CN102296117B - Rapid qualification and quantitation measurement method of saccharomyces cerevisiae in additive premix sample - Google Patents
Rapid qualification and quantitation measurement method of saccharomyces cerevisiae in additive premix sample Download PDFInfo
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Abstract
The invention belongs to the detection technical fields of feed science and feed additives, and more specifically relates to a rapid qualification and quantitation measurement method of saccharomyces cerevisiae in an additive premix sample. According the invention, cDNA gradient dilution is taken as an external standard substance used for distinguishing dead bacteria and live bacteria for accurately detecting the amount of the live bacteria in a sample to be measured. The invention is capable of measuring saccharomyces cerevisiae in premix sample with simple, rapid and accurate qualification and quantitation without pure culture of saacharomyces cereisiae, the whole process has the advantages of simple detection process, high detection efficiency, high accuracy and short detection period; and the invention provides a guarantee a long term development for a microecological preparation industry.
Description
Technical field
The invention belongs to forage science and fodder additives detection technique field, be specifically related to fast qualitative, the method for quantitatively determining of yeast saccharomyces cerevisiae in a kind of additive premix sample.
Background technology
Microbial forage additive becomes the focus that Preblend field people pay close attention to because of its abundant nutritive value and special prebiotic effect and environmental protection characteristic.Microbial forage additive is in the market of a great variety, and at full speed increases every year.Although the application of microbial forage additive on livestock breeding industry is increasingly extensive, but not yet form unified quality standard and perfect administrative mechanism, to the supervision of its quality and authentication, especially aspect qualitative and quantitative analysis, lack science, rationally, method fast, trace it to its cause is that fundamental research is weak, existing detection technique or sense cycle be long, it is high maybe can not to distinguish dead bacterium viable bacteria or cost, is difficult to Criterion detection technique.Qualitative and quantitative analysis to S. cervisiae in traditional method, still adopts the method for biochemical reactions and selective medium isolation of pure culture technology.Traditional method is subject to external environment factor and cultivates the impact of performance, detected result deficient in stability and reliability, and take time and effort.Present stage is badly in need of from producing reality, progressively sets up scientific and effective, to be easy to popularization rapidly and efficiently stdn detection technique of feeding bacterial classification, promotes the Sustainable development of microbial forage additive industry.
Round pcr is once occurring being just widely used in the fields such as molecular biology and microbiology.The appearance of real-time fluorescence quantitative PCR (Real-time PCR) has realized the leap of round pcr from qualitative to quantitative especially, has avoided the problem of crossed contamination in normal PCR Quantitative measurement.There is high specificity, the advantage such as reproducible, accurate, quick, become the important method of molecular biology and microorganism field detection by quantitative.
Summary of the invention
[technical problem that will solve]
The object of the present invention is to provide the fast qualitative measuring method of yeast saccharomyces cerevisiae in a kind of additive premix sample (Saccharomyces cerevisiae).
Another object of the present invention is to provide the quantitative determination method of yeast saccharomyces cerevisiae in a kind of additive premix sample (Saccharomyces cerevisiae).
The present invention adopts wine brewing species specificity PCR and Real-Time Fluorescent Quantitative PCR Technique, sets up easy, yeast saccharomyces cerevisiae rapid molecular detection method in additive premix fast and accurately, can simplify yeast saccharomyces cerevisiae trace routine, improve detection efficiency.
[technical scheme]
The present invention is achieved through the following technical solutions:
The invention provides the fast qualitative measuring method of yeast saccharomyces cerevisiae in a kind of additive premix sample (Saccharomyces cerevisiae), it is template that the method is used the DNA of yeast saccharomyces cerevisiae, species specificity primer with the 26s rDNA gene order of yeast saccharomyces cerevisiae carries out species specificity PCR reaction, amplification sheet degree length is 134bp, and then fast qualitative yeast saccharomyces cerevisiae; Described species specificity primer is as follows: upstream primer DZf5 '-CGAGAGACCGATAGCGAACA-3 ', downstream primer DZr5 '-AAGGAGCAGAGGGCACAAA-3 '.
