CN112695120A - Primer, kit and method for rapid identification and quantification of saccharomyces cerevisiae - Google Patents
Primer, kit and method for rapid identification and quantification of saccharomyces cerevisiae Download PDFInfo
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Abstract
The invention discloses a primer, a kit and a method for rapid identification and quantification of saccharomyces cerevisiae, belonging to the technical field of biological engineering. The invention designs specific primers aiming at the saccharomyces cerevisiae alcohol dehydrogenase ADH5 gene, can realize the rapid qualitative detection of the saccharomyces cerevisiae in samples such as Daqu, fermented grains and the like by a PCR technology, and can determine the content of the saccharomyces cerevisiae in the samples by fluorescent quantitative PCR; the method has the characteristics of high accuracy, good effectiveness and high sensitivity, and is suitable for identification and quantitative detection of the saccharomyces cerevisiae in fermented food or samples in the fermentation process.
Description
Technical Field
The invention relates to a primer, a kit and a method for rapid identification and quantification of saccharomyces cerevisiae, belonging to the technical field of biological engineering.
Background
At present, most famous Chinese white spirits are brewed by using a traditional Daqu method. The Daqu is prepared from barley, wheat, pea, etc. by crushing, adding water, stirring, pressing into brick-shaped fermented grains, and culturing at artificially controlled temperature and humidity. The yeast contains abundant microorganisms such as mould, yeast and bacteria and various enzymes produced by the microorganisms, and is a mixed crude enzyme preparation of multiple strains, wherein the action of fungi is not negligible. Because the Daqu inhabits abundant microorganisms, the Daqu not only directly transfers a large amount of beneficial brewing microorganism strains to fermented grains, but also provides various enzymes mainly comprising amylase and protease for the fermented grains, and simultaneously, the Daqu also brings considerable and various metabolites for the fermented grains, thereby playing an important role in endowing the Daqu with better taste and aroma.
Saccharomyces cerevisiae (also known as baker's yeast or brewer's yeast) is an important eukaryotic microorganism in cell biology, widely used in liquor brewing, and is a core yeast in liquor brewing systems. They are mainly involved in alcoholic fermentation, the action of which is to convert sugars into alcohol. The saccharomyces cerevisiae has the advantages of fast fermentation, high stability of finished wine and consistent quality. The existing method for identifying the saccharomyces cerevisiae mainly utilizes the physicochemical property of a strain or ITS4, and the identification method has the problems of long period, high cost, low success rate and the like.
In a white spirit fermentation system, Alcohol Dehydrogenase (ADH) plays an important role. Alcohol dehydrogenases are a class of oxidoreductases with highly conserved domains, typically a zinc-binding enzyme with a zinc-binding domain in the domain. The enzyme acts as a dimer and is dependent on NAD (P)+The cofactor can reversibly convert ethanol and acetaldehyde. The essential function of alcohol dehydrogenase is to convert acetaldehyde to ethanol in the last step of glycolysis during anaerobic respiration. The reaction can effectively reduce the toxic effect of the metabolite generated by anaerobic respiration on the cells. In addition, alcohol dehydrogenases catalyze the formation of ethanol from acetaldehydeThe process is also accompanied by the production of the energy molecule ATP. Production of NAD in NADH+Will be accompanied by the generation of an H+This energy is eventually converted to energy stored in high energy phosphate bonds in ATP via the electron transport chain. The alcohol dehydrogenase is believed to be primarily responsible for the regeneration of NAD from NADH by catalyzing the reduction of acetaldehyde to produce ethanol when Saccharomyces cerevisiae is grown on a fermentable carbon source such as glucose+. In addition, the gene sequences encoding alcohol dehydrogenase are different among different yeasts.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problems that the existing method for identifying the saccharomyces cerevisiae mainly utilizes the physicochemical properties of a strain or ITS4, and the identification method has the problems of long period, high cost, low success rate and the like.
[ solution ]
The invention provides a primer for identifying Saccharomyces cerevisiae (Saccharomyces cerevisiae), the sequence of which is shown as SEQ ID NO: 2 (primer ADH5F), SEQ ID NO: 3 (primer ADH 5R).
