CN103627805B - Method for rapidly detecting industrial saccharomyces pastorianus higher alcohol metabolizing genes - Google Patents
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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Abstract
The invention relates to biotechnologies and particularly relates to a method for rapidly detecting industrial saccharomyces pastorianus higher alcohol metabolizing genes. The method comprises the following steps: extracting RNA, preparing a cDNA template through reverse transcription reaction, performing multiple PCR, performing electrophoresis on blood capillaries, analyzing product segments, and metabolizing related genes BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc and BAT2-Sb for the industrial saccharomyces pastorianus higher alcohol. According the invention, the GeXP multiple-gene expression quantitative analysis technology is adopted, compared with the conventional gene expression quantitative analysis technology (fluorescent quantitative PCR), the GeXP multiple-gene expression quantitative analysis technology has the characteristics of relatively strong specificity and sensitivity, and high degree of automation, so that the reliability and repeatability of results are ensured, and the experimental period is shortened greatly. The metabolism and content of higher alcohol are controlled during the process of fermentation under such condition that beneficial to different productions, and the typical tastiness and uniformity of beer are improved; product features are highlighted and new products are developed by rapidly regulating tastiness of the higher alcohol in the beer.
Description
Technical field
The present invention relates to biotechnology, particularly a kind of method of rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene.
Background technology
Beer is a kind of low ethanol content of being loved by the people and nutritious beverage.Current global Beer is the beer of Lager type more than 90%, and the Saccharomyces uvarum (being commonly called as " Lager yeast " or " saccharomyces pastorianus ") therefore for brew Lager beer is widely used in brewing industry most.Lager yeast is also different from 1996 carry out genome sequencing yeast saccharomyces cerevisiae (S.cerevisiae) as model animals, but a kind of allopolyploid bacterial classification with highly coloured body ploidy, chromosome structure composition complexity, except carrying a part of genes of brewing yeast, also carry the genetic information of bayesian yeast (S.bayanus), be named as saccharomyces pastorianus (Saccharomyces pastorianus).Therefore all Sc-(S.cerevisiae is existed for the most gene in saccharomyces pastorianus) and Sb-(S.bayanus) two homologous genes.But, compared with the yeast saccharomyces cerevisiae entirely checked order, for gene and the expression of described gene in Process of Beer Brewing of bottom yeast specific participation brewing character, very limited at present.
In the polycomponent flavor systems of beer, higher alcohols is the essential substance forming the overall special favor of beer, plays critical effect to the quality of beer and the formation of local flavor typicalness.Higher alcohols can promote that wine body has plentiful fragrance and taste, and increases the Harmony of wine.Higher alcohols is produced by cereuisiae fermentum metabolism in beer fermentation process.Yeast cell participates in the yeast gene expression of relevant flavour substances synthesis by transcriptional control impact, finally causes the change of higher alcohols content.Therefore, to the research that the key gene participating in higher alcohols synthesis in industrial saccharomyces pastorianus fermenting process is expressed, be the key of Reasonable Regulation And Control higher alcohols content.
The synthesis of higher alcohols is directly related with amino acid whose metabolic process, has two pathways metabolisms: catabolism approach and metabolic pathway of synthesizing.Catabolism approach is also known as Ehrlich degradation pathway, and amino acid forms alpha-ketoacid under the effect of transaminase; Keto-acid decarboxylase becomes aldehyde; Aldehyde is reduced to higher alcohols further under the effect of desaturase.By Ehrlich approach, specific amino acid can form specific higher alcohols.Anabolism provides biosynthesizing amino acid whose carbon skeleton by carbohydrate, and in its synthetic mesophase stage, form alpha-ketoacid intermediate, decarboxylation and reduction thus, forms corresponding higher alcohols.