Further, the step of the method is as follows:
A. the preparation of template DNA
Adopt glass bead method to prepare Feed Sample template DNA:
(1) get Preblend sample that 1g contains S. cervisiae in 1.5mL centrifuge tube, add 500 μ LPBS suspended sample, the centrifugal 3min of 2500 * g;
(2) abandon supernatant, add the granulated glass sphere of appropriate pickling, concuss 2min;
(3) add 400 μ L TE and the saturated phenol of isopyknic Tris, upset mixes, the centrifugal 5min of 15000 * g;
(4) shift the new centrifuge tube of water to, add equal-volume phenol: chloroform: primary isoamyl alcohol=25: the mixture of 24: 1, put upside down and mix, the centrifugal 5min of 15000 * g;
(5) suct the new centrifuge tube of clearly to, add 1/10 volume 3mol/L NaAc and 2.5 times of volume 95% ice ethanol ,-20 ℃ are spent the night;
(6) 4 ℃ of centrifugal 10min of 15000 * g, abandon supernatant;
(7) add 1mL70% ethanol, the centrifugal 5min of 15000 * g, abandons supernatant;
(8) 37 ℃ of placement 15min are drying precipitated, and precipitation is dissolved in 60 μ L TE, and-20 ℃ save backup;
B.PCR amplification
Reaction system 25.0 μ L:12.5 μ L 2 * PCR-mix, each 1 μ L 10mmol/L upstream and downstream primer, 1 μ L 50ng/ μ L DNA profiling, redistilled water complement to 25.0 μ L;
PCR reaction conditions: 94 ℃ of denaturation 5min, 94 ℃ of 30s, 60 ℃ of 45s, 72 ℃ of 40s, carry out 35 circulations, 72 ℃ of final 7min that extend, the PCR product obtaining is preserved at 4 ℃ of temperature;
Get 5 μ LPCR products, 1% agarose gel electrophoresis detects;
C. result and judgement
During detection, establish and using the glue of object amplification sheet degree and reclaim product as the positive contrast of template, using aqua sterilisa as the negative contrast of template; According to occur expection feature band at 134bp place, determine in this Preblend sample and contain yeast saccharomyces cerevisiae, do not conform to and have yeast saccharomyces cerevisiae on the contrary.
The present invention also provides the quantitative determination method of yeast saccharomyces cerevisiae in a kind of additive premix sample (Saccharomyces cerevisiae), it is template that the method be take the cDNA of testing sample cDNA and standard substance, use upstream primer and the downstream primer described in qualitative test method, with identical system, carry out the fluorescent quantitative PCR of the gene fragment of yeast saccharomyces cerevisiae 26s rDNA D1/D2 simultaneously; By the Ct value of typical curve and testing sample, carry out the Quantitative detection of yeast saccharomyces cerevisiae in Preblend sample.
Further, the step of the method is as follows:
A. the preparation of the total RNA of sample
Adopt enzyme process to abolish brewing yeast cell wall, high-purity total RNA, rapid extraction test kit extracts the total RNA of sample: sample 0.5-1.0g joins in 1.5mL centrifuge tube, add 600 μ L sorbyl alcohol Buffer, add 50U Lyticase, fully mix 30 ℃ and process 30min, after the centrifugal 10min of 1500 * g, abandon supernatant, collecting precipitation, extract sample RNA, then with DNase I, process twice, obtain the RNA sample of purifying, use uv-spectrophotometric instrument to measure concentration, adopt 1% agarose gel electrophoresis to detect the integrity of RNA, then by Sample preservation in-80 ℃;
B. post transcription cloning
(1) in the centrifuge tube of the nuclease free of ice bath, add following reaction mixture:
The total RNA of 1-5 μ g, 2 μ L Oligo (dT) 15,2 μ LdNTP (2.5mM each), benefit RNase-freeddH2O are settled to 14.5 μ L;
After (2) 70 ℃ of heating 5min, rapidly at cooled on ice 2min, after centrifugal reaction solution, add fast following component: 4 μ L 5 * First-Strand Buffer (containing DTT); 0.5 μ L RNasin;
(3) add 1 μ L (200U) TIANScript M-MLV, with pipettor, mix gently;
(4) 42 ℃ of temperature are bathed 50min;
(5) 95 ℃ of heating 5min, termination reaction, puts and carries out subsequent experimental or freezing preservation on ice;
(6) with RNase-free ddH2O, reaction system is diluted to 50 μ L.-20 ℃ save backup;
C. quantitative fluorescent PCR
Reaction system 25.0 μ L:12.5 μ L 2 * SuperReal PreMix (containing SYBR Green I), each 0.75 μ L 10 μ mol/L upstream and downstream primer DZf and DZr, 2.0 μ L cDNA templates, 0.5 μ L 50 * ROXReference Dye, benefit RNase-free ddH2O to 25 μ L system;
