 70012 strain, the DNA sequence shown in SEQ ID No. 4 as shown in SEQ ID No. 7 is between different strains and between different strains within Aspergillus niger species has a particularly high specificity. Therefore, in a preferred embodiment of the present invention, the DNA barcode comprises a sequence of at least 500 bp selected from the DNA sequences shown in SEQ ID No.7.

Preferably, the DNA barcode sequence is shown in SEQ ID No.1, SEQ ID No.4 or SEQ ID No.7.

In this article, the term "missing annotation" means that after the species completes genome sequencing, gene prediction software (such as GeneMark, Augustus, Glimmer, etc.) fails to predict a gene or protein, and these genes or proteins are generally not highly expressed, It is only expressed under specific conditions, so it is difficult to be found in research.

The term "DNA barcoding" refers to a new molecular identification technology that uses a standard, short DNA fragment in the genome to identify species, which can quickly and accurately identify species.

The term "six box translation" is a known term in proteomics and genomics, and its principle is briefly described as follows: when DNA encodes proteins, triplet codons are used to encode proteins. Given a DNA sequence, there are three encoding possibilities. , plus the 3 coding possibilities on its complementary strand, there are 6 coding possibilities (+1, +2, +3, -3, -2, -1).

Another aspect of the present invention provides a primer pair for amplifying the DNA barcode of the present invention.

Those skilled in the art can understand that, according to the DNA barcode sequences for identifying A. adeninotropha strains provided by the present invention, corresponding primer pairs can be easily designed to amplify the desired DNA barcodes.

Preferably, the nucleotide sequence of the forward primer of the primer pair is the same as the sequence in the genome of the Aspergillus niger TMCC70012 strain: the sequence is the nuclear sequence shown in SEQ ID No. 4 from the genome of the TMCC 70012 strain The sequence in the region from the 1st position of the nucleotide sequence to the 3510th position of the nucleotide sequence shown in SEQ ID No.4, and the length of the forward primer is generally 15-30bp; The reverse complement of the sequence in the genome of the TMCC 70012 strain: the sequence from the 471st position of the nucleotide sequence shown in SEQ ID No. 4 in the genome of the TMCC 70012 strain to the position shown in SEQ ID No. 4 The sequence in the region of the last bit of the nucleotide sequence shown, and the length of the reverse primer is also generally 15-30 bp. The product amplified by the forward and reverse primers is a sequence of at least 500 bp selected from the nucleotide sequences shown in SEQ ID No. 4.

In a preferred embodiment, the nucleotide sequence of the forward primer of the primer pair is the same as the sequence in the genome of the Aspergillus niger TMCC 70012 strain: the sequence is obtained from the genome of the TMCC 70012 strain as SEQ ID No. 7 The sequence in the region from position 1 of the nucleotide sequence shown to position 369 of the nucleotide sequence shown in SEQ ID No. 7, and the length of the forward primer is generally 15-30 bp; The primers are reverse complementary to a sequence in the genome of the TMCC 70012 strain from position 471 of the nucleotide sequence shown in SEQ ID No. 7 to the nucleotide sequence shown in SEQ ID No. 7 in the genome of the TMCC 70012 strain The sequence in the region of the last digit of the nucleotide sequence shown in No. 7, and the length of the reverse primer is also generally 15-30 bp. The product amplified by the forward and reverse primers is a sequence of at least 500 bp selected from the nucleotide sequences shown in SEQ ID No. 7.

In a more preferred embodiment, the nucleotide sequences of the forward primer and the reverse primer are respectively as follows:

GLF A-F: 5'-ACGCCTCAGTAGAAGATAGT-3' (SEQ ID No. 5);

GLF A-R: 5'-GTGGATGGGACGTAAAGCAT-3' (SEQ ID No. 6).

The primers of the present invention enable specific amplification of the DNA barcode sequence.

The present invention also provides a kit for identifying Aspergillus niger strains, comprising the primer pair according to the present invention.

In another embodiment, the kit further comprises a DNA barcode according to the present invention. The DNA barcode may be present on a recording medium. The recording medium is, for example, an optical disc. The kit may also contain any tools and reagents for experimental manipulation.

Another aspect of the present invention provides a method for identifying Aspergillus niger strains, comprising the following steps:

a) provide the genomic DNA of the strain to be tested;

b) using the genomic DNA described in step a) as a template, using the primer pair according to the present invention to carry out PCR amplification to obtain a PCR product;

c) electrophoresis (such as agarose gel electrophoresis) to detect the PCR product, if there is no target band, it is determined that the strain to be tested is not Aspergillus niger strain TMCC 70012, if there is a target band, then proceed to step d);

d) Sequence the obtained PCR product to obtain the nucleotide sequence to be tested; compare the nucleotide sequence to be tested with the nucleotide sequence of the DNA barcode of the present invention for homology, if the homology If it is more than 97% (preferably more than 98%, more preferably more than 99%, more preferably 100%), it is determined that the strain to be tested is Aspergillus niger TMCC 70012 strain.

