CN112695121A - Primer, kit and method for quickly identifying and quantifying schizosaccharomyces pombe - Google Patents
Primer, kit and method for quickly identifying and quantifying schizosaccharomyces pombe Download PDFInfo
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Images
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 primer, a kit and a method for rapidly identifying and quantifying fission yeast schizosaccharomyces pombe, belonging to the technical field of biological engineering. Specific primers of SEQ ID NO. 2 and SEQ ID NO. 3 are designed aiming at the fission yeast schizosaccharomyces pombe alcohol dehydrogenase gene Adh1, and the rapid qualitative and quantitative detection of the fission yeast schizosaccharomyces pombe in samples such as Daqu, fermented grains and the like can be realized through a PCR technology; high accuracy, good effectiveness and high sensitivity. The technical scheme of the invention is suitable for the finished fermented food or the sample obtained from the fermentation process of the fermented food.
Description
Technical Field
The invention relates to a primer, a kit and a method for rapidly identifying and quantifying fission yeast schizosaccharomyces pombe, 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.
Schizosaccharomyces pombe (Schizosaccharomyces pombe) is a rod-shaped yeast of schizont, is widely applied to liquor brewing, is a core yeast in a liquor brewing system, has high ethanol fermentation capacity, can reduce the concentration of ethyl carbamate influencing the quality safety of products, and is a non-brewing yeast with great brewing potential. The screening, identification and analysis of the schizosaccharomyces pombe have important significance for improving the quality of the white spirit. The existing method for identifying the schizosaccharomyces pombe mainly utilizes the physicochemical property of a bacterial strain or ITS4, and the identification method has the problems of long period, high cost, low success rate and the like.
Alcohol Dehydrogenases (ADHs) 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 dehydrogenase catalyzes the production of ethanol from acetaldehyde with the production of ATP, an energy molecule. 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 schizosaccharomyces pombe 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.
[ solution ]
The invention provides a primer for identifying Schizosaccharomyces pombe (Schizosaccharomyces pombe), and the sequence of the primer is shown as SEQ ID NO:2 (primer Adh 1F: CAGTACTCGCAGTTACCGCA), SEQ ID NO:3 (primer Adh 1R: TTGATCGGTGGTCACGAAGG).
The invention also provides a method for identifying Schizosaccharomyces pombe (Schizosaccharomyces pombe) by applying 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, performing colony PCR by using the primer, observing a PCR product through gel electrophoresis, if the PCR product on an electrophoretogram has a band between 100 and 250bp, indicating that the sample contains Schizosaccharomyces pombe (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.
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 alcohol dehydrogenase encoding gene sequence of Schizosaccharomyces pombe.
The invention also provides a method for quantitatively detecting the Schizosaccharomyces pombe (Schizosaccharomyces pombe) by applying the primer, which comprises the following steps:
(1) preparing schizosaccharomyces pombe 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 3, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the quantity of the schizosaccharomyces pombe in the sample.
The present invention provides a method for differentiating Schizosaccharomyces pombe from Pichia kudriavzevii (Pichia kudriavzevii), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces bailini (Zygosaccharomyces bailii), Candida albicans (Candida humulis), and Saccharomyces pastorianus barnetii (Kazachstania barnetii), in which the nucleotide sequence of the nucleic acid sequence of SEQ ID NO: 2. SEQ ID NO:3 as a primer, performing colony PCR or performing PCR by respectively using the genomes of the yeasts as templates, observing PCR products through gel electrophoresis, and if the PCR products on an electrophoretogram have bands between 100-250bp, 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 contains SEQ ID NO: 2. SEQ ID NO:3, and further comprises DNA polymerase and a buffer solution.