Quantitative fluorescent PCR reaction parameter: 95 ℃ of denaturation 15min; 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, 40 circulations; 4 ℃ of preservations; Each sample repeats 3 times;
D. the preparation of outer standard substance and the drafting of typical curve
The preparation of outer standard substance: utilize spectrophotometer that the yeast saccharomyces cerevisiae fermented liquid of incubated overnight is diluted to 1OD, according to the total RNA step of said extracted and obtain cDNA by post transcription cloning, carry out 10 times of serial dilutions, gradient dilution is to the concentration of 107-1 yeast cDNA/ μ L, usings that this carries out quantitative fluorescent PCR reaction as outer standard substance;
The drafting of typical curve: the logarithm of template of different concns of take is X-coordinate, the typical curve of the S. cervisiae that the initial cycle number (Ct) that arrives fluorescence threshold in PCR reaction process of take obtains as ordinate zou, as the reference standard of testing sample quantitative assay;
E. result and judgement
The cDNA of testing sample cDNA and standard substance of take is template, with the species-specific primer of S. cervisiae, carries out the fluorescent quantitative PCR of the gene fragment of S. cervisiae 26s rDNA D1/D2 with identical system simultaneously; By the Ct value of typical curve and testing sample, carry out the Quantitative detection of S. cervisiae in Feed Sample.
[beneficial effect]
The invention has the beneficial effects as follows:
Primer provided by the present invention has high specificity, and the feature that amplification efficiency is high can be used for fast qualitative and identifies S. cervisiae.The present invention utilizes the grade dilution of cDNA can, for difference life or death bacterium, detect more accurately the viable count in testing sample as outer standard substance.The present invention can not carry out on the basis of S. cervisiae pure culture, S. cervisiae in easy, the sample of qualitative and quantitative analysis Preblend quickly and accurately, whole process is simple to the trace routine of yeast saccharomyces cerevisiae in Preblend sample, detection efficiency is high, accuracy good, the advantage that sense cycle is short; Thereby for the long term growth of probiotics industry provides technical guarantee.
Accompanying drawing explanation:
Fig. 1: through the species specificity primer PCR amplification of S. cervisiae, agarose gel electrophoresis detected result.
Fig. 2: the real time fluorescent quantitative that the DNA of take is template detects the typical curve of yeast saccharomyces cerevisiae.
Fig. 3: the real time fluorescent quantitative that the cDNA of take is template detects the typical curve of S. cervisiae.
Embodiment:
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1: in Preblend sample, the fast qualitative of yeast saccharomyces cerevisiae detects:
The glue of this embodiment application target amplified fragments reclaims product as positive control; Sterilized water is made negative control; Use in addition two parts of Feed Sample 1# and 2#, enterococcus faecalis, the Pichia yeast from market, collected to carry out as sample.
A. the preparation of template DNA
Adopt glass bead method to prepare Feed Sample template DNA, concrete steps are as follows:
(1) get Feed Sample that 1g contains S. cervisiae in 1.5mL centrifuge tube, add 500 μ LPBS suspended sample, the centrifugal 3min of 2500 * g;
(2) abandon supernatant, add the granulated glass sphere of appropriate pickling, concuss 2min;
(3) add 400 μ L TE and the saturated phenol of isopyknic Tris, upset mixes, the centrifugal 5min of 15000 * g;
(4) shift the new centrifuge tube of water to, add equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1), put upside down and mix, the centrifugal 5min of 15000 * g;
(5) suct the new centrifuge tube of clearly to, add 1/10 volume 3mol/L NaAc and 2.5 times of volume 95% ice ethanol ,-20 ℃ are spent the night;
(6) 4 ℃ of centrifugal 10min of 15000 * g, abandon supernatant;
(7) add 1mL70% ethanol, the centrifugal 5min of 15000 * g, abandons supernatant;
(8) 37 ℃ of placement 15min are drying precipitated, and precipitation is dissolved in 60 μ L TE, and-20 ℃ save backup.