The invention also provides a method for identifying saccharomyces cerevisiae by using the primer, which comprises the following steps: preparing a sample to be detected into bacterial suspension, diluting and coating the bacterial suspension to obtain a single colony, and performing sequencing on the single colony by using SEQ ID NO: 2. SEQ ID NO: 3 is a primer, colony PCR is carried out, a PCR product is observed through gel electrophoresis, if the PCR product on an electrophoretogram has a band between 100-250bp, the sample contains the saccharomyces cerevisiae, and if the PCR product is not between 100-250bp or has no band, the sample does not contain the saccharomyces cerevisiae.
In one embodiment, the sample contains a thallus, genome, or metagenome. Optionally, the sample is a fermented food finished product or a sample obtained in a fermented food fermentation process, or an environmental sample such as intestinal tract, soil, water body and the like; optionally, the sample to be tested is subjected to pretreatment such as centrifugation and collection of bacteria, and then subsequent measurement is performed. Optionally, the sample is fermented food or a sample obtained in a fermentation process of the fermented food, and the fermented food is any one or more of the following: white spirit, yellow wine, soy sauce, beer, wine, table vinegar, fermented tea, traditional fermented vegetables, fermented beverages, alcoholic drinks, yogurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd, fermented rice and flour foods and the like. Optionally, the sample comprises Daqu or fermented grains.
In one embodiment, the PCR product between 100-250bp can also be sequenced, and the sequencing result is aligned with the sequence of the alcohol dehydrogenase encoding gene of Saccharomyces cerevisiae.
The invention also provides a method for quantitatively detecting the saccharomyces cerevisiae by applying the primer, which comprises the following steps:
(1) preparing saccharomyces cerevisiae suspensions with different bacterial concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions;
(2) using SEQ ID NO: 2. SEQ ID NO: 3, respectively carrying out fluorescence quantitative PCR on the genome by using the primers shown in the specification, and drawing a standard curve by using a CT value as a horizontal coordinate and using the cell number or the Lg value of the cell number as a vertical coordinate;
(3) extracting a genome in a sample to be detected, and performing amplification reaction by using a nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3, carrying out fluorescence quantitative PCR on the genome of the sample to be detected by the primers shown in the specification, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the number of the saccharomyces cerevisiae in the sample.
The present invention provides a method for differentiating saccharomyces cerevisiae from Pichia kudriavzevii (Pichia kudriavzevii), Schizosaccharomyces pombe (Schizosaccharomyces pombe), saccharomyces bailii (Zygosaccharomyces bailii), Candida albicans (Candida humulis), and saccharomyces pastorianus (Kazachstania barnetii), wherein the sequence of the method is represented by SEQ ID NO: 2. SEQ ID NO: 3 as a primer, performing colony PCR or performing PCR by respectively using the genome of the yeast as a template, observing a PCR product through gel electrophoresis, and if the PCR product on the electrophoresis map has a band between 100-250bp, indicating that the corresponding strain is the saccharomyces cerevisiae.
The invention provides a qualitative and quantitative detection kit for saccharomyces cerevisiae, which contains SEQ ID NO: 2. SEQ ID NO: 3, and further comprises DNA polymerase and a buffer solution.
The method for qualitatively detecting the saccharomyces cerevisiae by using the kit comprises the following steps: preparing a sample to be detected into bacterial suspension, diluting and coating the bacterial suspension to obtain a single colony, and performing sequencing on the single colony by using SEQ ID NO: 2. SEQ ID NO: 3 as a primer, observing the PCR product through gel electrophoresis, and if the PCR product has a band between 100-250bp on an electrophoretogram, indicating that the sample contains the Saccharomyces cerevisiae (Saccharomyces cerevisiae).
The method for quantitatively detecting the saccharomyces cerevisiae by using the kit comprises the following steps: (1) preparing saccharomyces cerevisiae suspensions with different bacterial concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions; (2) using SEQ ID NO: 2. SEQ ID NO: 3, respectively carrying out fluorescence quantitative PCR on the genome by using the primers shown in the specification, and drawing a standard curve by using a CT value as a horizontal coordinate and using the cell number or the Lg value of the cell number as a vertical coordinate; (3) extracting a genome in a sample to be detected, and performing amplification reaction by using a nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3, carrying out fluorescence quantitative PCR on the genome of the sample to be detected by the primers shown in the specification, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the number of the saccharomyces cerevisiae in the sample.