As used herein, the terms "identity" and "homology" have the same meaning and are used interchangeably.

In a specific embodiment of the present invention, the PCR amplification procedure is: 1) pre-denaturation at 94°C for 5 minutes; 2) denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute, wherein the procedure 2) 30 cycles were performed; 3) 10 minutes extension at 72°C.

In yet another embodiment of the present invention, the method may further comprise performing cluster analysis (eg, a phylogenetic tree) on the tested nucleotide sequence obtained from the sequencing result and the DNA barcode of the present invention, if the tested sequence is related to the DNA The barcodes were clustered together, and the strain to be tested was determined to be Aspergillus niger strain TMCC 70012.

In a specific embodiment of the present invention, the genomic DNA extracted from the strain to be identified is amplified by PCR using the primer pair of the present invention, and then detected by agarose gel electrophoresis. Identify the strain based on the presence of PCR products: if the strain to be identified does not amplify the corresponding target band, it means that the strain is not TMCC 70012; if the corresponding target band is amplified, it proves that the strain may be TMCC 70012. For further identification, the PCR products were sequenced, and the DNA sequencing results were compared with the DNA barcode sequences for homology to obtain the similarity (ie homology) between sequences. If the sequence homology was less than 97%, it was judged The tested strain was not Aspergillus niger strain TMCC 70012. If the sequence homology is greater than or equal to 97%, the strain to be tested is determined to be Aspergillus niger strain TMCC 70012.

If a cluster analysis, such as a phylogenetic tree, is performed, the DNA barcodes and DNA sequencing results of each strain to be identified (ie, the sequence to be tested) are used to construct an NJ phylogenetic tree using MEGA 6 or PAUP software. If the sequence to be identified of the strain to be identified is clustered with the DNA barcode of Aspergillus niger TMCC 70012, it is identified as Aspergillus niger TMCC 70012.

The term "clustering" as used herein refers to being in the same branch and having the same evolutionary distance after phylogenetic tree analysis.

The present invention also provides the application of the DNA barcode of the present invention in identifying Aspergillus niger strains.

The present invention also provides the application of the primer pair of the present invention in identifying Aspergillus niger strains.

The present invention also provides the application of the kit of the present invention in identifying Aspergillus niger strains.

Example

The present invention will be further described below in conjunction with specific embodiments. The methods used in the examples all adopt conventional methods and known tools unless otherwise specified.

Example 1: Acquisition of GLF A gene and DNA barcode

1. Using high-coverage proteome technology, pFind and pAnno software were used to carry out a proteome in-depth coverage study of the industrial fermentation bacterium Aspergillus niger TMCC 70012 (Aspergillus niger TMCC 70012) in Pu'er tea, and to verify its genome annotation coding genes . In order to discover new protein coding regions, a six-frame translation database of Aspergillus niger TMCC 70012 genome data was obtained by using the Six Frame Translation strategy in systematic proteomics, and the six possible coding possibilities of the genome were exhausted (+ 1, +2, +3, -1, -2, -3), and the nucleic acid sequence is referred to as "six-frame translation nucleic acid sequence", and the protein sequence is referred to as "six-frame translation protein sequence". Generally speaking, a six-frame translation nucleic acid sequence is the sequence from one terminator to the next terminator, and is also referred to herein as a "protein-coding frame." Using this database, the high-coverage proteomic mass spectrometry data of total cellular proteins of the TMCC 70012 strain was used to identify novel peptides and novel proteins using pFind and pAnno software.

After identification, a peptide segment VPSFQDILQGR, which was not found in the annotated gene of Aspergillus niger TMCC 70012 in the prior art, was found, and the mass spectrum is shown in Figure 1.

Manual inspection of the mass spectrum showed that almost all the y-ion sequences of the peptide segment VPSFQDILQGR secondary mass spectrum (MS2) were detected, with good matching, strong signal, and credible results.

In order to further confirm the identification result, the peptide was chemically synthesized according to the amino acid sequence of the newly identified peptide VPSFQDILQGR, and the high-energy collision MS2 generated by the synthesized peptide was verified. value, indicating that the synthesized peptide sequence is correct, see Figure 2.

On this basis, the MS ₂ spectra of the synthesized peptides and the large-scale identification of the new peptide sequences identified from the large-scale proteomic data were manually checked, and the two were almost identical. The cosin value is as high as 0.99, which proves that the new peptide segment identified from the Pu-erh tea industrial fermentation bacteria Aspergillus niger TMCC 70012 is correct.

2. According to the position of the new peptide segment, and the region included by the former stop codon and the latter stop codon as the boundary, obtain a six-frame translation nucleic acid sequence (protein coding frame) SEQ ID NO.2.