The method for qualitatively detecting the schizosaccharomyces pombe 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, performing colony PCR by using the sequence shown in the figure as a primer, observing a PCR product through gel electrophoresis, and if the PCR product on an electrophoretogram has a band between 100 and 250bp, indicating that 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 schizosaccharomyces pombe 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 3, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the quantity of the schizosaccharomyces pombe 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 fission yeast schizosaccharomyces pombe alcohol dehydrogenase gene Adh1, and can realize the rapid qualitative and quantitative detection of the fission yeast schizosaccharomyces pombe in samples such as Daqu, fermented grains and the like by a PCR technology; high accuracy, good effectiveness and high sensitivity. Is particularly suitable for the identification and the quantification of the schizosaccharomyces pombe in a fermented food finished product or a sample obtained from the fermentation process of the fermented food.
The invention can distinguish the fission yeast schizosaccharomyces pombe from Pichia kudriavzevii (Pichia kudriavzevii), Saccharomyces cerevisiae (Saccharomyces cerevisiae), bayer combination yeast (Zygosaccharomyces bailii), Candida albicans (Candida miliis) and Saccharomyces pastorianus (Kazachstania barnetii) by using the specific primer, and the specific primer only specifically amplifies the alcohol dehydrogenase gene Adh1 of the fission yeast schizosaccharomyces pombe, but not amplifies the gene segments of other 5 yeasts.
Drawings
FIG. 1: the agarose gel electrophoresis identification results of the different primer pairs in example 2, wherein, 1: specific primers Adh1F, Adh1R, 2, 3, 4: primer pairs 2, 3, 4 designed with reference to a similar method.
FIG. 2: the results of agarose gel electrophoresis identification of different yeasts in example 3, wherein, SP: schizosaccharomyces pombe (Saccharomyces cerevisiae), SC: saccharomyces cerevisiae (Saccharomyces cerevisiae), 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: example 3 validation of the ethanol dehydrogenase gene of Schizosaccharomyces pombe.
FIG. 4: the agarose gel electrophoresis identification results of different strains of schizosaccharomyces pombe in example 4, wherein, the ratio of 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 AB 001834.
FIG. 5: a 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 Schizosaccharomyces pombe (Schizosaccharomyces pombe) specific primers