The sample contains a thallus, genome or metagenome. Optionally, the sample is a fermented food finished product or a sample obtained in a fermented food fermentation process, or an environmental sample such as intestinal tract, soil, water body and the like; optionally, the sample to be tested is subjected to pretreatment such as centrifugation and collection of bacteria, and then subsequent measurement is performed. Optionally, the sample is fermented food or a sample obtained in a fermentation process of the fermented food, and the fermented food is any one or more of the following: white spirit, yellow wine, soy sauce, beer, wine, table vinegar, fermented tea, traditional fermented vegetables, fermented beverages, alcoholic drinks, yogurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd, fermented rice and flour foods and the like. Optionally, the sample comprises Daqu or fermented grains.
[ advantageous effects ]
The invention designs specific primers aiming at the saccharomyces cerevisiae alcohol dehydrogenase ADH5 gene, and can realize the rapid qualitative and quantitative detection of the saccharomyces cerevisiae in samples such as Daqu, fermented grains and the like by a PCR technology; the method is suitable for identifying and quantifying the saccharomyces cerevisiae in finished fermented food products or samples taken from the fermentation process of the fermented food, or environmental samples such as intestinal tracts, soil, water bodies and the like.
The invention can distinguish the saccharomyces cerevisiae from Pichia kudriavzevii (Pichia kudriavzevii), Schizosaccharomyces Pombe (SP), saccharomyces bailii (Zygosaccharomyces pombe), Candida albicans (Candida humilis) and saccharomyces pastoria barnetii) by using specific primers, and the specific primers only specifically amplify the alcohol dehydrogenase ADH4 of the saccharomyces cerevisiae without amplifying gene fragments of other 5 yeasts.
Drawings
FIG. 1: and identifying the result of agarose gel electrophoresis of different primer pairs, wherein the ratio of 1: for the specific primers ADH5F/ADH5R, 2, 3, 4: pairs 2, 3, 4 designed for reference to a similar method.
FIG. 2: and (3) identifying the result of agarose gel electrophoresis of different yeasts, wherein the ratio of SC: saccharomyces cerevisiae (Saccharomyces cerevisiae), SP: schizosaccharomyces pombe (Schizosaccharomyces pombe), PK: pichia kudriavzevii (Pichia kudriavzevii), ZB: bayer associated yeast (Zygosaccharomyces bailii), CH: candida albicans (Candida humilis), KB: pasteurella pasteurella saxophone (Kazachstania barnettii), dd H2O: and (5) redistilling the water.
FIG. 3: and (3) verifying the saccharomyces cerevisiae ethanol dehydrogenase gene.
FIG. 4: and (3) identifying the agarose gel electrophoresis result of different strains of saccharomyces cerevisiae, wherein the agarose gel electrophoresis result of the different strains of saccharomyces cerevisiae comprises the following steps of C1: saccharomyces cerevisiae YJM143, C2: saccharomyces cerevisiae YJM1389, C3: saccharomyces cerevisiae YJM1083, C4: saccharomyces cerevisiae SK23 and C5: saccharomyces cerevisiae KSD-Yc.
FIG. 5: standard curve
Detailed Description
The invention is further illustrated with reference to specific examples.
The media involved in the following examples are as follows:
YPD liquid medium: 10g/L of yeast extract, 20g/L of tryptone and 20g/L of glucose.
YPD solid Medium: 10g/L of yeast extract, 20g/L of tryptone, 20g/L of glucose and 20g/L of agar.