At present, the portion gene participating in Ehrlich approach and higher alcohols synthesis has been identified.Branched-chain amino acid in yeast, comprises leucine, Isoleucine and α-amino-isovaleric acid, is all that the movement system mediated by the branched-chain amino acid permease of BAP2 genes encoding is transported in yeast cell.The disappearance of BAP2 gene reduces 20-50% by causing yeast to the absorbed dose of leucine, Isoleucine and α-amino-isovaleric acid.Because accumulation amino acid whose in cell is relevant with Ehrlich approach, therefore just there is dependency in the expression of BAP2 gene and the formation of higher alcohols.The primary rate-limiting step of branched-amino acid metabolic is exactly by the transamination being positioned at plastosome and the catalysis of intracellular branched-chain amino acid transaminase (BCAT) institute.This enzyme is encoded by BAT1 and BAT2 and is formed, and catalysis branched-chain amino acid is to the transamination of alpha-ketoacid, and wherein alpha-ketoacid is the precursor of higher alcohols synthesis.Research shows, in yeast, the overexpression of BAT1 gene and BAT2 gene all can cause the increase of higher alcohols output.The deletion mutantion strain of BAT2 gene causes primary isoamyl alcohol to decrease 40% and 72% with the content of isopropylcarbinol respectively compared with wild strain.Therefore, BAP2, BAT1, BAT2 gene is considered to the key gene in higher alcohols metabolic process, and the level detecting BAP2, BAT1, BAT2 genetic expression may be used for studying industrial saccharomyces pastorianus higher alcohols metabolic regulation mechanism.
At present, fluorescence quantifying PCR method is the method for now conventional quantification of mrna.But due to the limitation that the method flux is not high, therefore need one fast and accurately technology the multiple higher alcohols Metabolism-Related Genes Expression of saccharomyces pastorianus is detected simultaneously.Beckman Coulter company of the GenomeLab GeXP Genetic Analysis System(U.S.) be the technology platform being mainly used in studying multi-gene expression quantitative analysis, adopt the method for capillary electrophoresis and fluorescent mark amplified production, the gene expression abundance of nearly 30 genes can be detected simultaneously.Multi-gene expression regulation and control, medicine virulence Mechanism Study, early diagnosis of cancer research etc. are widely used at present.
Summary of the invention
The object of the invention is open one adopts GeXP multiple gene expression quantitative analysis tech to detect industrial saccharomyces pastorianus higher alcohols metabolism related gene (BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb) method expressed, and then be conducive under different working condition, control fermenting process Higher Alcohols metabolism and content, improve local flavor typicalness and the consistence of beer; And can rapid adjustment High Alcohols local flavor be passed through, save R&D costs, accelerate the exploitation of product innovation.
Technical scheme of the present invention is:
A kind of method of rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene, comprise the extraction of total serum IgE, reverse transcription reaction prepares cDNA template, multi-PRC reaction, capillary vessel electrophoresis, product sheet piecewise analysis, for industrial saccharomyces pastorianus higher alcohols metabolism related gene BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb, it is SEQ ID NO.2 that reverse transcription reaction prepares cDNA template application primer, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ IDNO.10, SEQ ID NO.12, primer SEQ ID NO.1 is applied in multi-PRC reaction, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11.
On the basis of above scheme, also comprise industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, the ACT1-Sb as internal reference gene, it is SEQ ID NO.14, SEQ ID NO.16 that reverse transcription reaction prepares cDNA template application primer; Primer SEQ ID NO.13, SEQ ID NO.15 is applied in multi-PRC reaction.
Higher alcohols in beer of the present invention, be commonly called as potato spirit, refer to the monovalence alcohols of more than 3 carbon atoms, mainly contain n-propyl alcohol, propyl carbinol, isopropylcarbinol, amylalcohol, primary isoamyl alcohol, phenylethyl alcohol, tryptophol, alcohol, sugar alcohol etc., wherein primary isoamyl alcohol accounts for more than 50% of total amount.
