CN112725496B - Method for rapidly identifying and quantifying schizosaccharomyces pombe in sample - Google Patents
Method for rapidly identifying and quantifying schizosaccharomyces pombe in sample Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a method for rapidly identifying and quantifying schizosaccharomyces pombe in a sample, belonging to the technical field of bioengineering. The specific primers SEQ ID NO. 2 and SEQ ID NO. 3 are designed aiming at the schizosaccharomyces pombe alcohol dehydrogenase gene Adh4, and the rapid qualitative and quantitative detection of the schizosaccharomyces pombe in a fermentation system in samples such as Daqu, fermented grains and the like can be realized by a PCR technology; high accuracy, good effectiveness and high sensitivity. The technical scheme of the invention is applicable to the finished fermented food or the sample obtained from the fermentation process of the fermented food.
Description
Technical Field
The invention relates to a method for rapidly identifying and quantifying schizosaccharomyces pombe in a sample, belonging to the technical field of bioengineering.
Background
At present, most of Chinese famous and excellent white spirits are brewed by using a traditional Daqu method. The Daqu is prepared from barley, wheat, pea, etc. by crushing, adding water, mixing, pressing into brick-shaped fermented grains, and culturing at artificially controlled temperature and humidity. The Daqu contains rich microorganisms such as mould, yeast, bacteria and the like and various enzymes produced by the microorganisms, and is a mixed crude enzyme preparation of multiple strains, wherein the action of the fungi cannot be ignored. As the yeast harbours abundant microorganisms, a great amount of beneficial brewing microorganism strains are directly transferred to fermented grains by adding the yeast, various enzymes mainly comprising amylase and protease are provided for the fermented grains, and meanwhile, the yeast harbour considerable and various metabolites are provided for the fermented grains, thereby playing an important role in endowing the yeast liquor with better taste and aroma.
The schizosaccharomyces pombe (Schizosaccharomyces pombe) is a rod-shaped yeast of schizosaccharomyces, is widely applied to white wine brewing, is a core yeast in a white wine brewing system, has higher ethanol fermentation capability, can reduce the concentration of the carbamate which affects the quality safety of products, and is non-saccharomyces cerevisiae with great brewing potential. Screening, identifying and analyzing schizosaccharomyces pombe has important significance for improving the quality of white spirit. The existing method for identifying schizosaccharomyces pombe mainly utilizes physicochemical properties of strains or ITS4, and the identification method has the problems of long period, high cost, low success rate and the like.
Alcohol dehydrogenase (Alcohol dehydrogenase, ADH for short) is a class of oxidoreductases with highly conserved domains, and a typical alcohol dehydrogenase is a zinc-binding enzyme with a zinc-binding domain in its domain. The enzyme acts as a dimer and relies on NAD (P) + cofactors to reversibly convert ethanol and acetaldehyde. The basic function of alcohol dehydrogenases is to convert acetaldehyde to ethanol in the last step of glycolysis in anaerobic respiration. The reaction can effectively reduce the toxic effect of metabolic products generated by anaerobic respiration on cells. In addition, the process of ethanol dehydrogenase catalyzing acetaldehyde to produce ethanol is accompanied by the production of energy molecule ATP. NAD production at NADH + Is accompanied by generation of an H + Through electron transfer chains, this energy is finally converted into high energy in ATPThe energy stored in the phosphate bond. When Saccharomyces cerevisiae is grown on fermentable carbon sources such as glucose, alcohol dehydrogenase is believed to be primarily responsible for regenerating NAD from NADH by catalyzing the reduction of acetaldehyde to ethanol + . Furthermore, the gene sequences of different yeasts encoding alcohol dehydrogenase are different.
Disclosure of Invention
[ technical problem ]
The invention aims to solve the technical problems that the existing method for identifying schizosaccharomyces pombe mainly utilizes physicochemical properties of strains or ITS4, and the identification method has the problems of long period, high cost, low success rate and the like.
Technical scheme
The invention provides a primer for identifying schizosaccharomyces pombe (Schizosaccharomyces pombe), the sequence of which is shown in SEQ ID NO:2 (primer Adh 4F), SEQ ID NO:3 (primer Adh 4R).