The design method of the specific primer pair comprises the following steps:
schizosaccharomyces pombe (Schizosaccharomyces pombe) is a core yeast in a liquor brewing system, and a 3463bp nucleotide sequence for coding a specific enzyme (Schizosaccharomyces pombe alcohol dehydrogenase) is obtained by searching the specific enzyme of the Schizosaccharomyces pombe through Kyoto gene and genome encyclopedia metabolic Pathway (KEGG Pathway), namely Schizosaccharomyces pombe alcohol dehydrogenase (Schizosaccharomyces pombe alcohol dehydrogenase Adh1), and the sequence is as follows:
GATGGATGGAAAGATTTGAAAATACATAGTGTTGGTTGAATTTGCATTGGGAGGCCGGC GGAGCATGCGAACCATTATAGAATAGGCAGTATGAAAATTTAGCAAGCGATAGCTCAAGT TTGCATGATCGAATGAGTTATCCAAAAAATAAACTGGAGTATTGTTGAATTCAGTTTTGA GCAACCGAATTCGCAGTACCTTCTTACAGTACCTCCTCACAGCCAAGTTTCTACCCTAGT AGAGAGTATCCAGTTTGTCGGTTTGTTGGTTGCGAAACCAGTATGGACGATGTTGGTTGT CAGATTAAGCAGTGCAAGTTTTGCAGCCCATGTTGGTAAGTGAAGAGAGACAAATAGGA TGACAACGAGCGGCAGGGCGAGAGCGAGGCAAGGATGAGAGTAGGAAGAGGGAAGA AGGACAAAAATTTGAAAAGGATGTGAATGTGAATGTGATTGAGTTGGATGAAAATTTCG TCTTTACATTTGCCCGTTATAAACGAGTGTTGAATTATTCCTATACTACAGATTTATATATCT ACATGATTCGATTCCTACAGGATTGTAAACCCACATGATTTGTATACACTATAATTTGTATA TACCTAGTAATGATTCTTTTATTCTCCACTCCACTAATATGAAAATATTCTTGTATGGTTATC CTTATGTGATGAAATATCTTTTAAACGAGGTCCACGCGGATCTCCATATCAGATAAAAAA AAATACTACGACAAAAGAAGGCTAAAGGAATAGGGTTGAGTGCTAGGTGGATGGTACGA TGTTGTTTGCCCTAATACGGCATACTACTAGGGAAATGTTGGGTGCATAGGCCTACAATG TTAGTCGTAAGAGACGAGACGAGACGAGACGAGATGAACGAAATAACCCGCTCCCAAC TCTATAAAGGGTTAAGGATAGAGAGAAATTGAGTGGTACGTTTGGTGTGCCGTTTGTCG AGCCGTCGACTGCTAGTAATATTGTCATTGTGATTACCATTGCCATTGTCAATGTCACTGC TATTGCTACTGACACTGCCGCTGTACTCTTCTTCCGATGTAGCTTTATTTACATTGTATCGG AAACCGTCCACACCCATGGTCGAGAGCTGTGGGTGACGCAGACATTCGAATGGCATGCC CTACAACAACTAAGAAAATGGCTATCATGCGGAAGCTGGTGAGAAGAACAGCATCGGG ACAAGGGAAGGAAGAACAAAGACAAAGAAAACAAAAGAAAGCAATTGAAAACAAAA CAAAACAATTTTCATTCCTTCTCTTATCATTCCTTTTCTTTTCTTTTCTCTCATTCAACGCA CTCCATCGTATCCGTATTCCTCTTATTTTTTCTCTTTCTCTATATCCATTTCTTTCTCTCTAG GTGTGTCCTCTCTCTCTCTTCAATTTCTCTACTCCGCATTCCAACGCATCCTTCCCCCAAC CTCCCATTTCCTCCTTACGGCCCGATAGCGATCGTCTTTCCCTCGCTATCACTCGCTACCG GCCCCTCCTCTGCACCGTAACCTCCTACGTATTTACCATATCATAAAGTTTTTTCCGACGC TTATCGCTGACCCCCTGTCGCCCTCCTATTGGCTTCCGGATTATCTTCTTGTCCATAAGGT GATCCATGCTTCCTGAAGATTCCCGAAATGTGTCCACTTTGGCGGGGAATCATTCCATCC ACTTCTTTCTCTCTCGCTTTCCTCATTCGGCGCTCCCCTTCCGCGTCTCATTGGTCTTCCG CTCCGTTTTTGCTTTGCCGATGTTACTTGGGGAGAGGTGCGATAATCCTTTCGCAAAAAC TCGGTTTGACGCCTCCCATGGTATAAATAGTGGGTGGTGGACAGGTGCCTTCGCTTTTCT TTAAGCAAGAGAATTCCATTGTCTTGACTATCACAAACTTTTAAGTCTTTTCTTTTTTCTA ACCACATAATGACTATTCCTGACAAGCAGTTGGCTGCCGTTTTCCACACCCACGGTGGTC CCGAGAACGTCAAGTTCGAGGAAGTCCCCGTCGCCGAGCCCGGTCAAGACGAGGTCTT GGTTAACATCAAGTACACCGGTGTCTGCCACACCGATTTACACGCTCTTCAAGGTGACT