Concrete detecting step is as follows:
(1) use following multi-primers to carry out the detection of industrial saccharomyces pastorianus higher alcohols metabolism key gene:
Design meets GeXP multi-PRC reaction feature, detects the specificity upstream and downstream primer of industrial saccharomyces pastorianus higher alcohols Metabolism-Related Genes Expression: and as the specificity upstream and downstream primer (seeing the following form) of internal reference gene:
The specificity upstream and downstream primer of table 1 industrial saccharomyces pastorianus higher alcohols metabolism related gene and internal reference gene
Wherein every bar primer is mixed with the storage liquid of 100 μMs respectively, and upstream primer working fluid is 200nM;
(2) Total RNAs extraction and purifying: adopt general RNA extraction method and purification process, extracts the yeast total serum IgE obtaining purifying from yeast cell;
(3) reverse transcription reaction prepares cDNA template: with yeast total serum IgE for templated synthesis cDNA first chain, above-mentioned (1) in the downstream primer of multi-primers be Auele Specific Primer, reaction system is 20 μ L; Wherein NTC and RT
-for negative control.
Table 2 reverse transcription reaction prepares the reaction conditions of cDNA template
The reverse primer concentration of each gene of table 3
It is as follows that reverse transcription reaction prepares cDNA template reaction optimum configurations:
48℃1minutes；42℃60minutes；95℃5minutes。
(4) multiplex PCR: with cDNA first chain of above-mentioned synthesis for template, above-mentioned (1) in the upstream primer of multi-primers be Auele Specific Primer, carry out multiplexed PCR amplification reaction, 3 parallel pipes established by each sample, and archaeal dna polymerase is purchased from BeckmanCoulter;
The reaction conditions of table 4 multiplex PCR
| Composition | Volume (μ L) |
| 25mMMgCl 2 | 4.0μL |
| 5 × PCR damping fluid | 4.0μL |
| Archaeal dna polymerase | 0.7μL |
| Forward primer (200nM) | 2μL |
| CDNA first chain | 9.3μL |
Pcr amplification optimum configurations is as follows:
95 DEG C of denaturation 10minutes; 94 DEG C of sex change 30seconds; Annealing temperature 56 DEG C, 30seconds; 71 DEG C extend 1minute, circulate 35 times.
(5) multiple PCR products capillary electrophoresis: get 1 μ lPCR multiple products and be added to and point be equipped with in the hole of 95% deionized formamide (SLS) of 39 μ l and the upper model of 400bp Marker mixed solution, cover a dropstone wax oil with after liquid-transfering gun mixing.The dissociating buffer of 250 μ l is added in addition in the every hole of damping fluid plate.After all finishing, upper machine carries out capillary electrophoresis.Separation gel, dissociating buffer are purchased from Beckman Coulter;
(6) product sheet piecewise analysis: utilize GeXP system parameter to analyze capillary electrophoresis result, record result.
The invention has the beneficial effects as follows:
(1) adopt test kit of the present invention and detection method thereof, can sensitive, special, detect BAP2-Sc exactly, the level of BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc and BAT2-Sb genetic expression.The change of higher alcohols metabolism key gene expression amount, has reacted the generation situation of yeast higher alcohols, therefore may be used for studying industrial saccharomyces pastorianus higher alcohols metabolic regulation mechanism.
(2) the GeXP multiple gene expression quantitative analysis tech that the present invention adopts has stronger specificity and susceptibility, level of automation high compared with conventional Quantitative analysis of gene expression technology (quantitative fluorescent PCR), ensure that reliability and the repeatability of result.In addition, because GeXP multiple gene expression quantitative analysis tech can detect the gene expression abundance of nearly 30 genes simultaneously, experimental period is substantially reduced.
(3) the present invention is conducive under different working condition, controls fermenting process Higher Alcohols metabolism and content, improves local flavor typicalness and the consistence of beer; And can rapid adjustment High Alcohols local flavor be passed through, outstanding selling point and carry out the exploitation of product innovation.