The invention also provides a method for identifying schizosaccharomyces pombe (Schizosaccharomyces pombe) by using the primer, which comprises the following steps: preparing a sample to be tested into bacterial suspension, diluting and coating the bacterial suspension to obtain single bacterial colony, and using SEQ ID NO: 2. SEQ ID NO:3, the sequence shown in the sequence 3 is a primer, colony PCR is carried out, the PCR product is observed through gel electrophoresis, if the PCR product has a band between 100 and 250bp on an electrophoresis pattern, the sample contains schizosaccharomyces pombe (Schizosaccharomyces pombe), and if the PCR product is not between 100 and 250bp or has no band, the sample does not contain schizosaccharomyces pombe.
In one embodiment, the sample contains a cell, genome or metagenome. Optionally, the sample is a finished fermented food or a sample obtained in the fermentation process of the fermented food, or an environmental sample such as intestinal tract, soil, water body and the like; optionally, the sample to be measured is subjected to pretreatment such as centrifugation and bacterial cell collection, and then subjected to subsequent measurement. Optionally, the sample is a fermented food or a sample taken from the 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 beverage, alcoholic beverage, yoghurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd, fermented rice flour food and the like. Optionally, the sample comprises Daqu or fermented grains.
In one embodiment, PCR products of between 100 and 250bp can also be sequenced and the sequencing result aligned with the gene sequence encoding the alcohol dehydrogenase of Schizosaccharomyces pombe.
The invention also provides a method for quantitatively detecting schizosaccharomyces pombe (Schizosaccharomyces pombe) by using the primer, which comprises the following steps:
(1) Preparing merozoite suspensions with different bacteria concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions;
(2) Using SEQ ID NO: 2. SEQ ID NO:3, respectively performing fluorescent quantitative PCR on the genome of the primer pair, and drawing a standard curve by taking a CT value as an abscissa and taking the number of cells or an Lg value of the number of cells as an ordinate;
(3) Extracting genome in a sample to be detected, and utilizing SEQ ID NO: 2. SEQ ID NO:3, carrying out fluorescent quantitative PCR on the genome of the sample to be detected by the primer, substituting the CT value of the fluorescent quantitative PCR into a standard curve, and converting to obtain the quantity of schizosaccharomyces pombe in the sample.
The invention provides a method for distinguishing schizosaccharomyces pombe from pichia kudriavzevii (Pichia kudriavzevii), saccharomyces cerevisiae (Saccharomyces cerevisiae), bayer combined yeast (Zygosaccharomyces bailii), candida albicans (Candida humilis) and kazakii yeast (Kazachstania barnettii), which is characterized in that the method comprises the following steps of: 2. SEQ ID NO:3, performing colony PCR or respectively performing PCR by using the genome of the yeast as a template, observing the PCR product by gel electrophoresis, and if the PCR product has a band between 100 and 250bp on an electropherogram, indicating that the corresponding strain is schizosaccharomyces pombe (Schizosaccharomyces pombe).
The invention provides a qualitative and quantitative detection kit for schizosaccharomyces pombe (Schizosaccharomyces pombe), which comprises the following components in percentage by weight: 2. SEQ ID NO:3, and also comprises DNA polymerase and buffer solution.
A method for qualitatively detecting schizosaccharomyces pombe using the kit, comprising: preparing a sample to be tested into bacterial suspension, diluting and coating the bacterial suspension to obtain single bacterial colony, and using SEQ ID NO: 2. SEQ ID NO:3 is a primer, colony PCR is carried out, the PCR product is observed through gel electrophoresis, and if the PCR product has a band between 100 and 250bp on an electrophoresis pattern, the sample contains schizosaccharomyces pombe (Schizosaccharomyces pombe).
The method for quantitatively detecting the schizosaccharomyces pombe by using the kit comprises the following steps: (1) Preparing merozoite suspensions with different bacteria concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions; (2) using SEQ ID NO: 2. SEQ ID NO:3, respectively performing fluorescent quantitative PCR on the genome of the primer pair, and drawing a standard curve by taking a CT value as an abscissa and taking the number of cells or an Lg value of the number of cells as an ordinate; (3) Extracting genome in a sample to be detected, and utilizing SEQ ID NO: 2. SEQ ID NO:3, carrying out fluorescent quantitative PCR on the genome of the sample to be detected by the primer, substituting the CT value of the fluorescent quantitative PCR into a standard curve, and converting to obtain the quantity of schizosaccharomyces pombe in the sample.
The sample contains a cell, genome or metagenome. Optionally, the sample is a finished fermented food or a sample obtained in the fermentation process of the fermented food, or an environmental sample such as intestinal tract, soil, water body and the like; optionally, the sample to be measured is subjected to pretreatment such as centrifugation and bacterial cell collection, and then subjected to subsequent measurement. Optionally, the sample is a fermented food or a sample taken from the 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 beverage, alcoholic beverage, yoghurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd, fermented rice flour food and the like. Optionally, the sample comprises Daqu or fermented grains.