B.PCR amplification
Reaction system 25.0 μ L:12.5 μ L 2 * PCR-mix, each 1 μ L 10mmol/L upstream and downstream primer, 1 μ L50ng/ μ L DNA profiling, redistilled water complement to 25.0 μ L;
PCR reaction conditions: 94 ℃ of denaturation 5min, 94 ℃ of 30s, 60 ℃ of 45s, 72 ℃ of 40s, carry out 35 circulations, 72 ℃ of final 7min that extend, the PCR product obtaining is preserved at 4 ℃ of temperature;
Get 5 μ LPCR products and use in 1% sepharose the voltage electrophoresis 20-30min with 5V/cm, EB dyeing, ultraviolet is taken pictures.
C. the glue of object fragment reclaims purifying
Adopt glue to reclaim test kit object fragment is cut to glue, recovery, purifying, obtain the object fragment of purifying.
D. result and judgement
Through the species specificity primer PCR amplification of S. cervisiae, agarose gel electrophoresis detected result is shown in Fig. 1.Swimming lane 1 is usingd the purified pcr product of object fragment as positive control, and swimming lane 2 is usingd aqua sterilisa as negative control.It is generally acknowledged that expection amplified band appears in positive feature at 134bp place, illustrate in this sample and contain S. cervisiae; Negative control is without feature band.The Feed Sample of swimming lane 3-4 for collecting from market, the positive property of result illustrates in these 2 parts of Feed Samples and contains S. cervisiae alive.Swimming lane 5-6 is pichia spp and the enterococcus faecalis outside S. cervisiae, and result is negative, illustrates that primer specificity is good, consistent with expected results.
Embodiment 2: the quantitative assay of yeast saccharomyces cerevisiae in Preblend sample
The different samples of mentioning in embodiment 1 are carried out to the quantitative assay of S. cervisiae.Its determination step is as follows:
A. the preparation of sample total DNA
Adopt enzyme process to abolish the high-purity total DNA rapid extraction test kit of yeast cells wall (centrifugal column type) and extract sample total DNA: sample 0.5-1.0g joins in 1.5mL centrifuge tube, add 600 μ L sorbyl alcohol Buffer, add 50U Lyticase, fully mix 30 ℃ and process 30min, after the centrifugal 10min of 1500 * g, abandon supernatant, collecting precipitation, by high-purity total DNA rapid extraction test kit specification sheets, extract sample DNA, use uv-spectrophotometric instrument to measure concentration, adopt 1% agarose gel electrophoresis to detect the integrity of DNA, then by Sample preservation in-20 ℃.
B. quantitative fluorescent PCR
Reaction system 25.0 μ L:12.5 μ L 2 * SuperReal PreMix (containing SYBR Green I), each 0.75 μ L 10 μ mol/L upstream and downstream primer DZf and DZr, 2.0 μ L DNA profilings, 0.5 μ L 50 * ROXReference Dye, benefit RNase-free ddH2O to 25 μ L system.
Quantitative fluorescent PCR reaction parameter: 95 ℃ of denaturation 15min; 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, 40 circulations; In 4 ℃ of preservations of temperature; Each sample repeats 3 times.
D. the preparation of outer standard substance and the drafting of typical curve
The preparation of outer standard substance: utilize spectrophotometer that the yeast saccharomyces cerevisiae fermented liquid of incubated overnight is diluted to 1OD (approximately 10
7cells), according to above-mentioned steps, extract total DNA, carry out 10 times of serial dilutions, gradient dilution to 10
7the concentration of-1 cerevisiae dna/μ L, usings that this carries out quantitative fluorescent PCR reaction as outer standard substance.The template of different concns of take is X-coordinate, and the typical curve of the S. cervisiae that the initial cycle number (Ct) that arrives fluorescence threshold in PCR reaction process of take obtains as ordinate zou, is shown in accompanying drawing 2.