GGCCTCTTCCCGCCAAGATGCCTTTGATCGGTGGTCACGAAGGTGCTGGTGTCGTCGTC AAGGTCGGTGCCGGTGTCACTCGTCTTAAGATTGGTGACCGTGTTGGTGTCAAGTGGAT GAACTCTTCTTGCGGTAACTGCGAGTACTGTATGAAGGCTGAGGAGACCATCTGCCCTC ACATTCAACTTTCCGGTTACACCGTTGACGGTACTTTCCAACACTACTGCATTGCCAATG CCACCCATGCTACCATCATCCCCGAGTCCGTTCCCCTCGAGGTTGCTGCTCCCATCATGT GCGCTGGTATCACTTGCTATCGTGCCTTGAAGGAATCCAAGGTCGGCCCTGGTGAGTGG ATCTGCATTCCCGGTGCCGGTGGTGGTCTTGGCCATCTTGCCGTCCAATACGCCAAGGCT ATGGCTATGCGTGTTGTTGCCATTGATACTGGTGATGACAAGGCTGAGCTCGTCAAGTCC TTTGGTGCTGAGGTCTTCCTTGACTTCAAGAAGGAAGCCGACATGATTGAGGCTGTCAA GGCTGCCACCAACGGTGGTGCCCACGGTACCTTGGTCTTATCCACCTCCCCCAAGTCTTA CGAGCAAGCTGCTGGCTTTGCCCGTCCCGGTTCCACCATGGTCACTGTTTCCATGCCTGC CGGTGCCAAGCTCGGTGCTGATATCTTCTGGTTGACCGTTAAGATGCTTAAGATCTGCGG TTCTCACGTCGGTAACCGTATTGACTCTATCGAGGCTCTTGAATACGTTTCCCGTGGTCTC GTCAAGCCTTACTACAAGGTCCAACCCTTCTCTACTCTTCCCGACGTCTACCGTCTCATG CATGAGAACAAGATTGCCGGCCGTATCGTCTTGGACCTTTCCAAGTAAGGGAATGAGAA TGTGATCCACTTTTAATTCCTAATGAATACATGCCTATAGTTCTTTTCTTTTGTTCTTTATGT CGTTTTTCGATGGTACGGCCGTTGTCAATCTCAGTTTGTGTGCTTGGTTGCAGCTTGGTT TCAAATCTGTTCATCTCATGAATCTTTTACCATTTCACCACACGTTTATACCATTCTCTCAT AGAATCTTCATCAAACCATCTCGGGGTTAGAGTGGAAAGAAAGTCTTGTTCTTTTATTTC CTTTTTTCCATCTTCAAGGCTTTTCTTTTCTTCCTCCTCCTCGTTCATCTTGAGGTTTGAC GTGTCTGTTTAGAATTTTGAGCTGTTGCAGCATCTTATTTTTTGTTTTGCGAAAACGAAG CGCTTTACTCTCTTCATCAGTTGGACGATTGTACCTTTGAAAACCAACTACTTTTGCATGT TTTGTATAGAAATCAATGATATTAGAATCCCATCCTTTAA). The nucleotide sequence was aligned by the National Center for Biotechnology Information (NCBI) using local alignment search tool (BLAST), and the alignment showed that the nucleotide sequence had 100% similarity only to Schizosaccharomyces pombe genomic alcohol dehydrogenase (Schizosaccharomyces pombe pochololar alcohol dehydrogenase Adh1), indicating that the 3463bp nucleotide sequence corresponding to Schizosaccharomyces pombe alcohol dehydrogenase (Schizosaccharomyces pombe pochololar alcohol dehydrogenase Adh1) is a highly specific sequence of Schizosaccharomyces pombe. 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 obtained primer pairs, the upstream primer Adh1F-CAGTACTCGCAGTTACCGCA and the downstream primer Adh1R-TTGATCGGTGGTCACGAAGG, which are not capable of self-looping (self-looping), were selected using DNMAN software. As a possible primer pair, the PCR product is 108bp in length (part of the gene coding for the alcohol dehydrogenase of Schizosaccharomyces pombe). BLAST was performed in NCBI using the potential target primer pair as the upstream and downstream primers, and the alignment showed 100% similarity only to Schizosaccharomyces pombe, indicating that the potential target primer pair of the selected primer pair was a specific primer pair suitable for this environment.