[ advantageous effects ]
The invention designs a specific primer aiming at the schizosaccharomyces pombe ethanol dehydrogenase Adh4 gene, and can realize the rapid qualitative and quantitative detection of the schizosaccharomyces pombe in samples such as Daqu, fermented grains and the like by a PCR technology; high accuracy, good effectiveness and high sensitivity. Is especially suitable for the identification and quantification of schizosaccharomyces pombe in the finished fermented food or the samples obtained from the fermentation process of the fermented food.
The invention utilizes specific primers which can distinguish schizosaccharomyces pombe from pichia kudriavzevii (Pichia kudriavzevii), saccharomyces cerevisiae (Saccharomyces cerevisiae), bayer-bound yeast (Zygosaccharomyces bailii), candida albicans (Candida humilis) and kazakii (Kazachstania barnettii), and the specific primers only specifically amplify ethanol dehydrogenase Adh4 of schizosaccharomyces pombe, but not the gene segments of the other 5 yeasts.
Drawings
Fig. 1: agarose gel electrophoresis identification results of different primer pairs, wherein 1: specific primers ADH4F, ADH4R,2, 3, 4 for use in the present invention: reference is made to primer pairs 2, 3, 4 designed in a similar way.
Fig. 2: agarose gel electrophoresis identification of different yeasts, wherein SP: schizosaccharomyces pombe (Saccharomyces cerevisiae), SC: saccharomyces cerevisiae (Saccharomyces cerevisiae), PK: pichia kudriavzevii (Pichia kudriavzevii), ZB: bayer-bound yeast (Zygosaccharomyces bailii), CH: candida albicans (Candida humilis), KB: kazakhstan yeast (Kazachstania barnettii), dd H 2 O: and (5) re-steaming water.
Fig. 3: verification of schizosaccharomyces pombe alcohol dehydrogenase gene.
Fig. 4: agarose gel electrophoresis identification results of different strains of merozoites pomica, wherein, P1: schizosaccharomyces pombe NM _001023234, P2: schizosaccharomyces pombe CU329672.1, P3: schizosaccharomyces pombe NM _001023235.2, P4: schizosaccharomyces pombe NR _150068.1 and P5: schizosaccharomyces pombe AB001834.
Fig. 5: a standard curve.
Detailed Description
The invention is further illustrated below in conjunction with specific examples.
The following examples relate to the following media:
YPD liquid medium: yeast extract 10g/L, tryptone 20g/L, glucose 20g/L.
YPD solid medium: yeast extract 10g/L, tryptone 20g/L, glucose 20g/L, agar 20g/L.
Example 1: design of primers specific to schizosaccharomyces pombe (Schizosaccharomyces pombe)
The design method of the specific primer pair comprises the following steps:
schizosaccharomyces pombe (Schizosaccharomyces pombe) is a core yeast in a white spirit brewing system, and a specific enzyme of the Schizosaccharomyces pombe, namely Schizosaccharomyces pombe alcohol dehydrogenase (Schizosaccharomyces pombe alcohol dehydrogenase Adh 4), is searched for through a metabolic Pathway (KEGG Pathway) of a complete book of Kyoto genes and genome, so that a 1850bp nucleotide sequence for encoding the specific enzyme is obtained, wherein the sequence is as follows:
AAATATTTGATCTTCTACTTTTTGGTAAGTAAAATTTATGATAATGGCGGAAAGAATGACAGTTTGGAGGTGATTTGATGTCGTTTGGAAAAGAGTTTTGGTGACAAGCAATTTTGTTTTGAAAATCATTTGAAGATCTTTTAATTCCTTAGCATTTATTTGACGTTGGAGTTGGCTGCAGATCTATTTAGCGAAATTGTTGTAAAAAGATCGATGTTCGATAAGTGTTCGTTAAGCGAGATAAGGAGTGGAAGTATCGAAATTTTAGACACTAATTGATTACACATTGATATTGGAGAAATTTTCGTACACTACTTAAGGCTCATTCCCCCTTGACTTTTTTGACCGATTGTTACATATTTCTTTTCCGTTTGGCTAATTATTTATTTTTTTTCATACTTTGTATATTATTGATTCTAAAAAATATCGGAGGTTAAGCTTTAGATGCGATTTTCTTTTTAACATGTCCATTCTTCGTTCTCCATTTCGTTTAATTCGATCGCCTGCTCGATTCTTTCCGTCACTCTTTCATTCCTCTTGTAATCAATCTTTTACTAATGGCTTAAAGCATCAATCAACCTCTTCGAAAGCCATGCCTGTTAGCGCATTCTACATACCTTCTTTCAACCTTTTCGGTAAAGGTTGTCTTGCTGAAGCCGCCAAGCAAATTAAGATGAGCGGCTTCAAAAACACCTTAATTGTTACCGATCCTGGTATTATTAAGGTCGGTTTGTATGACAAAGTTAAGGCTCTGTTGGAAGAACAAAGCATCACTGTTCACCTTTACGATGGTGTTCAACCTAATCCCACCGTTGGAAATGTCAACCAAGGTCTCGAAATCGTTAAGAAGGAAAACTGTGATTCTATGGTATCAATTGGTGGTGGTTCTGCTCACGATTGTGCCAAAGGTATTGCTTTGTTAGCTACTAATGGCGGTAAAATTGCGGATTATGAAGGTGTTGATAAATCCTCCAAACCTCAACTTCCCTTGATTGCAATCAATACGACTGCAGGAACTGCTTCTGAAATGACTCGTTTCGCAATTATCACCGAAGAGACCCGTCATATTAAAATGGCTATTATCGATAAGCATACTATGCCTATCCTTTCCGTTAACGATCCTGAAACAATGTATGGATTACCTCCTTCCTTGACCGCTGCTACTGGTATGGACGCTTTGACTCATGCTGTTGAAGCTTACGTCTCTACTGCTGCTAATCCCATCACCGATGCTTGTGCTGTCAAATGCATCGAACTCGTTAATAAGTATTTGAAGCGTGCTGTCGACAACGGCAAGGACGAAGAGGCTAGAGACAATATGGCTTATGCCGAATTCCTTGGTGGTATGGCATTTAACAACGCATCTTTGGGTTATGTTCATGCTATGGCTCATCAACTTGGTGGTTTCTACGGTATTCCACATGGCGTCTGTAACGCTGTTCTTCTTGCCCATGTACAGAAATTTAATTCTAGGGATCCAAGAGCAAATGCTCGCCTTGGTGATATTGCTTTTCACTTGGGTTGTGAAGAGCATACTGCCGAAGCAGCTCTTGACCGCATCAGTCAACTTGTTCTTGAAGTTAAAATTCGTCCTCATCTAGTTGATCTAGGTGTCAAGGAAAAAGATTTTGATGTCTTGGTTGACCACGCTATGAAGGATGCTTGTGGTGCTACTAACCCTATTCAGCCTACACATGACGAGGTAAAGGCTATTTTCAAATCGGCTATGTAAATAGAACGTTATCTCGATACCTAGAACAAATTATGAGCTTTTACATTTCACTGGCTTGAATGTTCACTTCAACATATCCTAAAGCATTTTTTTTGGCTTGAACCTTTTTTAGATTTGG。
the nucleotide sequence was aligned by the National Center for Biotechnology Information (NCBI) using the local alignment search tool (BLAST), and the alignment showed that the nucleotide sequence had 100% similarity only with the Schizosaccharomyces pombe genomic ethanol dehydrogenase (Schizosaccharomyces pombe alcohol dehydrogenase Adh 4) sequence, indicating that the 1850bp nucleotide sequence corresponding to Schizosaccharomyces pombe ethanol dehydrogenase (Schizosaccharomyces pombe alcohol dehydrogenase Adh 4) is a highly specific sequence of Schizosaccharomyces pombe. Setting Primer parameters using NCBI Primer-BLAST tool (Primer Parameters); a Polymerase Chain Reaction (PCR) product length (PCR product size) of 80 to 300; primer pair specificity verification parameters were set (Primer Pair Specificity Checking Parameters): database (Database) nr; the other parameters are set according to default; from the obtained pair primer pairs, upstream primer Adh4F-TGGACGCTTTGACTCATGCT and downstream primer Adh4R-ACCACCAAGGAATTCGGCAT which were unable to loop themselves (self-complexity) were selected using DNAMAN software. As a possible target primer pair, the PCR product was 160bp in length (part of the gene encoding Schizosaccharomyces pombe alcohol dehydrogenase). BLAST in NCBI with the potential target primer pair as the upstream and downstream primer, the comparison result showed 100% similarity with Schizosaccharomyces pombe only, indicating that the selected primer pair potential target primer pair was a specific primer pair suitable for this environment.