3. In order to further determine the coding start site and termination site of the gene encoding the protein, the above protein coding frame was extended upstream and downstream by 1000bp respectively, and GeneMark.hmm was used for gene prediction, and the reference species was Schizosaccharomyces pombe. (Schizosaccharomyces pombe). The existence of the protein-coding gene is predicted in this region, and its reading frame is consistent with the above-mentioned protein reading frame, and the gene starter is found in the ORF sequence, but the terminator of the ORF sequence is actually located in the intron part, so According to the gene prediction, the real terminator is found in the following sequence, so according to the prediction results, the possible coding start and termination sites of this gene are determined, and the complete gene sequence from the starter to the terminator is SEQ. ID NO.1, as shown in Figure 3.

The corresponding relationship between SEQ ID NO.1 and the amino acid sequence of the encoded protein is shown in FIG. 4 . The peptide VPSFQDILQGR is located near the N-terminus of the protein.

The nucleotide sequence of the gene (SEQ ID NO.1) is 1964 bp including the terminator, and the intron region is removed to encode a total of 547 amino acids, and the theoretical molecular weight is 61.0kDa. The theoretically encoded amino acid sequence is such as SEQ ID NO. 3 shown.

4. In order to further confirm the correctness of the identified sequence, it is necessary to determine the molecular weight of the protein encoded by the region where the ORF of the identified VPSFQDILQGR is located, so its experimental molecular weight was determined (Fig. 5). The TMCC70012 strain was cultured in YPD medium and the total protein was extracted, separated by SDS-PAGE, the electrophoresis gel was stained with Coomassie brilliant blue, and the molecular weight was verified according to the molecular weight characteristics of the proteome sample preparation. In SDS-PAGE, the protein migration distance The ratio of the migration distance to the dye front has a certain relationship with the logarithm of the protein molecular weight, as shown in Figure 5. The entire lane was cut into 30 fractions by molecular weight and protein abundance, and subjected to in-gel digestion and mass spectrometry analysis, in which GLF A protein was identified in the mass spectrometry data of the 13th strip, which was consistent with the predicted molecular weight. Size matches. The theoretical molecular weight is 61.0kDa, which is consistent with the position in the SDS-PAGE of the gel strip to which the protein belongs. The results showed that the experimental molecular weight of the identified GLF A was between 59.2-61.8 kDa, which was consistent with the theoretical molecular weight, which confirmed the correctness of the identified protein.

5. NCBI-BLASTP analysis was carried out with the amino acid sequence (SEQ ID NO. 3) of the theoretically encoded product of the gene, and a protein with high sequence similarity was retrieved. The Blastp results showed that the GLF A gene product we detected in the TMCC-70012 strain had a domain HemY superfamily, which indicated that the protein composed of this sequence had a specific structure and independent function in biological macromolecules. The above blastp alignment results are shown in Table 1.

Table 1 Sequences with higher homology to TMCC 70012 GLF A protein sequence

Table 2 Sequences with higher homology to TMCC 70012 GLF A gene sequence

6. Perform NCBI-BLASTN analysis on the sequence of the identified GLF A gene (ie, SEQ ID NO. 1), and the results are shown in Figure 6 . The BLASTN results are summarized in Table 2.

The results in Table 2 show that the sequence of the GLF A gene (ie, SEQ ID NO. 1) has a sequence with high homology to another strain in Aspergillus niger, but the difference between it and other species other than Aspergillus niger is relatively high. It can be used as a DNA barcode to distinguish TMCC-70012 strain from other strains. In addition, if a high homology alignment screening standard is set, for example, the homology is greater than or equal to 99%, the SEQ ID NO. 1 can also be used as a DNA barcode to distinguish the TMCC-70012 strain from other Aspergillus niger strains.

7. The sequence of the transcribed spacer region before and after the GLF A gene sequence (ie, SEQ ID NO. 1) was further considered, and SEQ ID NO. 4 was obtained, as shown in FIG. 7 . A homology comparison was performed on SEQ ID NO. 4, as shown in FIG. 8 . It can be known from Fig. 8 that SEQ ID NO. 4 has high specificity in the NCBI database, and the fragments after 2250 bp of SEQ ID NO. 4 have almost no homology matches. It is indicated that SEQ ID NO. 4 can effectively distinguish TMCC-70012 from near-derived strains, so it can be used as a DNA barcode.

Example 2. Identification of strains using DNA barcoding

According to the amplification results of the sample to be tested and the amplification results and sequence homology with the GLF A gene sequence of the Aspergillus niger TMCC 70012 strain, it is determined whether the sample to be tested is the industrial application strain of Aspergillus niger TMCC 70012.

(1) Based on SEQ ID NO.4, use NCBI primer design tool to design PCR primers, and obtain the forward and reverse primer sequences as follows:

GLF A-F: 5'-ACGCCTCAGTAGAAGATAGT-3';

GLF A-R: 5'-GTGGATGGGACGTAAAGCAT-3'.

