CN112725496A - 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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Abstract
The invention discloses a method for rapidly identifying and quantifying schizosaccharomyces pombe in a sample, 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 Adh4, and the rapid qualitative and quantitative detection of the fission yeast schizosaccharomyces pombe in a fermentation system 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 method for rapidly identifying and quantifying schizosaccharomyces pombe in a sample, 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 may act as a dimer and rely on nad (p) + cofactors for reversible conversion of 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, ethanol dehydrogenase catalyzes acetaldehyde generationThe ethanol 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 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 Adh4F), SEQ ID NO:3 (primer Adh 4R).
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 Adh4 gene, 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 Adh4 of the fission yeast schizosaccharomyces pombe, but not amplifies gene segments of other 5 yeasts.
Drawings
FIG. 1: and identifying the result of agarose gel electrophoresis of different primer pairs, wherein the ratio of 1: specific primers used for the present invention, ADH4F, ADH4R, 2, 3, 4: primer pairs 2, 3, 4 designed with reference to a similar method.
FIG. 2: and (3) identifying the result of agarose gel electrophoresis of different yeasts, wherein the ratio of 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: verification of Schizosaccharomyces pombe ethanol dehydrogenase gene.
FIG. 4: agarose gel electrophoresis identification results of different schizosaccharomyces pombe strains, 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 specific enzyme of the Schizosaccharomyces pombe, namely Schizosaccharomyces pombe ethanol dehydrogenase (Schizosaccharomyces pombe ethanol dehydrogenase), is searched through Kyoto gene and genome encyclopedia metabolic Pathway (KEGG Pathway), so that 1850bp nucleotide sequence for coding the specific enzyme is obtained, and the sequence is as follows:
AAATATTTGATCTTCTACTTTTTGGTAAGTAAAATTTATGATAATGGCGGAAAGAATGACAGTTTGGAGGTGATTTGATGTCGTTTGGAAAAGAGTTTTGGTGACAAGCAATTTTGTTTTGAAAATCATTTGAAGATCTTTTAATTCCTTAGCATTTATTTGACGTTGGAGTTGGCTGCAGATCTATTTAGCGAAATTGTTGTAAAAAGATCGATGTTCGATAAGTGTTCGTTAAGCGAGATAAGGAGTGGAAGTATCGAAATTTTAGACACTAATTGATTACACATTGATATTGGAGAAATTTTCGTACACTACTTAAGGCTCATTCCCCCTTGACTTTTTTGACCGATTGTTACATATTTCTTTTCCGTTTGGCTAATTATTTATTTTTTTTCATACTTTGTATATTATTGATTCTAAAAAATATCGGAGGTTAAGCTTTAGATGCGATTTTCTTTTTAACATGTCCATTCTTCGTTCTCCATTTCGTTTAATTCGATCGCCTGCTCGATTCTTTCCGTCACTCTTTCATTCCTCTTGTAATCAATCTTTTACTAATGGCTTAAAGCATCAATCAACCTCTTCGAAAGCCATGCCTGTTAGCGCATTCTACATACCTTCTTTCAACCTTTTCGGTAAAGGTTGTCTTGCTGAAGCCGCCAAGCAAATTAAGATGAGCGGCTTCAAAAACACCTTAATTGTTACCGATCCTGGTATTATTAAGGTCGGTTTGTATGACAAAGTTAAGGCTCTGTTGGAAGAACAAAGCATCACTGTTCACCTTTACGATGGTGTTCAACCTAATCCCACCGTTGGAAATGTCAACCAAGGTCTCGAAATCGTTAAGAAGGAAAACTGTGATTCTATGGTATCAATTGGTGGTGGTTCTGCTCACGATTGTGCCAAAGGTATTGCTTTGTTAGCTACTAATGGCGGTAAAATTGCGGATTATGAAGGTGTTGATAAATCCTCCAAACCTCAACTTCCCTTGATTGCAATCAATACGACTGCAGGAACTGCTTCTGAAATGACTCGTTTCGCAATTATCACCGAAGAGACCCGTCATATTAAAATGGCTATTATCGATAAGCATACTATGCCTATCCTTTCCGTTAACGATCCTGAAACAATGTATGGATTACCTCCTTCCTTGACCGCTGCTACTGGTATGGACGCTTTGACTCATGCTGTTGAAGCTTACGTCTCTACTGCTGCTAATCCCATCACCGATGCTTGTGCTGTCAAATGCATCGAACTCGTTAATAAGTATTTGAAGCGTGCTGTCGACAACGGCAAGGACGAAGAGGCTAGAGACAATATGGCTTATGCCGAATTCCTTGGTGGTATGGCATTTAACAACGCATCTTTGGGTTATGTTCATGCTATGGCTCATCAACTTGGTGGTTTCTACGGTATTCCACATGGCGTCTGTAACGCTGTTCTTCTTGCCCATGTACAGAAATTTAATTCTAGGGATCCAAGAGCAAATGCTCGCCTTGGTGATATTGCTTTTCACTTGGGTTGTGAAGAGCATACTGCCGAAGCAGCTCTTGACCGCATCAGTCAACTTGTTCTTGAAGTTAAAATTCGTCCTCATCTAGTTGATCTAGGTGTCAAGGAAAAAGATTTTGATGTCTTGGTTGACCACGCTATGAAGGATGCTTGTGGTGCTACTAACCCTATTCAGCCTACACATGACGAGGTAAAGGCTATTTTCAAATCGGCTATGTAAATAGAACGTTATCTCGATACCTAGAACAAATTATGAGCTTTTACATTTCACTGGCTTGAATGTTCACTTCAACATATCCTAAAGCATTTTTTTTGGCTTGAACCTTTTTTAGATTTGG。
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 Adh4), indicating that the 1850bp nucleotide sequence corresponding to Schizosaccharomyces pombe alcohol dehydrogenase (Schizosaccharomyces pombe pochololar alcohol dehydrogenase Adh4) 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 pair primer pairs obtained, the DNMAN software was used to select the upstream primer Adh4F-TGGACGCTTTGACTCATGCT and the downstream primer Adh4R-ACCACCAAGGAATTCGGCAT that were not capable of self-looping (self-completion). As a possible primer pair, the PCR product is 160bp 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 selected primer pair potential target primer pair was a specific primer pair suitable for this environment.
Example 2: optimization of Schizosaccharomyces pombe (Schizosaccharomyces pombe) 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 for shaking and mixing uniformly 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 preserving 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 model strain in an NCBI website system to obtain specific information of a target strain, and screening Schizosaccharomyces pombe (Schizosaccharomyces pombe) from a crowd of core yeasts.
Setting Primer Parameters (Primer Parameters) by using an NCBI Primer-BLAST tool with a target of 1850bp nucleotide sequence corresponding to Schizosaccharomyces pombe ethanol dehydrogenase (Schizosaccharomyces pombe pore dehydrogenase Adh 4); 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, other three pairs of primers which could not loop by themselves (self-completion) were selected by using DNAMAN software. The extracted genome of Schizosaccharomyces Pombe (SP) is used as a template, and the four pairs of primers (Adh4F/Adh4R, primer pair 2, primer pair 3 and primer pair 4) are respectively used for 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 was significantly less than that of the primer pair Adh4F/Adh4R of example 1.