Example 2: optimization of primers specific for Schizosaccharomyces pombe (Schizosaccharomyces pombe)
Firstly, screening core yeast from the fermentation process of Maotai-flavor liquor, weighing 10g of fermented grains sample in a sterilized 250mL triangular flask filled with 100mL of sterile PBS buffer solution and 3g of glass beads, then placing in a shaking table at 30 ℃ for shaking and mixing for 30min at 200r/min, and standing for 5min to obtain bacterial suspension. Diluting the bacterial suspension to obtain 10 -3 、10 -4 And 10 -5 Three dilutions of the sample bacterial suspension were plated on YPD medium plates and incubated in an incubator at 30℃for 72h, and single colonies were picked on the plates and numbered as core yeast-like strains.
Then, a slant culture medium is prepared by using sorghum leaching liquid for producing Maotai-flavor liquor, the screened core yeast similar strain is transferred to a test tube slant after being separated and purified, and is cultured at 30 ℃, and is preserved at 4 ℃ after the culture is finished.
Finally, the genome was extracted and PCR amplified using eukaryotic microorganism universal forward primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') to identify the strain. PCR reaction System (50. Mu.L): taq DNA Polymerase (5U/. Mu.L) 0.5. Mu.L, dNTP Mix (10 mmol/L) 1. Mu.L, each of the upstream and downstream primers (10. Mu. Mol/L) 2. Mu.L, 10 XTaq Buffer (M g 2+plus) 5. Mu.L, genomic template DNA 10ng, and ultrapure water to 50. Mu.L. PCR reaction conditions: 3min at 95 ℃;95℃15s,55℃30s,72℃45s, 30 cycles of 10min. Comparing the sequence obtained by PCR amplification with ITS amplicon sequence of model strain in NCBI website system to obtain specific information of target strain, and screening from the core yeast to obtain merozoite (Schizosaccharomyces pombe).
Setting Primer parameters (Primer Parameters) by NCBI Primer-BLAST tool with 1850bp nucleotide sequence corresponding to schizosaccharomyces pombe alcohol dehydrogenase (Schizosaccharomyces pombe alcohol dehydrogenase Adh) as target; a Polymerase Chain Reaction (PCR) product length (PCR product size) of 80 to 300; primer pair specificity verification parameters were set (Primer Pair Specificity Checking Parameters): database (Database) nr; the other parameters are set according to default; from the obtained pair primer pairs, three other pairs of primers that are not capable of self-priming (self-complete) were selected using DNAMAN software. The genome of the extracted Schizosaccharomyces Pombe (SP) is used as a template, and the four pairs of primers (Adh 4F/Adh4R, primer pair 2, primer pair 3 and primer pair 4) are used for PCR amplification (the PCR system is shown in Table 1). Reaction parameters: after 2min pre-denaturation at 94 ℃, 30s denaturation at 94 ℃, 45s annealing at 55 ℃, 30s extension at 72 ℃,25 cycles, 30s extension at 72 ℃, the product was identified by 2% agarose gel electrophoresis. The results (shown in FIG. 1) indicate that the other primer amplification was significantly less effective than the primer pair Adh4F/Adh4R of example 1.
