CN107012233A - A kind of fluorescence probe quantitative PCR quick determination method of bulk bacteria - Google Patents
A kind of fluorescence probe quantitative PCR quick determination method of bulk bacteria Download PDFInfo
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Abstract
The invention discloses the number detection of bulk bacteria in a kind of fluorescence probe quantitative PCR quick determination method of bulk bacteria, including (1) bacterial strain DNA extractions, (2) specific primer and fluorescence probe design, the detection of (3) primer specificity, the foundation of (4) standard curve, (5) tea sample DNA extractions and (6) tea sample.The inventive method improves that traditional quantitative PCR labor intensity is big, quantitative inaccurate, poor repeatability shortcoming, also overcome the limitation of classic flat-plate cultivation and time-consuming, the defect such as the cumbersome and workloads of DGCE PCR methods is big, expense is high, its application specific primer, by first order fluorescence quantitative PCR by the bacterial strain absolute quantitation of the corresponding kind in template, with high specificity, it is time-consuming short, easy to operate, quantitative accurate the characteristics of, the quantitative determination of bulk bacteria bacterial strain in tealeaves can be applied to well.
Description
Technical field
The present invention relates to technical field of microbial detection, more particularly to a kind of fluorescence probe quantitative PCR of bulk bacteria are quick
Detection method.
Background technology
Black tea is post-fermented tea, and typical process flow is to finish, just rub, pile fermentation, rub, bakee again.It is black that black tea includes Hunan
Tea, Hubei green brick tea, four Sichuan-Tibet tea (brick tea), the ancient Yi County black tea (peace tea) in Anhui, Yunnan black tea (Pu'er tea), Guangxi Liu Bao tea and
Shaanxi black tea (Fu tea)." golden flower " phenomenon occurs in some areas black tea during pile-fermentation, and this " golden flower " is to dissipate capsule
The golden yellow spore of bacterium.Eurotium can produce amylase, oxidizing ferment and other secondary metabolites in growth course, can
Improve color and luster, mouthfeel and the functional characteristic of tea.Golden flower (i.e. bulk bacteria) is more, and quality is better.At present on microorganism in tealeaves
Measure be confined to traditional colony counting method and gradient gel denaturing electrophoretic (DGGE-PCR) technology.
Determined for bulk bacteria microbe quantity purpose in tealeaves, there is following several method at present：
1. traditional colony counting method
This method operating process is：1. tealeaves surface microorganism is eluted with aqua sterilisa.2. above-mentioned eluent is put
Incubated a period of time in sterilizing flat board.3. treat that microorganism grows bacterium colony, counts colony counts, tealeaves table is calculated by formula
Contain micro organism quantity in face.This method is cumbersome, it is longer to take, can only calculate viable count, and subjectivity is strong in number bacterium colony engineering.
It is unable to the definite value situation of accurate response bacterial strain.
Gradient gel denaturing electrophoretic 2. (DGGE-PCR) technology
The principle of gradient gel denaturing electrophoretic (DGGE-PCR) technology is different double chain DNA fragments because its sequence is constituted not
Equally, so its melting domain and melting conditions denaturant concentration are also differed.The DNA pieces that length is identical, sequence is different
Section diverse location generating unit under the denaturant conditions of various concentrations decomposes chain, the diverse location of colloid is moved to, so that by area
Separate.The operating process of this method is：The extraction of microbial total gene -18SrRNA variable regions amplification-denaturing gradient gel
Electrophoresis-be coloured to picture-gel extraction-transformed clone-sequencing comparison, analyze data can obtain fungus strain in each Tea Samples
The relative amount of the bacterium of composition and colour developing band.DGCE-PCR lays particular emphasis on the analysis of microbial diversity in Tea Samples, can
Reflect that tealeaves flora is constituted and diversity, this method can be accurately qualitative, but it passes through the shading value sxemiquantitative point of comparing dna band
Analysis method can not reach the purpose of accurate quantitative analysis.Electrophoresis process is related to poisonous reagent, has a strong impact on experimenter healthy.Electricity
Swimming terminate after data analysis, complex disposal process, substantial amounts of expense can be produced by comparing this step in sequencing, not be a kind of accurate
Efficiently definite value ability assay method.
Protocols in Molecular Biology is a new technology for exempting from culture detection to microorganism, and real-time quantitative PCR refers in PCR
Fluorophor is added in reaction system, whole PCR processes are monitored in real time using fluorescence signal accumulation, finally by standard curve pair
The method that unknown template carries out quantitative analysis.Quantitative fluorescent PCR has following significant advantage：
1. it is specific good, quantitative molecular is identified using specific probe, with very high accuracy.Meanwhile, target
Sequence is by primer and probe double control, and specific good, false positive is low.
2. sensitivity is high, and fluorescent PCR detection technique is to combine round pcr, fluorescent labelling techniques, laser technology, number
The technology that visualization techniques are integrated, therefore its detection sensitivity is very high.