The positions of the primers are shown in Figure 7. The amplified sequence is SEQ ID NO. 7 (838 bp).

(2) Source of strain

The information of relevant strains selected in table 3

(3) Extracting strain DNA respectively: using the Yeast DNA kit of OMEGA EZNA ^TM to extract genomic DNA, and diluting the DNA concentration of the sample to 0.5 μg/μL with sterilized deionized water.

(4) Amplify DNA fragments, carry out polymerase chain (PCR) reaction, and the primer sequences used are respectively:

GLF A-F: 5'-ACGCCTCAGTAGAAGATAGT-3';

GLF A-R: 5'-GTGGATGGGACGTAAAGCAT-3'.

The PCR reaction system was 50 μL, and the PCR reagents were Taq DNA Polymerase (recombinant) from Thermo Scientific ^TM : ddH ₂ O 37.7 μL, MgCl ₂ 5 μL, dNTPs 4 μL, forward primer 1 μL, reverse primer 1 μL, Taq DNA polymerase 0.3 μL, 1 μL of DNA template without dye. The amplification program was: pre-denaturation at 94°C for 5 minutes; followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute; and a final extension at 72°C for 10 minutes.

(5) Detection of amplified products: use 1.0% agarose gel and 1×TBE electrophoresis solution for detection, and use DNA molecular weight marker to determine the size of PCR fragments. If the strain to be tested does not have the expected amplified band of 838bp, it means that the strain is not Aspergillus niger TMCC70012; if there is an obvious and clear band and no stray bands, send it to a biosequencing company for DNA fragment sequencing.

(6) This primer can only be amplified in Aspergillus niger TMCC 70012, but not in Aspergillus niger CBS 513.88, CBS 554.65T, CBS 120.49, CBS 101697, CBS 116681, CBS 119557, CBS 117984, CBS 118725 of the same species Amplification was achieved and the results are shown in Figure 8. The theoretical amplification sequence of the PCR primers is 838bp, and the actual amplification results are in line with expectations. To further verify the sequence of the amplified DNA, sequencing and homologous sequence comparisons were performed. This result proves that SEQ ID NO. 7 can be very specific and effective to distinguish A. niger TMCC 70012 strain, thereby serving as a DNA barcode for A. niger TMCC 70012 strain.

(7) For the sequencing results of the sequences with bands, first use the software Chromas to check the quality of the sequence peaks obtained after sequencing, and after confirming that the quality of the peaks meets the requirements of data analysis, use SeqMan in the DNASTAR software package to perform forward and reverse sequencing splicing. Perform manual proofreading and sequence splicing on the sequencing results. If the homology between the DNA fragment of the strain to be tested and the standard DNA barcode of Aspergillus niger TMCC70012 is more than 97%, it can be determined that the strain to be tested is likely to be the Aspergillus niger TMCC70012 strain.

(8) Further cluster analysis, such as a phylogenetic tree, can be further performed, which can further corroborate the judgment results obtained through the above steps.

SEQUENCE LISTING

<110> Menghai Tea Industry Co., Ltd.