Primer information is as follows:
primer pair 2:
F2：GAGGGATCGTTGACTGCAACA
R2：AATTGGAGATTACGAGGGGGTTA
primer pair 3:
F3：TGAATGAGCTAAACCAACCCT
R3：TCCAATGGGTAGTAGTGGTGC
primer pair 4:
F4：GGGCAACCCTACGGAAATGA
R4：TGCAGACGCAGAGAAGATGG
TABLE 1 PCR verification System
| Reaction mixture | Volume(μL) |
| Mixture | 10 |
| Upstream primer | 1 |
| Downstream primer | 1 |
| Template DNA | 1 |
| dd H 2 O | 7 |
| Total volume | 20 |
Example 3: verification of Schizosaccharomyces pombe (Schizosaccharomyces pombe) specific primer pairs
Verification of specific primer pairs:
the genome of the extracted Schizosaccharomyces Pombe (SP) is used as a template, and the specific primers Adh4F-TGGACGCTTTGACTCATGCT and Adh4R-ACCACCAAGGAATTCGGCAT are used. As primers, PCR was performed, negative controls were core yeast saccharomyces cerevisiae in the white spirit brewing system, respectively: pichia kudriavzevii (Pichia kudriavzevii, PK), saccharomyces cerevisiae (Saccharomyces cerevisiae, SC), saccharomyces cerevisiae (Zygosaccharomyces bailii, ZB), candida albicans (CH), kazakhstan yeast (Kazachstania barnettii, KB), and redistilled water (dd H) 2 O). The PCR system and PCR conditions were the same as in example 2. After the PCR was completed, the product was subjected to gel electrophoresis, and the band on the gel was observed by a gel imager to confirm the specificity of the primer, schizosaccharomyces pombe had a distinct target band around 100-250bp, and the remaining negative control had no band (as shown in FIG. 2), indicating that the designed specific primer pair had specificity for Schizosaccharomyces pombe (Schizosaccharomyces pombe). After sequencing the PCR product of Schizosaccharomyces pombe (Schizosaccharomyces pombe) (Schizosaccharomyces pombe), the PCR product was subjected to blast verification by NCBI website to confirm the PCR product as an alcohol dehydrogenase gene on Schizosaccharomyces pombe (Schizosaccharomyces pombe) (see FIG. 3).
Example 4: detection effectiveness and accuracy experiment of primer pair Adh4F/Adh4R
Five different strains Schizosaccharomyces pombe NM _001023234, schizosaccharomyces pombe CU329672.1, schizosaccharomyces pombe NM _001023235.2, schizosaccharomyces pombe NR _150068.1 and Schizosaccharomyces pombe AB001834 of Schizosaccharomyces pombe (Schizosaccharomyces pombe) were taken, and the respective genomes of the 5 strains of Schizosaccharomyces pombe (Schizosaccharomyces pombe) were separately extracted under the numbers P1, P2, P3, P4 and P5, respectively. PCR was performed on each genome using the above specific primers Adh4F-TGGACGCTTTGACTCATGCT, adh4R-ACCACCAAGGAATTCGGCAT as primers, and the negative control was distilled water (dd H 2 O) and then electrophoretically detected. The result shows that: the PCR products had bands in the range of about 100-250bp (as shown in FIG. 4), demonstrating that the primers were indeed able to amplify the alcohol dehydrogenase gene of Schizosaccharomyces pombe (Schizosaccharomyces pombe), thus allowing rapid identification of Schizosaccharomyces pombe (Schizosaccharomyces pombe) in the sample.
Example 5: quantification of alcohol dehydrogenase gene in sample
The genome concentration of Schizosaccharomyces pombe (Schizosaccharomyces pombe) obtained in example 2 was determined and shown to be 464 ng/. Mu.L and the number of cells was 9.9x10 by plate counting 7 cells/g, their genomes were diluted to a corresponding cell number of 10, respectively 6 、10 5 、10 4 、10 3 、10 2 After 10cells/g, fluorescent quantitative PCR (fluorescent quantitative PCR systems see Table 2) was performed by a StepOneP lus instrument (available from Simer's fly) using the diluted genomes as templates and the specific primers Adh4F-TGGACGCTTTGACTCATGCT and Adh4R-ACCACCAAGGAATTCGGCAT as primers, respectively; and drawing a standard curve by taking CT values measured by qPCR as an abscissa and cell values corresponding to dilution concentrations as an ordinate (the standard curve is shown in figure 5).
Table 2 qPCR validation System
| Reaction system | Volume (mu L) |
| Mixture | 10 |
| Upstream primer | 0.4 |
| Downstream primer | 0.4 |
| Template DNA | 1 |
| dd H 2 O | 8.2 |
| Total volume of | 20 |
Example 6: quantitative application of alcohol dehydrogenase gene in sample
10g of Daqu sample is weighed into a sterilized 250mL triangular flask filled with 100mL of sterile PBS buffer solution and 3g of glass beads, and then placed into a shaking table at 30 ℃ for shaking and mixing for 30min at 200r/min, and the mixture is kept stand for 5min to obtain bacterial suspension. Diluting the bacterial suspension to obtain 10 -3 、10 -4 And 10 -5 Three dilutions of the sample bacterial suspension were spread in YPD medium plates and incubated in a 30 ℃ incubator for 72h, single colonies were picked on plates and numbered, and colony PCR was performed using specific primers Adh4F, adh R. The experimental results show that the PCR products have bands between 100 and 250bp, which indicates that the Daqu sample contains schizosaccharomyces pombe (Schizosaccharomyces pombe).