3. linear relationship is good, the range of linearity is wide, because generation and each amplified production of fluorescence signal are into one-to-one
Relation, directly can be quantified by the detection of fluorescence signal to product；Quantification range can be in 0-1010Copies/ml.
4. simple to operate, safety, automaticity are high, anti-pollution.Amplification and detection can be detected in same pipe, be not required to
Uncap, be difficult pollution；Expand simultaneously and one step of detection is completed, it is not necessary to post-processing, it is no longer necessary to worry radioactive pollution.
In real-time quantitative fluorescence PCR reaction system, fluorophor or fluorescent dye are added, with the progress of reaction, PCR
Reaction product constantly adds up, and fluorescence signal intensity gradually increases.Often by a circulation, a fluorescence intensity signals are collected, are led to
The change of the variation monitoring product amount of fluorescence intensity is crossed, so as to obtain a fluorescent amplification curve figure.
In general, fluorescent amplification curve is segmented into three phases：Fluorescence background stage, fluorescence signal index increase
Long stage and non-exponential platform phase.In fluorescence signal exponential growth phase, the logarithm value and starting template amount of PCR primer amount it
Between there is linear relationship, qRT-PCR results are can to reflect the primary quantity of template in reaction system exactly.For the ease of to institute
Detection sample is compared, in fluorescence signal exponential growth phase, needs to set the thresholding of a fluorescence signal, this thresholding first
(threshold) it is usually that autofluorescent background signal (baseline) is used as using the fluorescence signal of PCR preceding 15 circulations reacted.Ct
Value is that the fluorescence signal of amplified production reaches the amplification cycles number passed through during the threshold value of setting.Ct values are main by amplified reaction body
The initial concentration of template is determined in system.Generally standard curve, Ran Houji are produced with the Ct values of the standard sample of various concentrations
Calculate relative equation.The gradient of equation can for check PCR efficiency.Real-time fluorescence quantitative PCR instrument has software, can be with
The original template amount of unknown sample is automatically calculated from standard curve.
Foreign scholar is carried out using multiple fluorescence quantitative PCR method to the diversity of the microorganism in air and shared ratio,
It was found that microbes in air is a lot, including Eurotium.Application probe method real-time fluorescence quantitative PCR skill is not yet found at present
Corresponding research or report that art is counted to Eurotium in tealeaves.
The content of the invention
It is an object of the invention to：For above-mentioned problem, there is provided a kind of fluorescence probe quantitative PCR of bulk bacteria is fast
Fast detection method, the present invention overcomes the limitation of classic flat-plate cultivation and time-consuming, the operation of DGCE-PCR methods is numerous
The trivial and defect such as workload is big, expense is high, the characteristics of with quick, accurate, high flux, high sensitivity and high specificity.
In order to realize foregoing invention purpose, the technical solution adopted by the present invention is as follows：
A kind of fluorescence probe quantitative PCR quick determination method of bulk bacteria, it is characterised in that：Comprise the following steps：
(1) bacterial strain DNA is extracted
Purebred culture Eurotium microorganism, collects its mycelia, carries out the extraction of Eurotium genomic DNA, obtains DNA
Solution；
(2) specific primer and fluorescence probe design
Forward primer Forward Primer EamstF1：5'-GTGGCGGCACCATGTCT
Reverse primer Reverse Primer EamstR1：5'-CTGGTTAAAAAGATTGGTTGCGA
The sequence Probe EamstP1 of fluorescence probe：5’-CAGCTGGACCTACGGGAGCGGG
Probe modification：6-FAM\Eclipse
(3) primer specificity is detected
Obtained DNA solution is extracted with step (1), with ddH2O is negative control, the specific primer of detecting step (2)
Specificity；
(4) foundation of standard curve
It is respectively 2.0 × 10 that the plasmid standard of concentration known is carried out into 10 times of gradient dilutions, i.e. diluted concentration7, 2.0 ×
106, 2.0 × 105, 2.0 × 104, 2.0 × 103, 2.0 × 102, 2.0 × 101copies/μL；Each dilution factor is parallel three times, together
When be used as negative control by template of sterilized water；Expanded by real-time fluorescence quantitative PCR, detection primer sensitivity, and to copy
Number logarithm value is x-axis, using Ct values as y-axis, builds real-time fluorescence quantitative PCR standard curve, and determine its sensitivity；
(5) tea sample DNA is extracted
(a) tea sample is pre-processed, obtains mycelium, it is standby；
(b) above-mentioned mycelium is added in the centrifuge tube equipped with 1mL lysates, stands 5min；Then concussion is mixed, 90
~100 DEG C of water bath with thermostatic control 8-15min；
(c) after water-bath, above-mentioned mixed liquor is centrifuged into 2min under conditions of 4 DEG C, 12000rpm；Then supernatant is taken, is obtained
To tea sample DNA extract solutions, saved backup under the conditions of -20 DEG C；
(6) number of bulk bacteria is detected in tea sample
Hypha,hyphae genomic DNA is template in the tea sample DNA extract solutions that step (5) is obtained, using in step (3)
Specific primer carries out fluorescent quantitative PCR, obtains the Ct values of different fluorescent quantitative PCRs, brings step (4) Plays into
In curvilinear equation, copy number is calculated, passes through conversion, you can obtain the number of Eurotium in tea sample.