Example 2: screening of Schizosaccharomyces pombe (Schizosaccharomyces pombe) specific primers
First, fromScreening 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, placing the triangular flask in a shaking table at 30 ℃ and 200r/min, shaking and uniformly mixing for 30min, and standing for 5min to obtain bacterial suspension. Diluting the bacterial suspension, and taking 10-3、10-4And 10-5The three dilutions of the bacterial suspension were spread on YPD medium plates and incubated at 30 ℃ for 72h, and individual 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 PCR amplified using the eukaryotic microorganism universal forward primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') 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 Schizosaccharomyces pombe model strain in an NCBI website system to obtain specific information of a target strain, and screening Schizosaccharomyces pombe (Schizosaccharomyces pombe) from a mass of core yeasts.
Setting Primer Parameters (Primer Parameters) by using an NCBI Primer-BLAST tool with a target nucleotide sequence of 3463bp corresponding to Schizosaccharomyces pombe ethanol dehydrogenase (Schizosaccharomyces pombe pore dehydrogenase Adh 1); 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 obtained primer pairs, three additional primer pairs that were not able to loop by themselves (self-completion) were selected using DNAMAN software: primer pair 2, primer pair 3 and primer pair 4. The extracted genome of Schizosaccharomyces Pombe (SP) is used as a template, and four pairs of primers (Adh1F/Adh1R, primer pair 2, primer pair 3 and primer pair 4) are respectively used for PCR amplification (the PCR system is shown in Table 1). PCR reaction parameters: 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 (shown in FIG. 1) indicate that the amplification effect of the other primers is significantly less than that of the primer pair Adh1F/Adh1R of example 1.
The primer information is as follows:
and (3) primer pair 2:
F2：TTCCTGACCCTCGTACTGGT
R2：TGGCCGGTTTATCAGGTGTC
and (3) primer pair:
F3:：CAAGACCGTATCAGTCAGAGACA
R3：GAGAGAGAAGCGGATGGCTG
and (3) primer pair 4:
F4：CAACTGTGTTGACAGGCACG
R4：TTTGTAGAACCCCAGCGACC
TABLE 1 PCR verification System
| Reaction mixture | Volume(μL) |
| Mixture | 10 |
| |
1 |
| |
1 |
| |
1 |
| dd H2O | 7 |
| |
20 |
Example 3: verification of Schizosaccharomyces pombe (Schizosaccharomyces pombe) specific primer pair
Verification of specific primer pairs:
PCR was carried out using the extracted genome of Schizosaccharomyces Pombe (SP) as a template and the above-mentioned specific primers Adh1F-CAGTACTCGCAGTTACCGCA and Adh1R-TTGATCGGTGGTCACGAAGG as primers, and negative controls were core yeasts in the liquor brewing system, Pichia Kudriavzevii (PK), Saccharomyces Cerevisiae (SC), Saccharomyces bayiensis (Zygosaccharomyces bailii, ZB), Candida albicans (Candida hugilis, CH), Saccharomyces pastorianus (Kazachstania barnetii, KB) and distilled water (dd H) respectively2O). The PCR system and PCR conditions were the same as in example 2. After the PCR is finished, performing gel electrophoresis on the product, and observing bands on the gel through a gel imager to confirm the specificity condition of the primers, wherein as shown in FIG. 2, Schizosaccharomyces pombe has obvious target bands near 100-250bp, and the rest negative controls have no bands, the designed specific primer pair has the specificity to Schizosaccharomyces pombe. After sequencing the PCR product of Schizosaccharomyces pombe by Kinzymenia corporation, blast verification was performed on NCBI website to confirm that it was indeed the alcohol dehydrogenase gene on Schizosaccharomyces pombe (Schizosaccharomyces pombe) (as shown in FIG. 3).