Obtaining genome by using "TIANGEN Plant Genomic DNA Kit" kit, and using the obtained genome as templateThe specific primer Adh4F-TGGACGCTTTGACTCATGCT, adh4R-ACCACCAAGGAATTCGGCAT is used as a primer for fluorescence quantitative PCR, the PCR reaction conditions are the same as those of example 5, the obtained CT value 23.989 is substituted into the standard curve of FIG. 5, and finally the content of Saccharomyces cerevisiae in the Daqu sample is: 10 5 cells/g。
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 Jiangnan
<120> a method for rapidly identifying and quantifying Schizosaccharomyces pombe in a sample
<130> BAA201562A
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 1850
<212> DNA
<213> schizosaccharomyces pombe
<400> 1
aaatatttga tcttctactt tttggtaagt aaaatttatg ataatggcgg aaagaatgac 60
agtttggagg tgatttgatg tcgtttggaa aagagttttg gtgacaagca attttgtttt 120
gaaaatcatt tgaagatctt ttaattcctt agcatttatt tgacgttgga gttggctgca 180
gatctattta gcgaaattgt tgtaaaaaga tcgatgttcg ataagtgttc gttaagcgag 240
ataaggagtg gaagtatcga aattttagac actaattgat tacacattga tattggagaa 300
attttcgtac actacttaag gctcattccc ccttgacttt tttgaccgat tgttacatat 360
ttcttttccg tttggctaat tatttatttt ttttcatact ttgtatatta ttgattctaa 420
aaaatatcgg aggttaagct ttagatgcga ttttcttttt aacatgtcca ttcttcgttc 480
tccatttcgt ttaattcgat cgcctgctcg attctttccg tcactctttc attcctcttg 540
taatcaatct tttactaatg gcttaaagca tcaatcaacc tcttcgaaag ccatgcctgt 600
tagcgcattc tacatacctt ctttcaacct tttcggtaaa ggttgtcttg ctgaagccgc 660
caagcaaatt aagatgagcg gcttcaaaaa caccttaatt gttaccgatc ctggtattat 720
taaggtcggt ttgtatgaca aagttaaggc tctgttggaa gaacaaagca tcactgttca 780
cctttacgat ggtgttcaac ctaatcccac cgttggaaat gtcaaccaag gtctcgaaat 840
cgttaagaag gaaaactgtg attctatggt atcaattggt ggtggttctg ctcacgattg 900
tgccaaaggt attgctttgt tagctactaa tggcggtaaa attgcggatt atgaaggtgt 960
tgataaatcc tccaaacctc aacttccctt gattgcaatc aatacgactg caggaactgc 1020
ttctgaaatg actcgtttcg caattatcac cgaagagacc cgtcatatta aaatggctat 1080
tatcgataag catactatgc ctatcctttc cgttaacgat cctgaaacaa tgtatggatt 1140
acctccttcc ttgaccgctg ctactggtat ggacgctttg actcatgctg ttgaagctta 1200
cgtctctact gctgctaatc ccatcaccga tgcttgtgct gtcaaatgca tcgaactcgt 1260
taataagtat ttgaagcgtg ctgtcgacaa cggcaaggac gaagaggcta gagacaatat 1320
ggcttatgcc gaattccttg gtggtatggc atttaacaac gcatctttgg gttatgttca 1380
tgctatggct catcaacttg gtggtttcta cggtattcca catggcgtct gtaacgctgt 1440
tcttcttgcc catgtacaga aatttaattc tagggatcca agagcaaatg ctcgccttgg 1500
tgatattgct tttcacttgg gttgtgaaga gcatactgcc gaagcagctc ttgaccgcat 1560
cagtcaactt gttcttgaag ttaaaattcg tcctcatcta gttgatctag gtgtcaagga 1620
aaaagatttt gatgtcttgg ttgaccacgc tatgaaggat gcttgtggtg ctactaaccc 1680
tattcagcct acacatgacg aggtaaaggc tattttcaaa tcggctatgt aaatagaacg 1740
ttatctcgat acctagaaca aattatgagc ttttacattt cactggcttg aatgttcact 1800
tcaacatatc ctaaagcatt ttttttggct tgaacctttt ttagatttgg 1850
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tccgtaggtg aacctgcgg 19
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aattggagat tacgaggggg tta 23
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tccaatgggt agtagtggtg c 21
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gggcaaccct acggaaatga 20
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Claims (7)
1. Identification of schizosaccharomyces pombeSchizosaccharomyces pombe) Is characterized by comprising the steps of: preparing a sample to be tested into bacterial suspension, diluting and coating the bacterial suspension to obtain single bacterial colony, and using SEQ ID NO: 2. SEQ ID NO:3, performing colony PCR by using the primer, observing a PCR product through gel electrophoresis, if the PCR product has a band between 100 and 250bp on an electropherogram, indicating that the sample contains schizosaccharomyces pombe, and if the PCR product is not between 100 and 250bp or has no band, indicating that the sample does not contain schizosaccharomyces pombe;
the sample is a finished fermented food product or a sample taken from the fermentation process of the fermented food, and comprises Daqu or fermented grains;
the sample contains Pichia kudriavzeviiPichia kudriavzevii) Saccharomyces cerevisiaeSaccharomyces cerevisiae) Bayer combined yeastZygosaccharomyces bailii) Kazakhstan yeastKazachstania barnettii）。
2. The method according to claim 1, wherein the PCR product of between 100 and 250bp is further sequenced and the sequencing result is aligned with the gene sequence encoding the alcohol dehydrogenase of Schizosaccharomyces pombe.