Example 1: design of specific primer of Saccharomyces cerevisiae
The design method of the specific primer pair comprises the following steps:
saccharomyces cerevisiae (Saccharomyces cerevisiae) is a core yeast in a liquor brewing system, and specific enzyme of the Saccharomyces cerevisiae, namely Saccharomyces cerevisiae alcohol dehydrogenase (Saccharomyces cerevisiae alcohol dehydrogenase ADH5), is searched through Kyoto gene and genome encyclopedia metabolic Pathway (KEGG Pathway), so that a 1056bp nucleotide sequence for coding the specific enzyme is obtained, and the sequence is as follows:
ATGCCTTCGCAAGTCATTCCTGAAAAACAAAAGGCTATTGTCTTTTATGAGACAGA TGGAAAATTGGAATATAAAGACGTCACAGTTCCGGAACCTAAGCCTAACGAAATTTTA GTCCACGTTAAATATTCTGGTGTTTGTCATAGTGACTTGCACGCGTGGCACGGTGATTG GCCATTTCAATTGAAATTTCCATTAATCGGTGGTCACGAAGGTGCTGGTGTTGTTGTTA AGTTGGGATCTAACGTTAAGGGCTGGAAAGTCGGTGATTTTGCAGGTATAAAATGGTT GAATGGGACTTGCATGTCCTGTGAATATTGTGAAGTAGGTAATGAATCTCAATGTCCTT ATTTGGATGGTACTGGCTTCACACATGATGGTACTTTTCAAGAATACGCAACTGCCGAT GCCGTTCAAGCTGCCCATATTCCACCAAACGTCAATCTTGCTGAAGTTGCCCCAATCTT GTGTGCAGGTATCACTGTTTATAAGGCGTTGAAAAGAGCCAATGTGATACCAGGCCAA TGGGTCACTATATCCGGTGCATGCGGTGGCTTGGGTTCTCTGGCAATCCAATACGCCCT TGCTATGGGTTACAGGGTCATTGGTATCGATGGTGGTAATGCCAAGCGAAAGTTATTTG AACAATTAGGCGGAGAAATATTCATCGATTTCACGGAAGAAAAAGACATTGTTGGTGC TATAATAAAGGCCACTAATGGCGGTTCTCATGGAGTTATTAATGTGTCTGTTTCTGAAG CAGCTATCGAGGCTTCTACGAGGTATTGTAGGCCCAATGGTACTGTCGTCCTGGTTGGT ATGCCAGCTCATGCTTACTGCAATTCCGATGTTTTCAATCAAGTTGTAAAATCAATCTC CATCGTTGGATCTTGTGTTGGAAATAGAGCTGATACAAGGGAGGCTTTAGATTTCTTCG CCAGAGGTTTGATCAAATCTCCGATCCACTTAGCTGGCCTATCGGATGTTCCTGAAATT TTTGCAAAGATGGAGAAGGGTGAAATTGTTGGTAGATATGTTGTTGAGACTTCTAAAT GA。
the nucleotide sequence is aligned by the National Center for Biotechnology Information (NCBI) of the United states using the local alignment search tool (BLAST), and the alignment result shows that the nucleotide sequence has 100% similarity with the Saccharomyces cerevisiae genome alcohol dehydrogenase (ADH5) sequence only, which indicates that the 173bp nucleotide sequence corresponding to the alcohol dehydrogenase (ADH5) is the highly specific sequence of the Saccharomyces cerevisiae.
Setting Primer Parameters (Primer Parameters) using the NCBI Primer-BLAST tool; the length (PCR product size) of a Polymerase Chain Reaction (PCR) product is 80-300; primer Pair Specificity Checking Parameters (Primer Pair Specificity Checking Parameters) were set: database (Database) nr; setting other parameters according to defaults; from the pair primer pairs obtained, DNMAN software was used to select an upstream primer ADH5F-TGTCGTCCTGGTTGGTATGC and a downstream primer ADH5R-GCCAGCTAAGTGGATCGGAG, which were not capable of self-looping (self-completion), as potential target primer pairs, and the PCR product length was 173 bp. BLAST was performed in NCBI using the potential target primer pair as the upstream and downstream primers, and the alignment result was only 100% similar to Saccharomyces cerevisiae, indicating that the selected primer pair potential target primer pair is a specific primer pair suitable for this environment.