Accompanying drawing explanation
Accompanying drawing 1 is the amplified production of ACT1-Sb gene and KANr gene;
Accompanying drawing 2 is the amplified production of BAT2-Sb gene and KANr gene;
Accompanying drawing 3 is the amplified production of ACT1-Sc gene and KANr gene;
Accompanying drawing 4 is the amplified production of BAP2-Sb gene and KANr gene;
Accompanying drawing 5 is the amplified production of BAT1-Sc gene and KANr gene;
Accompanying drawing 6 is the amplified production of BAP2-Sc gene and KANr gene;
Accompanying drawing 7 is the amplified production of BAT1-Sb gene and KANr gene;
Accompanying drawing 8 is the amplified production of BAT2-Sc gene and KANr gene;
Accompanying drawing 9 is the multiplex amplification product of higher alcohols genes involved and KANr gene;
Accompanying drawing 10 is the change of higher alcohols genes involved expression amount during the fermentation;
Embodiment
The specific embodiment of the present invention is as follows:
Embodiment 1
A kind of method of rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene, comprise the extraction of total serum IgE, reverse transcription reaction prepares cDNA template, multi-PRC reaction, capillary vessel electrophoresis, product sheet piecewise analysis, for industrial saccharomyces pastorianus higher alcohols metabolism related gene BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb, it is SEQ ID NO.2 that reverse transcription reaction prepares cDNA template application primer, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ IDNO.10, SEQ ID NO.12, primer SEQ ID NO.1 is applied in multi-PRC reaction, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11.Also comprise industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, the ACT1-Sb as internal reference gene, it is SEQ ID NO.14, SEQ IDNO.16 that reverse transcription reaction prepares cDNA template application primer; Primer SEQ ID NO.13, SEQ ID NO.15 is applied in multi-PRC reaction.
According to the full length sequence of BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb gene and reference gene ACT1-Sc, ACT1-Sb, the Auele Specific Primer using Primer Premier5 software design to be applicable to GeXP to detect, primer sequence is as follows:
Design meets GeXP multi-PRC reaction feature, detects the specificity upstream and downstream primer of industrial saccharomyces pastorianus higher alcohols Metabolism-Related Genes Expression: and as the specificity upstream and downstream primer (seeing the following form 1) of internal reference gene:
The specificity upstream and downstream primer of table 1 industrial saccharomyces pastorianus higher alcohols metabolism related gene and internal reference gene
Product length after single primer capillary electrophoresis result display amplification is consistent with expected product length, and is single band, and result as shown in figures 1-8.Illustrate that the primer of design has very strong specificity, be suitable for GeXP and detect.
2000L pilot scale fermentation:
Carry out 13 degree of wort fermentation tests of 2000L scale.
Canful yeast number 22.0 × 10
6individual/ml, canful temperature 8.5 DEG C, canful 24 hours deslagginves once, are warming up to 9.5 DEG C of fermentations naturally.When pol is down to 5.2 ° of BX, be warming up to 12 DEG C, carry out di-acetyl reduction.When pol is down to 3.8 ° of BX, sealed cans pressurize 0.1Mpa, deslagging once.When di-acetyl is down to 0.05mg/L, be cooled to 5 DEG C, after 24 hours, thoroughly drain the yeast at the bottom of clean tank and bits, then be cooled to 0 DEG C, during storage wine, arrange a yeast every three days.Get yeast cell sample in different time sections, extract RNA rapidly.