E. result and judgement
As seen from Figure 2, the template concentrations of Ct value and different gradient dilutions is good linear relationship, the template of different concns of take is X-coordinate, Ct value is ordinate zou, the quantitative fluorescent PCR typical curve equation of drawing out: Y=-1.802X+26.115 (Y represents Ct value, and X represents the initial DNA concentration of template), between the concentration of the initial DNA of template and Ct value, linearly dependent coefficient is 0.994, slope is-1.802, and the typical curve of setting up meets the quantitative requirement of Real-Time PCR.
DNA and the standard substance DNA of testing sample of take is template, and the species-specific primer of the S. cervisiae of describing with embodiment 1 carries out the gene fragment fluorescent quantitative PCR of S. cervisiae 26s rDNA D1/D2 simultaneously with identical system.By typical curve with adopt the computer software software v2.0.1 of 7500 real-time fluorescence quantitative PCR instrument of ABI company to carry out the Quantitative detection of S. cervisiae in Feed Sample.The measurement result of these samples and analytical error result are listed in the table below in 1 in the lump.
The Real-time measurement result of table 1 sample
Embodiment 3: the quantitative assay of yeast saccharomyces cerevisiae in Feed Sample
The different samples of mentioning in embodiment 1 are carried out to the quantitative assay of S. cervisiae.Its determination step is as follows:
A. the preparation of the total RNA of sample
Adopt enzyme process to abolish the high-purity total RNA rapid extraction test kit of yeast cells wall (centrifugal column type) and extract the total RNA of sample: sample 0.5-1.0g joins in 1.5mL centrifuge tube, add 600 μ L sorbyl alcohol Buffer, add 50U Lyticase, fully mix 30 ℃ and process 30min, after the centrifugal 10min of 1500 * g, abandon supernatant, collecting precipitation, by high-purity total RNA rapid extraction test kit specification sheets, extract sample RNA, then with DNaseI, process twice, the DNA that guarantees to eliminate completely in RNA pollutes, obtain the RNA sample of purifying, use uv-spectrophotometric instrument to measure concentration, adopt 1% agarose gel electrophoresis to detect the integrity of RNA, then by Sample preservation in-80 ℃,
B. post transcription cloning
(1) in the centrifuge tube of the nuclease free of ice bath, add following reaction mixture:
The total RNA of 1-5 μ g, 2 μ L Oligo (dT) 15,2 μ LdNTP (2.5mM each), benefit RNase-freeddH2O are settled to 14.5 μ L;
(2) 70 ℃ heating 5min after rapidly at cooled on ice 2min.After brief centrifugal secretary's reaction solution, add following component: 4 μ L5 * First-Strand Buffer (containing DTT); 0.5 μ L RNasin;
(3) add 1 μ L (200U) TIANScript M-MLV, with pipettor, mix gently;
(4) 42 ℃ of temperature are bathed 50min;
(5) 95 ℃ of heating 5min termination reactions, put and carry out subsequent experimental or freezing preservation on ice;
(6) with RNase-free ddH2O, reaction system is diluted to 50 μ L.-20 ℃ save backup.
C. quantitative fluorescent PCR
Reaction system 25.0 μ L:12.5 μ L 2 * SuperReal PreMix (containing SYBR Green I), each 0.75 μ L 10 μ mol/L upstream and downstream primer DZf and DZr, 2.0 μ L cDNA templates, 0.5 μ L 50 * ROXReference Dye, benefit RNase-free ddH2O to 25 μ L system.
Quantitative fluorescent PCR reaction parameter: 95 ℃ of denaturation 15min; 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, 40 circulations; In 4 ℃ of preservations of temperature; Each sample repeats 3 times.
D. the preparation of outer standard substance and the drafting of typical curve
The preparation of outer standard substance: utilize spectrophotometer that the yeast saccharomyces cerevisiae fermented liquid of incubated overnight is diluted to 1OD (approximately 10
7cells), according to above-mentioned steps, extract total RNA and obtain cDNA by post transcription cloning, carrying out 10 times of serial dilutions, gradient dilution 10
7the concentration of-1 yeast cDNA/ μ L, usings that this carries out quantitative fluorescent PCR reaction as outer standard substance.The template of different concns of take is X-coordinate, and the typical curve of the S. cervisiae that the initial cycle number (Ct) that arrives fluorescence threshold in PCR reaction process of take obtains as ordinate zou, is shown in accompanying drawing 3.