Example 2: screening of Saccharomyces cerevisiae specific primers
Firstly, screening core yeast in the fermentation process of the Maotai-flavor liquor, weighing 10g of fermented grain sample in a sterilized 250mL triangular flask filled with 100mL sterile PBS buffer solution and 3g glass beads, then placing the triangular flask in a shaking table at 30 ℃ and 200r/min to uniformly mix for 30min, and standing for 5min to obtain bacterial suspension. Diluting the bacterial suspension, and taking 10-3、10-4And 10-5The sample bacterial suspensions of the three dilutions were spread on YPD medium plates and cultured in an incubator at 30 ℃ for 72h, and single colonies on the plates were picked and numbered as core yeast-like strains.
Then, preparing a slant culture medium by using sorghum lixivium for producing the Maotai-flavor liquor, separating and purifying the screened core yeast similar strains, transferring the strains to a test tube slant, culturing at 30 ℃, and storing at 4 ℃ after the culture is finished.
Finally, the genome was extracted and the universal forward primer ITS1 for eukaryotic microorganisms was used
(5'-TCCGTAGGTGAACCTGCGG-3') and the reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used to PCR amplify the genome to identify the strain. PCR reaction (50. mu.L): taq DNA Polymerase (5U/. mu.L) 0.5. mu.L, dNTP Mix (10mmol/L) 1. mu.L, upstream and downstream primers (10. mu. mol/L) each 2. mu.L, 10 XTaq Buffer (M g2+ plus) 5. mu.L, genomic template DNA 10ng, and ultrapure water to 50. mu.L. And (3) PCR reaction conditions: 3min at 95 ℃; 95 ℃ for 15s, 55 ℃ for 30s and 72 ℃ for 45s, and 30 cycles of 72 ℃ for 10 min. And comparing the sequence obtained by PCR amplification with an ITS amplicon sequence of a model strain in an NCBI website system to obtain specific information of a target strain, and finally obtaining the yeast Saccharomyces cerevisiae.
The invention adopts a method similar to that of the embodiment 1, designs other three pairs of primers, and takes the genome of the extracted Saccharomyces Cerevisiae (SC) as a template and respectively takes the four pairs of primers (ADH5F/ADH5R, primer pair 2, primer pair 3 and primer pair 4) to carry out PCR amplification (the PCR system is shown in Table 1). Reaction parameters are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 30s, 25 cycles, extension at 72 ℃ for 30s, and identification of the product by 2% agarose gel electrophoresis. The results (as shown in FIG. 1) indicate that the amplification effect of the other primers is significantly less than that of the primer pair of example 1.
The primer information is as follows:
and (3) primer pair 2:
F2：CGTCAAACGGTTTTGGCTTG
R2：AGAACAGCATAGGGCTCGTA
and (3) primer pair:
F3：TTCCAGCGGCTGTTATCGAG
R3：CTGGCAGACCAACTAGCACA
and (3) primer pair 4:
F4：CGCTGGTGGTCTAGGTTCTC
R4：CCAAACCGTTAGTTGCAGCC
TABLE 1 PCR verification System
| Reaction mixture | Volume(μL) |
| Mixture | 10 |
| Upstream primer | 1 |
| Downstream primer | 1 |
| Template DNA | 1 |
| dd H2O | 7 |
| |
20 |
Example 3: verification of specific primer pair of Saccharomyces cerevisiae
Verification of specific primer pairs:
the extracted genome of the Saccharomyces Cerevisiae (SC) is taken as a template, and the specific primer is
ADH5F-TGTCGTCCTGGTTGGTATGC, ADH5R-GCCAGCTAAGTGGATCGGAG as primers, PCR was performed, and negative controls were core yeast Saccharomyces cerevisiae in the white spirit brewing system: pichia Kudriavzevii (PK), Schizosaccharomyces Pombe (SP), Saccharomyces bailii (Zygosaccharomyces bailii, ZB), Candida albicans (Candida humilis, CH), Saccharomyces pastorianus (Kazachstania barnetii, KB) and redistilled water (dd H2O). The PCR system and PCR conditions were the same as in example 2. After the PCR is finished, performing gel electrophoresis on the product, observing a band on the gel through a gel imager to confirm the specificity condition of the primer,the designed specific primer pair has specificity to the Saccharomyces cerevisiae if the Saccharomyces cerevisiae has an obvious target band near 100-250bp and the rest negative controls have no band (as shown in figure 2). After sequencing the PCR product of Saccharomyces cerevisiae by Jinzhi corporation, blast validation of NCBI website was performed to confirm that it was the alcohol dehydrogenase gene on Saccharomyces cerevisiae (as shown in FIG. 3).