<120> DNA barcodes, primers, kits, methods and applications

<130> FI-190367-59:52/C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1964

<212> DNA

<213> Aspergillus niger

<400> 1

atgctcagcc tcgcccgcag gactttgaac cgtgtcccca gctttcaaga tattctacaa 60

ggcaggatga cccaccccga tatgtaagga gacctttccc cctccctcaa acggcaagct 120

tgccttcact acaacaagaa gctcattgga acaattgctg accgttgtct ttgtgcttga 180

attcagctcc gtcgacgttc tcgtcattgg tgccggccct actggtctcg gtgccgcgaa 240

gcgtcttaac cagattgtac gcattgcgcg ccccctatcg tataccttca ctcgaataaa 300

ggctaattgg acaattcgcg ggtgtaggat ggcccttctt ggttgatcgt tgacagcaac 360

gagacccctg gtggtcttgc ttccaccgat gtgacccccg aaggcttcgt atgttgattc 420

ccactttccc ctcaatgggc aggcattgca ctggtcaagc aaacagcgac tgactcgtga 480

caatcacagc tcttcgatgt tggtggtcac gtcatcttct cccactacaa gtacttcgac 540

gactgcatca acgaggctct ccccaaggat gacgactggt acacccacca gcgtatctcc 600

tacgttcgct gccagggcca atgggttccc taccccttcc agaacaacat ctccatgctt 660

cccaagaatg agcaggtccg ctgtatcgat ggcctgatcg acgctgccct tgaggctcgt 720

gtcgccaaca ccaagcccca gaacttcgat gagtggattg ttcgccagat gggtgtcggt 780

atcgccgacc tcttcatgag accctacaac ttcaaggttt gggctgtgcc tacgaccaag 840

gtaagatacg atgtaccaca tggcgagtaa cggcgtgctg attctcatca gatgcaatgt 900

gcctggttgg gtgagcgtgt tgctgcccct aacgtcaagg ccgtgacgac caacgtcatc 960

cttaacaaga ccgctggtaa ctggggtcct aacgctactt tccgtttccc cgcccgtggt 1020

ggtaccggtg gtatctggat cgctgtcgcc gacactatcc ccaaggagaa gactcgcttc 1080

ggtgagaagg gcaaggtcgt caaggtcaat gccaacaaca agaccgtcac cctgggtgat 1140

ggcaccactg tcggctacaa gaagctcgtc tccaccatgg ctgtcgactt cctcgctgag 1200

cagatcggcg accaggagct cgttggcctc accaagcagc tcttctactc ctccactcac 1260

gtcattggtg tcggtatccg tggtacccgc cccgagagaa tcggtgacaa gtgctgggta 1320

agttgacctt ggatggaaac catgaatgac ttcaaaacta acatgctcgt tactagctct 1380

acttccccga ggacaactgc cccttctacc gtgccaccat cttctccaac tactcccccc 1440

acaaccagcc cgagggctcc aagaagcttc ccactctgca gcttgcggat ggctctaagc 1500

cccagagcac tgaggctcag gagggtcctt actggtccat catgttggag gtttccgagt 1560

cttcgatgaa gcctgtcaac cacgagactc tcctggctga ttgcatccag ggtctcgtca 1620

acaccgagat gctgaagccc accgatgaga ttgtctccac ctaccaccgc cgcttcgacc 1680

acggctaccc caccccctcc ctggagcgtg agggtgctct tacccagatc ctgcccagac 1740

tccaggagaa ggacatctgg acccgtggtc gcttcggtag ctggcgctac gaggtcggta 1800

accaggacca ctctttcatg ctcggtgttg aggctgttga caacattgtc aacggcgctg 1860

tcgagctgac ccttaactac cctgacttcg tcaacggtag acagaacacc gagcgtcggc 1920

tggttgacgg cgcccaggtt ttcgccaaga gccaggcgca gtaa 1964

<210> 2

<211> 294

<212> DNA

<213> Aspergillus niger

<400> 2

tgaaaccgat tccaagcgcc ccccaccgct tgcttgtcac gtttcgcttt cccgaccgac 60

cgcaggacgg atacacctat accttcccgc ccatctcccc ttcttgctcc ctccatcacc 120

aacctctctt ctctctctct cttcctcccc tctcatctct ctccttataa ctctgctgtc 180

cctgcagctt cacaaggctc gttttctatg ctcagcctcg cccgcaggac tttgaaccgt 240

gtccccagct ttcaagatat tctacaaggc aggatgaccc accccgatat gtaa 294

<210> 3

<211> 547

<212> PRT

<213> Aspergillus niger

<400> 3

Met Leu Ser Leu Ala Arg Arg Thr Leu Asn Arg Val Pro Ser Phe Gln

1 5 10 15

Asp Ile Leu Gln Gly Arg Met Thr His Pro Asp Ile Ser Leu Glu Gln

20 25 30

Leu Leu Thr Val Val Phe Val Leu Glu Phe Ser Ser Val Asp Val Leu

35 40 45

Val Ile Gly Ala Gly Pro Thr Gly Leu Gly Ala Ala Lys Arg Leu Asn

50 55 60

Gln Ile Asp Gly Pro Ser Trp Leu Ile Val Asp Ser Asn Glu Thr Pro

65 70 75 80

Gly Gly Leu Ala Ser Thr Asp Val Thr Pro Glu Gly Phe Leu Phe Asp

85 90 95

Val Gly Gly His Val Ile Phe Ser His Tyr Lys Tyr Phe Asp Asp Cys

100 105 110

Ile Asn Glu Ala Leu Pro Lys Asp Asp Asp Trp Tyr Thr His Gln Arg

115 120 125

Ile Ser Tyr Val Arg Cys Gln Gly Gln Trp Val Pro Tyr Pro Phe Gln