1, the extraction and purification (RiboPure of total serum IgE
tM-YeastKit, Ambion, production code member AM1926):
(1) get 750 μ L zirconium oxide beads in 1.5mL centrifuge tube (band screw-cap), about 2.5cm eminence;
(2) in yeast collection tube, be sequentially added into lysate (480 μ LLysis Buffer+48 μ L10%SDS+480 μ LPhenol:Cloroform:IAA), resuspended.Vortex oscillation 10-15s;
(3) mixed solution is transferred in ready 750 μ L zirconia bead centrifuge tubes, cover tightly;
(4) centrifuge tube is placed in turbula shaker concussion 10min(maximum speed of revolution), lysing cell;
(5) the centrifugal 5min of 16000g room temperature, by upper water phase transition in 10mL centrifuge tube;
(6) add 1.9mLBinding Buffer, mixing;
(7) add 1.25mL100% ethanol, mixing;
(8) get 700 μ L mixed solutions in Filter Cartridge+Collection Tube, the centrifugal 1min of 12000g, abandons collection liquid; Repeat this step, all shift to above-mentioned mixed solution;
(9) on Filter Cartridge, add 700 μ LWash Solution1, the centrifugal 1min of 12000g, abandons collection liquid;
(10) add 500 μ LWash Solution2/3, the centrifugal 1min of 12000g, abandons collection liquid.Repeat this step;
(11) the centrifugal 1min of 12000g, thoroughly removes liquid on film, is transferred to by Filter Cartridge in new Collection Tube;
(12) add the Elution Solution25-50 μ L of 95-100 DEG C of preheating to film central authorities, the centrifugal 1min of 12000g.Add 25-50 μ LElution Solution again to film central authorities, the centrifugal 1min of 12000g, namely obtain RNA and collect liquid;
(13) DNaseI process
50-100 μ LRNA Sample+1/10thvol10 × DNase I Buffer+4 μ LDNaseI, 37 DEG C of reaction 30min.Add 1/10th vol DNase Inactivation Reagent(and use front first vortex), mixing after vortex concussion, after room temperature places 5min, the centrifugal 2-3min of 12000g, DNase Inactivation Reagent is precipitated, RNA supernatant liquor is transferred in new pipe and preserves, the total serum IgE of purifying can be obtained.
2, reverse transcription reaction prepares cDNA template:
With the yeast cell RNA of purifying described above for template, use the GenomeLab of Beckman Coulter company
tMgeXP starts test kit, and the downstream primer of the multi-primers in above-mentioned table 1 is Auele Specific Primer, and with the total serum IgE of yeast cell for templated synthesis cDNA first chain, reaction system is 20 μ L.
GenomeLab
tMgeXP starts test kit (production code member: PNA85017), containing reverse transcription reaction damping fluid, ThermoScript II, PCR reaction buffer, KAN
rrNA, the water without DNA enzymatic/RNA enzyme, DNAMarker (DSS-400), mineral oil, GeXP sample-loading buffer; Archaeal dna polymerase (production code member: PNA85022); Separation gel (production code member: PNA608010); Dissociating buffer (production code member: PNA608012), all purchased from Beckman Coulter.
CDNA first chain synthesis reaction system arranges as follows, and wherein NTC and RT-is negative control:
Table 2 reverse transcription reaction prepares the reaction conditions of cDNA template
| Often pipe composition | NTC | RT - | Standard reaction |
| Without DNA enzymatic/RNA enzyme water | 8μL | 4μL | 3μL |
| 5 × reverse transcription buffer | 4μL | 4μL | 4μL |
| KAN rRNA (1:50 dilution) | 5μL | 5μL | 5μL |
| ThermoScript II | 1μL | 0 | 1μL |
| Downstream primer (according to concentration in table 2) | 2μL | 2μL | 2μL |
| RNA template (5-20ng/ μ L) | 0 | 5μL | 5μL |
The reverse primer concentration of each gene of table 3
| Gene | Product size (bp) | Reverse primer concentration (nM) |
| BAT1-sb | 286 | 500 |
| BAT1-sc | 233 | 125 |
| BAT2-sb | 142 | 500 |
| BAT2-sc | 332 | 500 |
| BAP2-sb | 225 | 500 |
| BAP2-sc | 280 | 500 |
| ACT1-sb | 135 | 0.5 |
| ACT1-sc | 176 | 7.8125 |
| KAN r | 325 | 50 |
CDNA first chain synthesis reaction optimum configurations is as follows:
48℃ 1minutes；
42℃ 60minutes；
95℃ 5minutes。
3、RT-PCR：
RT-PCR adopts archaeal dna polymerase and the GenomeLab of BeckmanCoulter company
tMgeXP starts test kit and carries out.With cDNA first chain of synthesis in above-mentioned 2 for template, the upstream primer of the multi-primers in above-mentioned table 1 is Auele Specific Primer, carries out RT-PCR amplified reaction, and 3 parallel pipes established by each sample.