Example 4: test on detection effectiveness and accuracy of primer pair ADH5F/ADH5R
Five different strains of Saccharomyces cerevisiae YJM1434, Saccharomyces cerevisiae YJM1389, Saccharomyces cerevisiae YJM1083, Saccharomyces cerevisiae SK23 and Saccharomyces cerevisiae KSD-Yc are respectively numbered C1, C2, C3, C4 and C5, and the respective genomes of the 5 strains of Saccharomyces cerevisiae are separately extracted. PCR was performed on each genome using the above-mentioned specific primer ADH5F-TGTCGTCCTGGTTGGTATGC, ADH5R-GCCAGCTAAGTGGATCGGAG as a primer, and the negative control was redistilled water (dd H)2O), and then detecting by electrophoresis. As a result, it was found that: the PCR product has a band (shown in figure 4) in a range of about 100-250bp, which indicates that the primer can be used for amplifying the ethanol dehydrogenase gene of the saccharomyces cerevisiae, so that the saccharomyces cerevisiae in the sample can be rapidly identified.
Example 5: quantification of alcohol dehydrogenase Gene in sample
Yeast (Saccharomyces cerevisiae) obtained in example 2 was checked to have a cell number of 1.1X10 by a hemacytometer method8cells/g, which were diluted to 10cells7、106、105、104、103、10210cells/g, respectively extracting the genome, and performing fluorescent quantitative PCR (the fluorescent quantitative PCR system is shown in table 2) by using the specific primers ADH5F-TGTCGTCCTGGTTGGTATGC, ADH5R-GCCAGCTAAGTGGATCGGAG as primers and a StepOnePlus instrument (purchased from Saimer fly); the CT value measured by qPCR is used as the abscissa, and the cell value corresponding to the dilution concentration is used as the ordinate, and a standard curve is drawn (see FIG. 5 for the standard curve).
TABLE 2 qPCR validation System
Example 6: application of quantification of alcohol dehydrogenase gene in sample
Weighing 10g of Daqu sample in a sterilized 250mL triangular flask filled with 100mL sterile PBS buffer solution and 3g glass beads, placing the flask in a shaking table at 30 ℃ and 200r/min, shaking and mixing for 30min, and standing for 5min to obtain a bacterial suspension. Diluting the bacterial suspension, and taking 10-3、10-4And 10-5The sample bacterial suspension with 3 dilutions was spread on YPD medium plates, cultured in an incubator at 30 ℃ for 72 hours, individual colonies on the plates were picked and numbered, and colony PCR was performed using specific primers ADH5F and ADH 5R. The experimental result shows that the PCR product has a band between 100 bp and 250bp, which indicates that the yeast sample contains Saccharomyces cerevisiae.