E. result and judgement
As seen from Figure 3, the template concentrations of Ct value and different gradient dilutions is good linear relationship, the template of different concns of take is X-coordinate, Ct value is ordinate zou, the quantitative fluorescent PCR typical curve equation of drawing out: Y=-3.661X+29.837 (Y represents Ct value, and X represents the initial cDNA concentration of template), between the concentration of the initial cDNA of template and Ct value, linearly dependent coefficient is 0.993, slope is-3.661, and the typical curve of setting up meets the quantitative requirement of Real-Time PCR.
CDNA and the standard cDNA of testing sample of take is template, and the species-specific primer of the S. cervisiae of describing with embodiment 1 carries out the gene fragment fluorescent quantitative PCR of S. cervisiae 26s rDNA D1/D2 simultaneously with identical system.By typical curve with adopt the computer software software v2.0.1 of 7500 real-time fluorescence quantitative PCR instrument of ABI company to carry out the Quantitative detection of yeast saccharomyces cerevisiae in Feed Sample.The measurement result of these samples and analytical error result are listed in the table below in 2 in the lump.
The Real-time measurement result of table 2 sample
4: twice fluorescent quantitation count results of embodiment and the experiment of plate count results relevance
The different Preblend samples of mentioning in embodiment 1 are carried out to gradient dilution plate count.Its step is as follows:
A. gradient dilution is coated with dull and stereotyped
(1) with aseptic technique, the sample 25g through fully shaking up is moved in the sterilizing triangular flask that contains 225ml sterile saline and makes the even diluent of 1: 10, mix;
(2) get 1: 10 diluent 10.0ml and be added in the dilution bottle that contains 90.0ml stroke-physiological saline solution, fully shake up the diluent of making 1: 100;
(3) with the diluent 1.0ml that aseptic straw is drawn 1: 10, be added in the test tube that contains 9.0ml stroke-physiological saline solution band granulated glass sphere, carry out 10 times of gradient dilutions, and on microoscillator, mix 3min.Each extent of dilution is used a 1.0ml sterilizing suction pipe instead;
(4) select three suitable serial dilution degree, respectively when doing 10 times of dilutions, respectively get 0.1ml and be added to respectively counting substratum plate, evenly coating, each extent of dilution is made two plates, preferably make 2～3 kinds of substratum (wort agar substratum, bean sprout juice glucose agar medium, respectively by this standard Appendix B .2, B.3 regulation execution) simultaneously, make sterile saline blank simultaneously.More than operating whole process need complete in 20min;
(5) thermostat container that flat-plate inverted is placed in to 28 ℃ after agar solidifies is cultivated, and cultivates and observes colonial morphology after 2～3 days.Yeast saccharomyces cerevisiae is poor growth on bean sprout juice nutrient agar, well-grown on wort agar substratum, and 25～30 ℃ of optimal temperatures, bacterium colony is oyster white, circle, is easily provoked at glossy, neat in edge, thickness.Micro-Microscopic observation, somatic cells ovalize, 5.4 * (3.86～7.85) μ m, sprout in two ends.
B. enumeration
Choose and there are 30～200 typical cases or suspicious yeast saccharomyces cerevisiae lawn carries out enumeration on flat board, and calculate two dull and stereotyped average colony numbers under same extent of dilution.Pro rata is calculated the yeast saccharomyces cerevisiae bacterium colony number in this plate, then takes advantage of its extension rate to be multiplied by 10 again, is every gram of contained yeast saccharomyces cerevisiae number of sample.
C. result and judgement
Fluorescent quantitation result comparative studies to testing sample in the plate count result of the fermentation pure growth that spends the night of two parts of Feed Sample 1# and 2# and yeast saccharomyces cerevisiae and embodiment 2 and embodiment 3, the quantitative result of embodiment 3 and plate count result are good linear relationship, the results are shown in following table 3.The relation conefficient of three kinds of samples is respectively 0.983,0.991 and 0.987.And in embodiment 2, the actual plate count results of two testing sample quantitative results and sample has differed 3 orders of magnitude, its reason may also be counted out for the fluorescent quantitation that the dead bacterium in Feed Sample is cooked template by DNA, therefore the quantitative fluorescent PCR counting of doing template with DNA can not well be distinguished bacterium anyway, and take typical curve that the cDNA of gradient dilution makes as outer standard substance and the cDNA of testing sample, it is template, counting testing sample is accurately credible, differentiation that can be correct is bacterium anyway, and omnidistance only with about 5 hours, the live bacterial count method of comparing traditional has shortened detection time greatly.The quantitative fluorescent PCR method of counting that the cDNA of take of the present invention is template can be used for replacing traditional enumeration, and has the detection time of shortening and count accurately.