Example 4: detection effectiveness and accuracy experiment of primer pair Adh1F/Adh1R
Five different strains of Schizosaccharomyces pombe (Schizosaccharomyces pombe) Schizosaccharomyces pombe NM-001023234, Schizosaccharomyces pombe CU329672.1, Schizosaccharomyces pombe NM-001023235.2, Schizosaccharomyces pombe NR-150068.1 and Schizosaccharomyces pombe AB001834, numbered P1, P2, P3, P4 and P5, were taken, and the respective genomes of the above 5 strains of Schizosaccharomyces pombe (Schizosaccharomyces pombe) were separately extracted. PCR was performed on each genome using the above-mentioned specific primers Adh1F-CAGTACTCGCAGTTACCGCA and Adh1R-TTGATCGGTGGTCACGAAGG as primers, 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 in the range of about 100-250bp (as shown in FIG. 4), which indicates that the primer can be used for really amplifying the ethanol dehydrogenase gene of Schizosaccharomyces pombe (Schizosaccharomyces pombe), thereby realizing the rapid identification of the Schizosaccharomyces pombe (Schizosaccharomyces pombe) in the sample.
Example 5: quantification of alcohol dehydrogenase Gene in sample
Schizosaccharomyces pombe (Schizosaccharomyces pombe) obtained in example 2 the number of cells was 9.9X 10 by the hemacytometer method7cells/mL, diluted to 10cells6、105、104、103、102After 10cells/mL, the genome is respectively extracted, and fluorescent quantitative PCR is carried out by using the specific primers Adh1F-CAGTACTCGCAGTTACCGCA and Adh1R-TTGATCGGTGGTCACGAAGG as primers and a StepOnePlus instrument (purchased from Saimer fly) (the fluorescent quantitative PCR system is shown in Table 2); the CT value measured by qPCR is used as an abscissa, the value of the cell corresponding to the dilution concentration is used as an ordinate, and a standard curve (the standard curve is shown in figure 5) is drawn, wherein y is-0.3020 x +12.5585, and R is20.9948, y represents Lg value of cell number, and x represents CT value of fluorescent quantitative PCR.
TABLE 2 qPCR validation System
| Reaction system | Volume (μ L) |
| Mixture | 10 |
| Upstream primer | 0.4 |
| Downstream primer | 0.4 |
| |
1 |
| dd H2O | 8.2 |
| |
20 |
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 Adh1F and Adh 1R. The experimental result shows that the PCR product has a band between 100 bp and 250bp, which indicates that the Daqu sample contains Schizosaccharomyces pombe (Schizosaccharomyces pombe).
Using "TIThe ANGEN Plant Genomic DNA Kit obtains the genome in the Daqu sample, takes the obtained genome as a template, takes the specific primers Adh1F-CAGTACTCGCAGTTACCGCA and Adh1R-TTGATCGGTGGTCACGAAGG as primers to carry out fluorescence quantitative PCR, the PCR reaction conditions are the same as example 5, the obtained CT value is substituted into the standard curve of figure 5, and the content of the saccharomyces cerevisiae in the Daqu sample is finally obtained as follows: 105cells/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> probes, kits and methods for rapid identification and quantification of Schizosaccharomyces pombe
<130> BAA201561A
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 3463
<212> DNA
<213> Schizosaccharomyces pombe
<400> 1
gatggatgga aagatttgaa aatacatagt gttggttgaa tttgcattgg gaggccggcg 60
gagcatgcga accattatag aataggcagt atgaaaattt agcaagcgat agctcaagtt 120
tgcatgatcg aatgagttat ccaaaaaata aactggagta ttgttgaatt cagttttgag 180
caaccgaatt cgcagtacct tcttacagta cctcctcaca gccaagtttc taccctagta 240
gagagtatcc agtttgtcgg tttgttggtt gcgaaaccag tatggacgat gttggttgtc 300