The primer information is as follows:
and (3) primer pair 2:
F2：GAGGGATCGTTGACTGCAACA
R2：AATTGGAGATTACGAGGGGGTTA
and (3) primer pair:
F3：TGAATGAGCTAAACCAACCCT
R3：TCCAATGGGTAGTAGTGGTGC
and (3) primer pair 4:
F4：GGGCAACCCTACGGAAATGA
R4：TGCAGACGCAGAGAAGATGG
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:
the extracted genome of Schizosaccharomyces Pombe (SP) is taken as a template, and the specific primers Adh4F-TGGACGCTTTGACTCATGCT and Adh4R-ACCACCAAGGAATTCGGCAT are adopted. As primers, PCR was performed, and negative controls were core yeast Saccharomyces cerevisiae in the white spirit brewing system: pichia Kudriavzevii (PK), Saccharomyces Cerevisiae (SC), Saccharomyces cerevisiae (Zygosaccharomyces bailii, Zb), Candida albicans (Candida hugilis, CH), Saccharomyces pastorianus (Kazachstania barnetii, KB) and redistilled water (dd H)2O). The PCR system and PCR conditions were the same as in example 2. After the PCR is finished, the product is subjected to gel electrophoresis, and the specificity of the primer is confirmed by observing bands on the gel through a gel imager, wherein Schizosaccharomyces pombe has a remarkable target band in the vicinity of 100-250bp, and the rest negative controls have no bands (as shown in figure 2), which indicates that the designed specific primer pair has the specificity to Schizosaccharomyces pombe. The PCR product of Schizosaccharomyces pombe is sequenced by Jinzhi company, then blast verification of NCBI website is carried out, and the result is verified to be Schizosaccharomyces pombe (Schizosaccharomyces pombe)Ethanol dehydrogenase gene from Schizosaccharomyces pombe) (see FIG. 3).
Example 4: detection effectiveness and accuracy experiment of primer pair Adh4F/Adh4R
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 Adh4F-TGGACGCTTTGACTCATGCT and Adh4R-ACCACCAAGGAATTCGGCAT 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
The concentration of the genome of Schizosaccharomyces pombe (Schizosaccharomyces pombe) obtained in example 2 was measured, and as a result, it was shown that the concentration was 464 ng/. mu.L and the number of cells was 9.9X10 when examined by plate counting method7cells/g, the genome was diluted to 10cells6、105、104、103、102After 10cells/g, fluorescence quantitative PCR was performed by using the diluted genome as a template, the specific primers Adh4F-TGGACGCTTTGACTCATGCT and Adh4R-ACCACCAAGGAATTCGGCAT as primers and a StepOnePlus apparatus (purchased from Saimer fly) (the fluorescence quantitative PCR system is shown in Table 2); 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
| 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 suspensions of the three dilutions are thinly spread on YPD medium plates and cultured in an incubator at 30 ℃ for 72h, single colonies on the plates are selected and numbered, and colony PCR is performed by using specific primers Adh4F and Adh 4R. The experimental result shows that the PCR product has a band between 100 bp and 250bp, which indicates thatThe Daqu sample contains Schizosaccharomyces pombe (Schizosaccharomyces pombe).
Obtaining a 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 primers Adh4F-TGGACGCTTTGACTCATGCT and Adh4R-ACCACCAAGGAATTCGGCAT as primers, wherein the PCR reaction conditions are the same as example 5, and substituting the obtained CT value 23.989 into a standard curve shown in a figure 5 to finally obtain the content of the saccharomyces cerevisiae in the Daqu sample, wherein the content of the saccharomyces cerevisiae in the Daqu sample is 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> method for rapidly identifying and quantifying schizosaccharomyces pombe in 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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<213> Artificial sequence
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<210> 6
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<213> Artificial sequence
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gagggatcgt tgactgcaac a 21
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<213> Artificial sequence
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aattggagat tacgaggggg tta 23
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tgaatgagct aaaccaaccc t 21
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tccaatgggt agtagtggtg c 21
<210> 10
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<211> 20
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<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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| 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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| 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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| BROWN, STEVEN RICHARD: "A Design of Experiments Approach for Engineering Carbon Metabolism in the Yeast Saccharomyces cerevisiae", UNIVERSITY OF EXETER, pages 67 * |
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