RT-PCR amplification reaction system arranges as follows:
The reaction conditions of table 4 multiplex PCR
| Composition | Volume (μ L) |
| 25mMMgCl 2 | 4.0μL |
| 5 × PCR damping fluid | 4.0μL |
| Archaeal dna polymerase | 0.7μL |
| Forward primer (200nM) | 2μL |
| CDNA first chain | 9.3μL |
RT-PCR Amplification arranges as follows:
95 DEG C of denaturation 10minutes; 94 DEG C of sex change 30seconds; Annealing temperature 56 DEG C, 30seconds; 71 DEG C extend 1minute, circulate 35 times.
4, multiple PCR products capillary electrophoresis: get 1 μ lPCR multiple products and be added to and point be equipped with in the hole of 95% deionized formamide (SLS) of 39 μ l and the upper model of 400bpMarker mixed solution, cover a dropstone wax oil with after liquid-transfering gun mixing.The dissociating buffer of 250 μ l is added in addition in the every hole of damping fluid plate.After all finishing, upper machine carries out capillary electrophoresis.Separation gel, dissociating buffer are purchased from Beckman Coulter.
5, product sheet piecewise analysis: utilize GeXP system parameter to analyze capillary electrophoresis result, record result, is shown in Fig. 9.
Figure 10 is the expression change during the fermentation of industrial saccharomyces pastorianus higher alcohols metabolism key gene.The expression amount of BAP2-Sc and BAP2-Sb gene slowly increases with fermentation time as seen from the figure, but the expression amount of the two significantly increases after 86h, especially BAP2-Sb gene.The expression amount of BAT1-Sc and BAT1-Sb gene continues to increase during the fermentation, but the expression amount of BAT1-Sc is higher than the expression amount of BAT1-Sb.The expression of BAT2-Sc and BAT2-Sb gene continued to increase in early stage, and the latter two expression amount of 110h all starts to reduce.
According to method provided by the invention, industrial saccharomyces pastorianus higher alcohols metabolism key gene (BAP2-Sc, BAP2-Sb can be detected fast simultaneously, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb) expression level, for studying industrial saccharomyces pastorianus higher alcohols metabolic regulation mechanism.The present invention is conducive under different working condition, controls fermenting process Higher Alcohols metabolism and content, improves local flavor typicalness and the consistence of beer; And can rapid adjustment High Alcohols local flavor be passed through, outstanding selling point and carry out the exploitation of product innovation.
Sequence table
<110> Qingdao Beer Co., Ltd.