Obtaining genome by using a 'TIANGEN Plant Genomic DNA Kit' Kit, carrying out fluorescent quantitative PCR by using the obtained genome as a template and the specific primer ADH5F-TGTCGTCCTGGTTGGTATGC, ADH5R-GCCAGCTAAGTGGATCGGAG as a primer, wherein the PCR reaction conditions are the same as those of example 5, substituting the obtained CT value 27.08 into the standard curve shown in the figure 5, and finally obtaining the content of the saccharomyces cerevisiae in the Daqu sample as follows: 102cells/g。
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> primer, kit and method for rapid identification and quantification of saccharomyces cerevisiae
<130> BAA201564A
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 1056
<212> DNA
<213> Saccharomyces cerevisiae
<400> 1
atgccttcgc aagtcattcc tgaaaaacaa aaggctattg tcttttatga gacagatgga 60
aaattggaat ataaagacgt cacagttccg gaacctaagc ctaacgaaat tttagtccac 120
gttaaatatt ctggtgtttg tcatagtgac ttgcacgcgt ggcacggtga ttggccattt 180
caattgaaat ttccattaat cggtggtcac gaaggtgctg gtgttgttgt taagttggga 240
tctaacgtta agggctggaa agtcggtgat tttgcaggta taaaatggtt gaatgggact 300
tgcatgtcct gtgaatattg tgaagtaggt aatgaatctc aatgtcctta tttggatggt 360
actggcttca cacatgatgg tacttttcaa gaatacgcaa ctgccgatgc cgttcaagct 420
gcccatattc caccaaacgt caatcttgct gaagttgccc caatcttgtg tgcaggtatc 480
actgtttata aggcgttgaa aagagccaat gtgataccag gccaatgggt cactatatcc 540
ggtgcatgcg gtggcttggg ttctctggca atccaatacg cccttgctat gggttacagg 600
gtcattggta tcgatggtgg taatgccaag cgaaagttat ttgaacaatt aggcggagaa 660
atattcatcg atttcacgga agaaaaagac attgttggtg ctataataaa ggccactaat 720
ggcggttctc atggagttat taatgtgtct gtttctgaag cagctatcga ggcttctacg 780
aggtattgta ggcccaatgg tactgtcgtc ctggttggta tgccagctca tgcttactgc 840
aattccgatg ttttcaatca agttgtaaaa tcaatctcca tcgttggatc ttgtgttgga 900
aatagagctg atacaaggga ggctttagat ttcttcgcca gaggtttgat caaatctccg 960
atccacttag ctggcctatc ggatgttcct gaaatttttg caaagatgga gaagggtgaa 1020
attgttggta gatatgttgt tgagacttct aaatga 1056
<210> 2
<211> 20
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<213> Artificial sequence
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<212> DNA
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tccgtaggtg aacctgcgg 19
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<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
agaacagcat agggctcgta 20
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cgctggtggt ctaggttctc 20
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Claims (10)
1. A primer for identifying Saccharomyces cerevisiae (Saccharomyces cerevisiae) is characterized in that the sequence of the primer is shown as SEQ ID NO: 2. SEQ ID NO: 3, respectively.
2. A method for identifying Saccharomyces cerevisiae (Saccharomyces cerevisiae) using the primer of claim 1, comprising the steps of: preparing a sample to be detected into bacterial suspension, diluting and coating the bacterial suspension to obtain a single colony, and performing sequencing on the single colony by using SEQ ID NO: 2. SEQ ID NO: 3 is a primer, colony PCR is carried out, a PCR product is observed through gel electrophoresis, if the PCR product on an electrophoretogram has a band between 100-250bp, the sample contains the saccharomyces cerevisiae, and if the PCR product is not between 100-250bp or has no band, the sample does not contain the saccharomyces cerevisiae.
3. The method of claim 2, wherein the sample is a finished fermented food or a sample obtained from a fermentation process of a fermented food, and comprises Daqu or fermented grains.
4. The method according to claim 2 or 3, wherein the sample contains Pichia kudriavzevii (Pichia kudriavzevii), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Saccharomyces bailii (Zygosaccharomyces bailii), Candida albicans (Candida hugilis) and Saccharomyces pastorianus (Kazachstania barnetii).
5. The method as claimed in claim 2, wherein the PCR product of between 100 and 250bp is also sequenced and the sequencing result is aligned with the sequence of the alcohol dehydrogenase encoding gene of Schizosaccharomyces pombe.
6. The method for quantitatively detecting Saccharomyces cerevisiae (Saccharomyces cerevisiae) by using the primer as claimed in claim 1, which comprises the steps of:
(1) preparing saccharomyces cerevisiae suspensions with different bacterial concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions;
(2) using SEQ ID NO: 2. SEQ ID NO: 3, respectively carrying out fluorescence quantitative PCR on the genome by using the primers shown in the specification, and drawing a standard curve by using a CT value as a horizontal coordinate and using the cell number or the Lg value of the cell number as a vertical coordinate;
(3) extracting a genome in a sample to be detected, and performing amplification reaction by using a nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3, carrying out fluorescence quantitative PCR on the genome of the sample to be detected by the primers shown in the specification, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the number of the saccharomyces cerevisiae in the sample.