The plate count of table 3 yeast saccharomyces cerevisiae sample and fluorescent quantitation count detection result
Claims (2)
- Yeast saccharomyces cerevisiae in an additive premix sample ( saccharomyces cerevisiae) fast qualitative measuring method, it is characterized in that using the DNA of yeast saccharomyces cerevisiae is template, species specificity primer with the 26s rDNA gene order of yeast saccharomyces cerevisiae carries out species specificity PCR reaction, and amplification sheet degree length is 134bp, and then fast qualitative yeast saccharomyces cerevisiae; Described species specificity primer is as follows: upstream primer DZf 5 '-CGAGAGACCGATAGCGAACA-3 ', downstream primer DZr 5 '-AAGGAGCAGAGGGCACAAA-3 '.
- 2. fast qualitative measuring method as claimed in claim 1, is characterized in that the step of the method is as follows:A. the preparation of template DNAAdopt glass bead method to prepare Feed Sample template DNA:(1) get Preblend sample that 1g contains S. cervisiae in 1.5mL centrifuge tube, add 500 μ L phosphate buffered saline buffer suspended sample, the centrifugal 3min of 2500 * g;(2) abandon supernatant, add the granulated glass sphere of appropriate pickling, concuss 2min;(3) add 400 μ L TE damping fluids and the saturated phenol of isopyknic Tris, upset mixes, the centrifugal 5min of 15000 * g;(4) shift the new centrifuge tube of water to, add equal-volume phenol: chloroform: primary isoamyl alcohol=25: the mixture of 24: 1, put upside down and mix, the centrifugal 5min of 15000 * g;(5) suct the new centrifuge tube of clearly to, add 1/10 volume 3mol/L sodium-acetate and 2.5 times of volume 95% ice ethanol ,-20 ℃ are spent the night;(6) 4 ℃ of centrifugal 10min of 15000 * g, abandon supernatant;(7) add 1mL70% ethanol, the centrifugal 5min of 15000 * g, abandons supernatant;(8) 37 ℃ of placement 15min are drying precipitated, and precipitation is dissolved in 60 μ L TE damping fluids, and-20 ℃ save backup;B.PCR amplification reaction system 25.0 μ L:12.5 μ L 2 * PCR-mix, each 1 μ L 10mmol/L upstream and downstream primer, 1 μ L 50ng/ μ L DNA profiling, redistilled water complement to 25.0 μ L;PCR reaction conditions: 94 ℃ of denaturation 5min, 94 ℃ of 30s, 60 ℃ of 45s, 72 ℃ of 40s, carry out 35 circulations, 72 ℃ of final 7min that extend, the PCR product obtaining is preserved at 4 ℃ of temperature; Get 5 μ LPCR products, 1% agarose gel electrophoresis detects;C. result and judgementDuring detection, establish and using the glue of object amplification sheet degree and reclaim product as the positive contrast of template, using aqua sterilisa as the negative contrast of template; According to occur expection feature band at 134bp place, determine in this Preblend sample and contain yeast saccharomyces cerevisiae, do not contain on the contrary yeast saccharomyces cerevisiae.Yeast saccharomyces cerevisiae in an additive premix sample ( saccharomyces cerevisiae) quantitative determination method, the cDNA that it is characterized in that take testing sample cDNA and standard substance is template, right to use requires upstream primer and the downstream primer described in 1, carries out the fluorescent quantitative PCR of the gene fragment of yeast saccharomyces cerevisiae 26s rDNA D1/D2 with identical system simultaneously; By the Ct value of typical curve and testing sample, carry out the Quantitative detection of yeast saccharomyces cerevisiae in Preblend sample.4. quantitative determination method as claimed in claim 3, is characterized in that the step of the method is as follows:A. the preparation of the total RNA of sampleAdopt enzyme process to abolish brewing yeast cell wall, high-purity total RNA rapid extraction test kit extracts the total RNA of sample: sample 0.5-1.0g joins in 1.5mL centrifuge tube, add 600 μ L sorbyl alcohol damping fluids, add 50U lywallzyme, fully mix 30 ℃ and process 30min, after the centrifugal 10min of 1500 * g, abandon supernatant, collecting precipitation, extracts sample RNA, then with DNase I, processes twice, obtain the RNA sample of purifying, use uv-spectrophotometric instrument to measure concentration, adopt 1% agarose gel electrophoresis to detect the integrity of RNA, then by Sample preservation in-80 ℃;B. post transcription cloning(1) in the centrifuge tube of the nuclease free of ice bath, add following reaction mixture: the total RNA of 1-5 μ g, 2 μ L Oligo (dT) 15,2 μ LdNTP, mend RNase-free ddH 2o is settled to 14.5 μ L;After (2) 70 ℃ of heating 5min, rapidly at cooled on ice 2min, after centrifugal reaction solution, add fast following component: 5 * First-Strand Buffer that 4 μ L contain DTT; 0.5 μ L RNasin;(3) add 1 μ L TIANScript M-MLV, with pipettor, mix gently;(4) 42 ℃ of temperature are bathed 50min;(5) 95 ℃ of heating 5min, termination reaction, puts and carries out subsequent experimental or freezing preservation on ice;(6) use RNase-free ddH 2o is diluted to 50 μ L by reaction system, and-20 ℃ save backup;C. quantitative fluorescent PCRReaction system 25.0 μ L:12.5 μ L 2 * SuperReal PreMix, each 0.75 μ L 10 μ mol/L upstream and downstream primer DZf and DZr, 2.0 μ L cDNA templates, 0.5 μ L 50 * ROX Reference Dye, benefit RNase-free ddH 2o to 25 μ L system; Quantitative fluorescent PCR reaction parameter: 95 ℃ of denaturation 15min; 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, 40 circulations; 4 ℃ of preservations; Each sample repeats 3 times;D. the preparation of outer standard substance and the drafting of typical curveThe preparation of outer standard substance: utilize spectrophotometer that the yeast saccharomyces cerevisiae fermented liquid of incubated overnight is diluted to 1OD, according to the total RNA step of said extracted and obtain cDNA by post transcription cloning, carry out 10 times of serial dilutions, gradient dilution to 10 7the concentration of-1 yeast cDNA/ μ L, usings that this carries out quantitative fluorescent PCR reaction as outer standard substance; The drafting of typical curve: the logarithm of template of different concns of take is X-coordinate, the typical curve of the S. cervisiae that the initial cycle number that arrives fluorescence threshold in PCR reaction process of take obtains as ordinate zou, as the reference standard of testing sample quantitative assay;E. result and judgementThe cDNA of testing sample cDNA and standard substance of take is template, with the species-specific primer of S. cervisiae, carries out the fluorescent quantitative PCR of the gene fragment of S. cervisiae 26s rDNA D1/D2 with identical system simultaneously; By the Ct value of typical curve and testing sample, carry out the Quantitative detection of S. cervisiae in Feed Sample.
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| CN112553362B (en) * | 2020-12-11 | 2023-07-04 | 江南大学 | Absolute quantitative probe for saccharomyces cerevisiae and application thereof |
| CN112646923A (en) * | 2021-01-11 | 2021-04-13 | 江南大学 | Primer, kit and method for rapidly identifying and quantifying saccharomyces cerevisiae |
| CN112695121A (en) * | 2021-01-11 | 2021-04-23 | 江南大学 | Primer, kit and method for quickly identifying and quantifying schizosaccharomyces pombe |
| CN112695120A (en) * | 2021-01-11 | 2021-04-23 | 江南大学 | Primer, kit and method for rapid identification and quantification of saccharomyces cerevisiae |
| CN115029476A (en) * | 2022-06-27 | 2022-09-09 | 江苏今世缘酒业股份有限公司 | Primer, kit and method for detecting saccharomycetes |
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