3. The primer of claim 1 for quantitatively detecting schizosaccharomyces pombeSchizosaccharomyces pombe) Is characterized by comprising the steps of:
(1) Preparing merozoite suspensions with different bacteria concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions;
(2) Using SEQ ID NO: 2. SEQ ID NO:3, respectively performing fluorescent quantitative PCR on the genome of the primer pair, and drawing a standard curve by taking a CT value as an abscissa and taking the number of cells or an Lg value of the number of cells as an ordinate;
(3) Extracting genome in a sample to be detected, and utilizing SEQ ID NO: 2. SEQ ID NO:3, carrying out fluorescent quantitative PCR on the genome of the sample to be detected by the primer, substituting the CT value of the fluorescent quantitative PCR into a standard curve, and converting to obtain the quantity of schizosaccharomyces pombe in the sample.
4. Differentiation of Schizosaccharomyces pombe and Pichia kudriavzeviiPichia kudriavzevii) Saccharomyces cerevisiaeSaccharomyces cerevisiae) Bayer combined yeastZygosaccharomyces bailii) Kazakhstan yeast @ PasteurKazachstania barnettii) Characterized in that the sequence represented by SEQ ID NO: 2. SEQ ID NO:3, performing colony PCR or respectively performing PCR with genome of the yeast as template, observing PCR product by gel electrophoresis, if there is a band of 100-250bp on the electrophoresis pattern, indicating that the corresponding strain is Schizosaccharomyces pombeSchizosaccharomyces pombe）。
5. Schizosaccharomyces pombe used for schizosaccharomyces pombeSchizosaccharomyces pombe) The qualitative and quantitative detection kit is characterized by comprising a nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO:3, and also comprises DNA polymerase and buffer solution.
6. A method for qualitative detection of schizosaccharomyces pombe using the kit of claim 5, comprising: preparing a sample to be tested into bacterial suspension, diluting and coating the bacterial suspension to obtain single bacterial colony, and using SEQ ID NO: 2. SEQ ID NO:3, the sequence shown as the primer is used for colony PCR, the PCR product is observed through gel electrophoresis, if the PCR product has a band between 100 and 250bp on the electropherogram, the sample is indicated to contain schizosaccharomyces pombe @ theSchizosaccharomyces pombe）。
7. A method for quantitatively detecting schizosaccharomyces pombe using the kit of claim 5, comprising: (1) Preparing merozoite suspensions with different bacteria concentrations and known concentrations, and respectively extracting genomes in the bacterial suspensions; (2) using SEQ ID NO: 2. SEQ ID NO:3, respectively performing fluorescent quantitative PCR on the genome of the primer pair, and drawing a standard curve by taking a CT value as an abscissa and taking the number of cells or an Lg value of the number of cells as an ordinate; (3) Extracting genome in a sample to be detected, and utilizing SEQ ID NO: 2. SEQ ID NO:3, carrying out fluorescent quantitative PCR on the genome of the sample to be detected by the primer, substituting the CT value of the fluorescent quantitative PCR into a standard curve, and converting to obtain the quantity of schizosaccharomyces pombe in the sample.
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| CN104126011A (en) * | 2011-11-30 | 2014-10-29 | 帝斯曼知识产权资产有限公司 | Yeast strains engineered to produce ethanol from acetic acid and glycerol |
| 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 |
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| 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 |
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