agattaagca gtgcaagttt tgcagcccat gttggtaagt gaagagagac aaataggatg 360
acaacgagcg gcagggcgag agcgaggcaa ggatgagagt aggaagaggg aagaaggaca 420
aaaatttgaa aaggatgtga atgtgaatgt gattgagttg gatgaaaatt tcgtctttac 480
atttgcccgt tataaacgag tgttgaatta ttcctatact acagatttat atatctacat 540
gattcgattc ctacaggatt gtaaacccac atgatttgta tacactataa tttgtatata 600
cctagtaatg attcttttat tctccactcc actaatatga aaatattctt gtatggttat 660
ccttatgtga tgaaatatct tttaaacgag gtccacgcgg atctccatat cagataaaaa 720
aaaatactac gacaaaagaa ggctaaagga atagggttga gtgctaggtg gatggtacga 780
tgttgtttgc cctaatacgg catactacta gggaaatgtt gggtgcatag gcctacaatg 840
ttagtcgtaa gagacgagac gagacgagac gagatgaacg aaataacccg ctcccaactc 900
tataaagggt taaggataga gagaaattga gtggtacgtt tggtgtgccg tttgtcgagc 960
cgtcgactgc tagtaatatt gtcattgtga ttaccattgc cattgtcaat gtcactgcta 1020
ttgctactga cactgccgct gtactcttct tccgatgtag ctttatttac attgtatcgg 1080
aaaccgtcca cacccatggt cgagagctgt gggtgacgca gacattcgaa tggcatgccc 1140
tacaacaact aagaaaatgg ctatcatgcg gaagctggtg agaagaacag catcgggaca 1200
agggaaggaa gaacaaagac aaagaaaaca aaagaaagca attgaaaaca aaacaaaaca 1260
attttcattc cttctcttat cattcctttt cttttctttt ctctcattca acgcactcca 1320
tcgtatccgt attcctctta ttttttctct ttctctatat ccatttcttt ctctctaggt 1380
gtgtcctctc tctctcttca atttctctac tccgcattcc aacgcatcct tcccccaacc 1440
tcccatttcc tccttacggc ccgatagcga tcgtctttcc ctcgctatca ctcgctaccg 1500
gcccctcctc tgcaccgtaa cctcctacgt atttaccata tcataaagtt ttttccgacg 1560
cttatcgctg accccctgtc gccctcctat tggcttccgg attatcttct tgtccataag 1620
gtgatccatg cttcctgaag attcccgaaa tgtgtccact ttggcgggga atcattccat 1680
ccacttcttt ctctctcgct ttcctcattc ggcgctcccc ttccgcgtct cattggtctt 1740
ccgctccgtt tttgctttgc cgatgttact tggggagagg tgcgataatc ctttcgcaaa 1800
aactcggttt gacgcctccc atggtataaa tagtgggtgg tggacaggtg ccttcgcttt 1860
tctttaagca agagaattcc attgtcttga ctatcacaaa cttttaagtc ttttcttttt 1920
tctaaccaca taatgactat tcctgacaag cagttggctg ccgttttcca cacccacggt 1980
ggtcccgaga acgtcaagtt cgaggaagtc cccgtcgccg agcccggtca agacgaggtc 2040
ttggttaaca tcaagtacac cggtgtctgc cacaccgatt tacacgctct tcaaggtgac 2100
tggcctcttc ccgccaagat gcctttgatc ggtggtcacg aaggtgctgg tgtcgtcgtc 2160
aaggtcggtg ccggtgtcac tcgtcttaag attggtgacc gtgttggtgt caagtggatg 2220
aactcttctt gcggtaactg cgagtactgt atgaaggctg aggagaccat ctgccctcac 2280
attcaacttt ccggttacac cgttgacggt actttccaac actactgcat tgccaatgcc 2340
acccatgcta ccatcatccc cgagtccgtt cccctcgagg ttgctgctcc catcatgtgc 2400
gctggtatca cttgctatcg tgccttgaag gaatccaagg tcggccctgg tgagtggatc 2460
tgcattcccg gtgccggtgg tggtcttggc catcttgccg tccaatacgc caaggctatg 2520
gctatgcgtg ttgttgccat tgatactggt gatgacaagg ctgagctcgt caagtccttt 2580
ggtgctgagg tcttccttga cttcaagaag gaagccgaca tgattgaggc tgtcaaggct 2640
gccaccaacg gtggtgccca cggtaccttg gtcttatcca cctcccccaa gtcttacgag 2700
caagctgctg gctttgcccg tcccggttcc accatggtca ctgtttccat gcctgccggt 2760
gccaagctcg gtgctgatat cttctggttg accgttaaga tgcttaagat ctgcggttct 2820
cacgtcggta accgtattga ctctatcgag gctcttgaat acgtttcccg tggtctcgtc 2880
aagccttact acaaggtcca acccttctct actcttcccg acgtctaccg tctcatgcat 2940
gagaacaaga ttgccggccg tatcgtcttg gacctttcca agtaagggaa tgagaatgtg 3000
atccactttt aattcctaat gaatacatgc ctatagttct tttcttttgt tctttatgtc 3060
gtttttcgat ggtacggccg ttgtcaatct cagtttgtgt gcttggttgc agcttggttt 3120
caaatctgtt catctcatga atcttttacc atttcaccac acgtttatac cattctctca 3180
tagaatcttc atcaaaccat ctcggggtta gagtggaaag aaagtcttgt tcttttattt 3240
ccttttttcc atcttcaagg cttttctttt cttcctcctc ctcgttcatc ttgaggtttg 3300
acgtgtctgt ttagaatttt gagctgttgc agcatcttat tttttgtttt gcgaaaacga 3360
agcgctttac tctcttcatc agttggacga ttgtaccttt gaaaaccaac tacttttgca 3420
tgttttgtat agaaatcaat gatattagaa tcccatcctt taa 3463
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence
<400> 4
tccgtaggtg aacctgcgg 19
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
tggccggttt atcaggtgtc 20
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence
<400> 8
caagaccgta tcagtcagag aca 23
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
Claims (10)
1. A primer for identifying Schizosaccharomyces pombe (Schizosaccharomyces pombe) is characterized by having a sequence shown in SEQ ID NO: 2. SEQ ID NO:3, respectively.
2. A method for identifying Schizosaccharomyces pombe (Schizosaccharomyces pombe) 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 to carry out colony PCR, 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 Schizosaccharomyces pombe, and if the PCR product is not between 100-250bp or has no band, the sample does not contain the Schizosaccharomyces pombe.
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 comprises Pichia kudriavzevii (Pichia kudriavzevii), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces bailii (Zygosaccharomyces bailii), Candida albicans (Candida hugalis), and Saccharomyces pastorianus barnetii (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 quantitative determination of Schizosaccharomyces pombe (Schizosaccharomyces pombe) using the primer according to claim 1, comprising the steps of:
(1) preparing schizosaccharomyces pombe 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 3, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the quantity of the schizosaccharomyces pombe in the sample.
7. A method for distinguishing schizosaccharomyces pombe from Pichia kudriavzevii (Pichia kudriavzevii), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces bailii (Zygosaccharomyces bailii), Candida albicans (Candida humulis), and Saccharomyces pastorianus barnetii (Kazachstania barnetii), characterized in that the sequence of the sequence given in SEQ ID NO: 2. SEQ ID NO:3 as a primer, performing colony PCR or performing PCR by respectively using the genomes of the yeasts as templates, observing PCR products through gel electrophoresis, and if the PCR products on an electrophoretogram have bands between 100-250bp, indicating that the corresponding strain is Schizosaccharomyces pombe (Schizosaccharomyces pombe).
8. A qualitative and quantitative detection kit for Schizosaccharomyces pombe (Schizosaccharomyces pombe), which 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 the qualitative detection of schizosaccharomyces pombe 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, performing colony PCR by using the sequence shown in the figure as a primer, observing a PCR product through gel electrophoresis, and if the PCR product on an electrophoretogram has a band between 100 and 250bp, indicating that the sample contains Schizosaccharomyces pombe (Schizosaccharomyces pombe).
10. A method for quantitative detection of schizosaccharomyces pombe using the kit of claim 8, comprising: (1) preparing schizosaccharomyces pombe 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 3, substituting the CT value of the fluorescence quantitative PCR into the standard curve, and converting to obtain the quantity of the schizosaccharomyces pombe in the sample.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| 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 |
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Application publication date: 20210423 |