130 135 140

Asn Asn Ile Ser Met Leu Pro Lys Asn Glu Gln Val Arg Cys Ile Asp

145 150 155 160

Gly Leu Ile Asp Ala Ala Leu Glu Ala Arg Val Ala Asn Thr Lys Pro

165 170 175

Gln Asn Phe Asp Glu Trp Ile Val Arg Gln Met Gly Val Gly Ile Ala

180 185 190

Asp Leu Phe Met Arg Pro Tyr Asn Phe Lys Val Trp Ala Val Pro Thr

195 200 205

Thr Lys Met Gln Cys Ala Trp Leu Gly Glu Arg Val Ala Ala Pro Asn

210 215 220

Val Lys Ala Val Thr Thr Asn Val Ile Leu Asn Lys Thr Ala Gly Asn

225 230 235 240

Trp Gly Pro Asn Ala Thr Phe Arg Phe Pro Ala Arg Gly Gly Thr Gly

245 250 255

Gly Ile Trp Ile Ala Val Ala Asp Thr Ile Pro Lys Glu Lys Thr Arg

260 265 270

Phe Gly Glu Lys Gly Lys Val Val Lys Val Asn Ala Asn Asn Lys Thr

275 280 285

Val Thr Leu Gly Asp Gly Thr Thr Val Gly Tyr Lys Lys Leu Val Ser

290 295 300

Thr Met Ala Val Asp Phe Leu Ala Glu Gln Ile Gly Asp Gln Glu Leu

305 310 315 320

Val Gly Leu Thr Lys Gln Leu Phe Tyr Ser Ser Thr His Val Ile Gly

325 330 335

Val Gly Ile Arg Gly Thr Arg Pro Glu Arg Ile Gly Asp Lys Cys Trp

340 345 350

Leu Tyr Phe Pro Glu Asp Asn Cys Pro Phe Tyr Arg Ala Thr Ile Phe

355 360 365

Ser Asn Tyr Ser Pro His Asn Gln Pro Glu Gly Ser Lys Lys Leu Pro

370 375 380

Thr Leu Gln Leu Ala Asp Gly Ser Lys Pro Gln Ser Thr Glu Ala Gln

385 390 395 400

Glu Gly Pro Tyr Trp Ser Ile Met Leu Glu Val Ser Glu Ser Ser Met

405 410 415

Lys Pro Val Asn His Glu Thr Leu Leu Ala Asp Cys Ile Gln Gly Leu

420 425 430

Val Asn Thr Glu Met Leu Lys Pro Thr Asp Glu Ile Val Ser Thr Tyr

435 440 445

His Arg Arg Phe Asp His Gly Tyr Pro Thr Pro Ser Leu Glu Arg Glu

450 455 460

Gly Ala Leu Thr Gln Ile Leu Pro Arg Leu Gln Glu Lys Asp Ile Trp

465 470 475 480

Thr Arg Gly Arg Phe Gly Ser Trp Arg Tyr Glu Val Gly Asn Gln Asp

485 490 495

His Ser Phe Met Leu Gly Val Glu Ala Val Asp Asn Ile Val Asn Gly

500 505 510

Ala Val Glu Leu Thr Leu Asn Tyr Pro Asp Phe Val Asn Gly Arg Gln

515 520 525

Asn Thr Glu Arg Arg Leu Val Asp Gly Ala Gln Val Phe Ala Lys Ser

530 535 540

Gln Ala Gln

545

<210> 4

<211> 3979

<212> DNA

<213> Aspergillus niger

<400> 4

atgctcagcc tcgcccgcag gactttgaac cgtgtcccca gctttcaaga tattctacaa 60

ggcaggatga cccaccccga tatgtaagga gacctttccc cctccctcaa acggcaagct 120

tgccttcact acaacaagaa gctcattgga acaattgctg accgttgtct ttgtgcttga 180

attcagctcc gtcgacgttc tcgtcattgg tgccggccct actggtctcg gtgccgcgaa 240

gcgtcttaac cagattgtac gcattgcgcg ccccctatcg tataccttca ctcgaataaa 300

ggctaattgg acaattcgcg ggtgtaggat ggcccttctt ggttgatcgt tgacagcaac 360

gagacccctg gtggtcttgc ttccaccgat gtgacccccg aaggcttcgt atgttgattc 420

ccactttccc ctcaatgggc aggcattgca ctggtcaagc aaacagcgac tgactcgtga 480

caatcacagc tcttcgatgt tggtggtcac gtcatcttct cccactacaa gtacttcgac 540

gactgcatca acgaggctct ccccaaggat gacgactggt acacccacca gcgtatctcc 600

tacgttcgct gccagggcca atgggttccc taccccttcc agaacaacat ctccatgctt 660

cccaagaatg agcaggtccg ctgtatcgat ggcctgatcg acgctgccct tgaggctcgt 720

gtcgccaaca ccaagcccca gaacttcgat gagtggattg ttcgccagat gggtgtcggt 780

atcgccgacc tcttcatgag accctacaac ttcaaggttt gggctgtgcc tacgaccaag 840

gtaagatacg atgtaccaca tggcgagtaa cggcgtgctg attctcatca gatgcaatgt 900

gcctggttgg gtgagcgtgt tgctgcccct aacgtcaagg ccgtgacgac caacgtcatc 960

cttaacaaga ccgctggtaa ctggggtcct aacgctactt tccgtttccc cgcccgtggt 1020

ggtaccggtg gtatctggat cgctgtcgcc gacactatcc ccaaggagaa gactcgcttc 1080

ggtgagaagg gcaaggtcgt caaggtcaat gccaacaaca agaccgtcac cctgggtgat 1140

ggcaccactg tcggctacaa gaagctcgtc tccaccatgg ctgtcgactt cctcgctgag 1200

cagatcggcg accaggagct cgttggcctc accaagcagc tcttctactc ctccactcac 1260

gtcattggtg tcggtatccg tggtacccgc cccgagagaa tcggtgacaa gtgctgggta 1320