The method of <120> rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 35
<212> primer
<213> BAP2-Sc
<400> 1
aggtgacact atagaatagc tggtccaaga ctgat 35
<210> 2
<211> 35
<212> primer
<213> BAP2-Sc
<400>2
gtacgactca ctatagggat tgtctgaatc gcagatg 37
<210> 3
<211> 36
<212> primer
<213> BAP2-Sb
<400>3
aggtgacact atagaatagt agtatttttg gcaccg 36
<210> 4
<211> 42
<212> primer
<213> BAP2-Sb
<400>4
gtacgactca ctatagggaa gctttatatc ctaactcatc ta 42
<210> 5
<211> 36
<212> primer
<213> BAT1-Sc
<400>5
aggtgacact atagaatatc agaacactcg ctactg 36
<210> 6
<211> 39
<212> primer
<213> BAT1-Sc
<400>6
gtacgactca ctatagggaa gagaaagatt accgtaagg 39
<210> 7
<211> 37
<212> primer
<213> BAT1-Sb
<400>7
aggtgacact atagaatagc cttatgctaa tttatcg 37
<210> 8
<211> 37
<212> primer
<213> BAT1-Sb
<400>8
gtacgactca ctatagggac cattcccttc aggaact 37
<210> 9
<211> 37
<212> primer
<213> BAT2-Sc
<400>9
aggtgacact atagaatacc gaacagtgag ttagtgt 37
<210> 10
<211> 40
<212> primer
<213> BAT2-Sc
<400>10
gtacgactca ctatagggat tagggtaatc aactcttctg 40
<210> 11
<211> 36
<212> primer
<213> BAT2-Sb
<400>11
aggtgacact atagaataga cgaatttgtc tgccag 36
<210> 12
<211> 37
<212> primer
<213> BAT2-Sb
<400>12
gtacgactca ctatagggaa agagtagccc tttccct 37
<210> 13
<211> 36
<212> primer
<213> ACT1-Sc
<400>13
aggtgacact atagaatata acgttccagc cttcta 36
<210> 14
<211> 39
<212> primer
<213> ACT1-Sc
<400>14
gtacgactca ctatagggag tgaggtagag agaaaccag 39
<210> 15
<211> 37
<212> primer
<213> ACT1-Sb
<400>15
aggtgacact atagaatagt atcttgacat tgcgtta 37
<210> 16
<211> 40
<212> primer
<213> ACT1-Sb
<400>16
gtacgactca ctatagggat ctcaactcgt tgtaaaaggt 40
Claims (4)
1. the method for a rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene, comprise the extraction of total serum IgE, reverse transcription reaction prepares cDNA template, multi-PRC reaction, capillary electrophoresis, product sheet piecewise analysis, it is characterized in that for industrial saccharomyces pastorianus higher alcohols metabolism related gene BAP2-Sc, BAP2-Sb, BAT1-Sc, BAT1-Sb, BAT2-Sc, BAT2-Sb, it is SEQ ID NO.2 that reverse transcription reaction prepares cDNA template application primer, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.12 synthesizes cDNA first chain, in multi-PRC reaction with described cDNA first chain for template, application primer be SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.11.
2. the method for rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene according to claim 1, characterized by further comprising industrial saccharomyces pastorianus β-actin Gene A CT1-Sc, the ACT1-Sb as internal reference gene, it is that SEQ ID NO.14 and SEQ ID NO.16 synthesize cDNA first chain that reverse transcription reaction prepares cDNA template application primer; In multi-PRC reaction with described cDNA first chain for template, application primer be SEQ ID NO.13 and SEQ ID NO.15.
3. the method for rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene according to claim 2, it is characterized in that described reverse transcription reaction is prepared in cDNA template reaction system, described application primer SEQ ID NO.2 concentration is 500nM, SEQ IDNO.4 concentration be 500nM, SEQ ID NO.6 concentration be 125nM, SEQ ID NO.8 concentration be 500nM, SEQ ID NO.10 concentration be 500nM, SEQ ID NO.12 concentration be 500nM, SEQ ID NO.14 concentration be 7.8125nM, SEQ ID NO.16 concentration is 0.5nM; In the reaction system of described multi-PRC reaction, described application primer SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15 concentration are 200nM.
4. the method for rapid detection industry saccharomyces pastorianus higher alcohols metabolic gene according to claim 3, it is characterized in that in the reaction system of described multi-PRC reaction, total reaction system is 20 μ L, wherein, 25mM MgCl
24.0 μ L, 5 × PCR damping fluid 4.0 μ L, application primer 2 μ L, archaeal dna polymerase 0.7 μ L, cDNA the first chain 9.3 μ L; 95 DEG C of denaturation 10minutes; 94 DEG C of sex change 30seconds; Annealing temperature 56 DEG C, 30seconds; 71 DEG C extend 1minute, circulate 35 times.
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