7. A method for differentiating Saccharomyces cerevisiae (Saccharomyces cerevisiae) from Pichia kudriavzevii (Pichia kudriavzevii), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Saccharomyces bailii (Zygosaccharomyces bailii), Candida albicans (Candida humulis), and Saccharomyces pastoria (Kazachstania barnetii), characterized in that the expression of the amino acid sequence of Saccharomyces cerevisiae (Saccharomyces cerevisiae) is expressed as SEQ ID NO: 2. SEQ ID NO: 3 as a primer, performing colony PCR or performing PCR by respectively using the genome of the yeast as a template, observing a PCR product through gel electrophoresis, and if the PCR product on the electrophoresis map has a band between 100-250bp, indicating that the corresponding strain is the saccharomyces cerevisiae.
8. A qualitative and quantitative detection kit for Saccharomyces cerevisiae (Saccharomyces cerevisiae) is characterized by comprising the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3, and further comprises DNA polymerase and a buffer solution.
9. A method for qualitative detection of Saccharomyces cerevisiae using the kit of claim 8, comprising: preparing a sample to be detected into bacterial suspension, diluting and coating the bacterial suspension to obtain a single colony, and performing sequencing on the single colony by using SEQ ID NO: 2. SEQ ID NO: 3 is used as a primer to carry out colony PCR, PCR products are observed through gel electrophoresis, and if the PCR products on an electrophoretogram have bands between 100-250bp, the sample contains the saccharomyces cerevisiae.
10. A method for quantitative detection of Saccharomyces cerevisiae using the kit of claim 8, comprising: (1) preparing saccharomyces cerevisiae suspensions with different bacterial concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions; (2) using SEQ ID NO: 2. SEQ ID NO: 3, respectively carrying out fluorescence quantitative PCR on the genome by using the primers shown in the specification, and drawing a standard curve by using a CT value as a horizontal coordinate and using the cell number or the Lg value of the cell number as a vertical coordinate; (3) extracting a genome in a sample to be detected, and performing amplification reaction by using a nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 3, carrying out fluorescence quantitative PCR on the genome of the sample to be detected by the primers shown in the specification, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the number of the saccharomyces cerevisiae in the sample.
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|---|---|---|---|---|
| CN113637788A (en) * | 2021-07-30 | 2021-11-12 | 贵州茅台酒股份有限公司 | Method for absolute quantification of fungi in sample by constructing recombinant plasmid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296117A (en) * | 2011-09-06 | 2011-12-28 | 北京大北农科技集团股份有限公司 | Rapid qualification and quantitation measurement method of saccharomyces cerevisiae in additive premix sample |
| CN107523625A (en) * | 2017-09-20 | 2017-12-29 | 贵州茅台酒股份有限公司 | The quantitative analysis method of producing and ethanol and lactic acid producing key microorganisms in more micro- solid state fermentation |
-
2021
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296117A (en) * | 2011-09-06 | 2011-12-28 | 北京大北农科技集团股份有限公司 | Rapid qualification and quantitation measurement method of saccharomyces cerevisiae in additive premix sample |
| CN107523625A (en) * | 2017-09-20 | 2017-12-29 | 贵州茅台酒股份有限公司 | The quantitative analysis method of producing and ethanol and lactic acid producing key microorganisms in more micro- solid state fermentation |
Non-Patent Citations (4)
| Title |
|---|
| CLAUDIA E. THOMPSON等: "Molecular evolution and functional divergence of alcohol dehydrogenases in animals, fungi and plants", 《GENET. MOL. BIOL》 * |
| DE SMIDT O等: "The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review", 《FEMS YEAST RES》 * |
| QIAN CHEN等: "Transcriptional analysis of Saccharomyces cerevisiae during high-temperature fermentation", 《ANNALS OF MICROBIOLOGY》 * |
| ZHENGUO LIN等: "Comparative Genomics and Evolutionary Genetics of Yeast Carbon Metabolism", 《MOLECULAR MECHANISMS IN YEAST CARBON METABOLISM》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113637788A (en) * | 2021-07-30 | 2021-11-12 | 贵州茅台酒股份有限公司 | Method for absolute quantification of fungi in sample by constructing recombinant plasmid |
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