agttgacctt ggatggaaac catgaatgac ttcaaaacta acatgctcgt tactagctct 1380

acttccccga ggacaactgc cccttctacc gtgccaccat cttctccaac tactcccccc 1440

acaaccagcc cgagggctcc aagaagcttc ccactctgca gcttgcggat ggctctaagc 1500

cccagagcac tgaggctcag gagggtcctt actggtccat catgttggag gtttccgagt 1560

cttcgatgaa gcctgtcaac cacgagactc tcctggctga ttgcatccag ggtctcgtca 1620

acaccgagat gctgaagccc accgatgaga ttgtctccac ctaccaccgc cgcttcgacc 1680

acggctaccc caccccctcc ctggagcgtg agggtgctct tacccagatc ctgcccagac 1740

tccaggagaa ggacatctgg acccgtggtc gcttcggtag ctggcgctac gaggtcggta 1800

accaggacca ctctttcatg ctcggtgttg aggctgttga caacattgtc aacggcgctg 1860

tcgagctgac ccttaactac cctgacttcg tcaacggtag acagaacacc gagcgtcggc 1920

tggttgacgg cgcccaggtt ttcgccaaga gccaggcgca gtaaagggtt gaaatgttaa 1980

tagaattaca cgggttttaa ttttttttctt gaatgattcc ggggggcaaa agatttgttt 2040

agagatctga ttacgatcaa cgggagccat atttcgtcat ttttttactt cttgtcctat 2100

ctatttaact tattttacca attttctttt tgctctttgg ctcattgaaa gcgatgtatg 2160

ggagatgttc ttggcatttt tgtgatgcgt atcatcatcc actttaccag ccgtcttttg 2220

ctccatctac tagacatgaa tagaagttgc ttatcaaacg cgtgtctttc tgttggggac 2280

ttaatttgct gatttacgta tgccggttac atgtctcgcc gcgaccccgt ggagatcgaa 2340

tccagcttca tgacagatga cacatgtatc acccaccgaa gtgaggcagt gtgtgcagca 2400

catcagccca tgcagggcgt ttctaaattc gctcacttct gtcccgatca agtatagcaa 2460

tgtgatatat agcagcatcg caacaactga gtatggagta tggagtatgc agaacggagt 2520

agtatggaat acatcaaacc cacacatatg gagtcatcta ctactacttc gtacttatgt 2580

acaccaatca tctagagaat aaatactact gcagtggtcc aatcggtatt ccgtgtaaga 2640

catactaccg tacgtagtga aacatgggat ctaccgttag ctattaatca atatctgaca 2700

tactatctat ctatcctacc gtgtgataac tccatattct cctcagtact aacccacatc 2760

ccccccatcc acaacccacc cacccgccgt ctcataaggc tgagtgagtg agctcgagct 2820

cgagctcgga gggtatggat gaagaagaga gtgcactgac tatgtcggaa attttcttta 2880

ctcattgttc tactactgta tatctgagta tgctagtaac tactagtagt aatcatgcaa 2940

gtatcccata gttgagcatg tgaattttat atcagtagta gtatcacagc agttactata 3000

tatgatcatg ctgcaggcat acttacatac atatattcac atgtacatac tatggggacc 3060

atcatgtgaa tactacttac atagatacta ctaagtacgt agtacttaac ttagcattcg 3120

cctccaatac tagagaccac cacgcctcag tagaagatag taatttaccg tgataatatt 3180

attaaccacc aaccttacta gtacttactt actatcctcc ccccggaaca tccattaccc 3240

gcaaccaacc atgccaagaa ggggaaccac cccacacacc taaagcggta tagcgcactg 3300

gggcaggacg cgtaagtaga actaaaccca agaaaggaaa aaaaaaaaaa gaatgaaaat 3360

tgggagggag agtcgtgcag aaaaatatgt atccactatt tatccagcat ccacaggcag 3420

aggaaaatta attcccaatg taaaaataga ttttgattat tagtagtagt ctagcccagg 3480

taggacaggt ggtcagaact atcttactct attatttttg cccctcatgg atggccgatg 3540

acgattaaat ggagtgtgat ggcgatgagg gtggtttgtt tcatggatag tagatactct 3600

gtagtactct aggattatgc tagtattgag gtgatactga ggtaataatg gtgatgtcta 3660

atttgttatt tgcagtaatg atagtatctt tcagggaaga gggataccta catacacatg 3720

gttacataac gcaagttggc agctttggat tccatccgct tcagccttat tgtcggtcgg 3780

ttttgctttc ttatttgagc tccccagtct acaccgtcac tcctctacat tacactacac 3840

tacaccatca ttactgaatt ttctgcagct tgcgtgtcga ctgggctcat tgggatggat 3900

gacgaatagc ctcacgagat ctcacccttc attgggtccg gacaccaact ccgcccccca 3960

tgctttacgt cccatccac 3979

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

acgcctcagt agaagatagt 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

gtggatggga cgtaaagcat 20

<210> 7

<211> 838

<212> DNA

<213> Aspergillus niger

<400> 7

acgcctcagt agaagatagt aatttaccgt gataatatta ttaaccacca accttactag 60

tacttactta ctatcctccc cccggaacat ccattacccg caaccaacca tgccaagaag 120

gggaaccacc ccacacacct aaagcggtat agcgcactgg ggcaggacgc gtaagtagaa 180

ctaaacccaa gaaaggaaaa aaaaaaaaag aatgaaaatt gggagggaga gtcgtgcaga 240

aaaatatgta tccactattt atccagcatc cacaggcaga ggaaaattaa ttcccaatgt 300

aaaaatagat tttgattatt agtagtagtc tagcccaggt aggacaggtg gtcagaacta 360

tcttactcta ttatttttgc ccctcatgga tggccgatga cgattaaatg gagtgtgatg 420

gcgatgaggg tggtttgttt catggatagt agatactctg tagtactcta ggattatgct 480

agtattgagg tgatactgag gtaataatgg tgatgtctaa tttgttattt gcagtaatga 540

tagtatcttt cagggaagag ggatacctac atacacatgg ttacataacg caagttggca 600

gctttggatt ccatccgctt cagccttatt gtcggtcggt tttgctttct tatttgagct 660

ccccagtcta caccgtcact cctctacatt acactacact acaccatcat tactgaattt 720

tctgcagctt gcgtgtcgac tgggctcatt gggatggatg acgaatagcc tcacgagatc 780

tcacccttca ttgggtccgg acaccaactc cgccccccat gctttacgtc ccatccac 838

Claims (10)

1. A DNA barcode for identifying a strain of Aspergillus niger, the DNA barcode being derived from the genome of Aspergillus niger TMCC 70012 strain, and comprising a sequence of at least 500bp selected from the DNA sequence shown in SEQ ID No.4, and the The length of the DNA barcode is 500bp-4000bp, preferably 500bp-900bp. 2 . The DNA barcode according to claim 1 , wherein the nucleotide sequence is shown in SEQ ID No. 1, SEQ ID No. 4 or SEQ ID No. 7. 3 . 3. A primer pair for amplifying the DNA barcode of claim 1 or 2. 4. primer pair according to claim 3, the nucleotide sequence of its forward primer is identical with such sequence in aspergillus niger TMCC 70012 strain genome: this sequence is from described TMCC 70012 strain genome as SEQ ID No. .4 a sequence in the region from position 1 of the nucleotide sequence shown in SEQ ID No. 4 to position 3510 of the nucleotide sequence shown in SEQ ID No. 4, and the length of the forward primer is 15-30 bp; its The reverse primer is reverse complementary to a sequence in the genome of the TMCC 70012 strain from position 471 of the nucleotide sequence shown in SEQ ID No. 4 to the nucleotide sequence shown in SEQ ID No. 4 in the genome of the TMCC 70012 strain The sequence in the region of the last digit of the nucleotide sequence shown in ID No. 4, and the length of the reverse primer is 15-30 bp. 5. The primer pair according to claim 4, the nucleotide sequences of the forward primer and the reverse primer are respectively as follows: Forward primer: 5'-ACGCCTCAGTAGAAGATAGT-3'; Reverse primer: 5'-CTTGCCATGGTGGTGGGATA-3'. 6. A kit for identifying a strain of Aspergillus niger, comprising a primer pair according to any one of claims 3-5. 7. A method for identifying Aspergillus niger bacterial strains, comprising the following steps: a) provide the genomic DNA of the strain to be tested; b) using the genomic DNA described in step a) as a template, using the primer pair according to any one of claims 3-5 to carry out PCR amplification to obtain a PCR product; c) Detect the PCR product by agarose gel electrophoresis, if there is no target band, then determine that the strain to be tested is not Aspergillus niger TMCC 70012 strain, if there is a target band, then proceed to step d); d) Sequence the obtained PCR product to obtain a nucleotide sequence to be tested; compare the sequence to be tested with the nucleotide sequence of the DNA barcode according to claim 1 or 2 for homology, if the homology If more than 97%, it is determined that the strain to be tested is Aspergillus niger TMCC 70012 strain. 8. The application of DNA barcoding according to claim 1 or 2 in identifying strains of Aspergillus niger. 9. The use of the primer pair according to any one of claims 3-5 in identifying Aspergillus niger strains. 10. The application of the test kit according to claim 6 in identifying Aspergillus niger strains.

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