It is preferred that in step (1), the bacterial strain DNA is extracted, comprised the following steps：
(a) liquid nitrogen is poured into through high-temperature sterilization, the mortar of precooling, adds 0.05~0.15g bulk bacteria mycelial samples,
After after liquid nitrogen completely volatilization, quick ground sample obtains mycelia powder to powdered；
(b) mycelia powder is put in centrifuge tube, adds 150~250 μ L Buffer Digestion, 1~3 μ L β-mercapto
Base ethanol and 10~30 μ L Proteinase K solution, after concussion is mixed, 50~60 DEG C of 0.5~1.5h of water bath with thermostatic control；
(c) toward 80~120 μ L Buffer PF of addition in the centrifuge tube after above-mentioned water-bath, mix after -15~-25 DEG C
Under the conditions of stand 3-6min；Then room temperature 10000rpm centrifuges 5min, takes supernatant；
(d) toward take out supernatant in add 150~250 μ L Buffer BD mix after, add 150~250 μ L without
Water-ethanol, after mixing, solution and translucent fibre shape suspension are all moved in the adsorption column of collecting pipe, stand 1-5min,
Then 10000rpm room temperatures centrifugation 1min, outwells the waste liquid in collecting pipe；
(e) adsorption column is put back into collecting pipe, adds 400~600 μ L PW Solution, 10000rpm centrifugations 30s and outwell
Waste liquid in collecting pipe；Add 400~600 μ L Wash Solution, 10000rpm centrifugations 30s and outwell giving up in collecting pipe
Liquid, then centrifuges 2min, the Wash Solution for residual of leaving away in room temperature 12000rpm, cleans the mouth of pipe；
(f) above-mentioned adsorption column is taken out, is put into new centrifuge tube, 40-60 μ l TE Buffer is added and stands 3min,
12000rpm room temperatures centrifuge 2min, then collect DNA solution, are saved backup under the conditions of -20 DEG C.
It is preferred that in step (3), the reaction system and reaction condition of the primer specificity detection are：
(a) reaction system：Carried out using Eppendorf quantitative fluorescent PCRs instrument, each sample takes 1 μ L as template；25
It is as follows that μ L quantitative fluorescent PCR reaction system contains composition：premiex TaqTM, 12.5 μ L；Primers F, 0.5 μ L；Primer R, 0.5
μL；DNA profiling, 2 μ L；6-FAM modifies probe, 0.1 μ L；dH2O, 25 μ L；
(b) reaction condition：95 DEG C of pre-degeneration 30s, 1 circulation：95 DEG C of denaturation 5s, 55 DEG C of annealing 10s, 72 DEG C of extension 20s,
45 circulations.
It is preferred that in step (4), the preparation method of the plasmid standard is：
Above-mentioned Eurotium genome is used for template, using specific primer in step (3), regular-PCR is carried out, obtains
Product carries out gel extraction, and the target DNA fragment of gel extraction is converted by transduction, plasmid standard is prepared into, and determine
Plasmid concentration, calculates plasmid standard copy number.
It is preferred that the calculation formula of the plasmid standard copy number is：Copy number=6.02 × 1023× concentration × 10-9/ (amplification base number × 660).
It is preferred that in step (5), the tea sample pretreatment is comprised the steps of：
Weigh each 20g of Tea Samples to pour into sterilized conical flask, 150rpm vibrates 30min at room temperature, liquid is turned
Move in 50mI centrifuge tubes, under the conditions of 4 DEG C, 10000r/min centrifugation 10min remove supernatant, collect precipitation, given birth to sterilizing
Manage tealeaves in normal saline washing conical flask；Repeat the above steps 4 times, obtained sediment suspended with 1.0mL sterile salines,
Mycelium is obtained, it is standby.
It is preferred that the sterilizing is to be added into conical flask after physiological saline and bead, high pressure steam sterilization is carried out.
It is preferred that the tea sample is Fu tea, thousand tael tea, black brick tea, green brick tea, brick tea, peace tea, Pu'er tea or six fort tea.
The present invention be applicant on the basis of early-stage Study, identified from six fort tea of Guangxi Zhuang Autonomous Region Wuzhou
Many plants of " golden flower " bacterium, it was demonstrated that it all belongs to Eurotium, rather than single coronoid process dissipate capsule bacterium together, so as to set up according to the fort tea of golden flower six
In Eurotium be characterized index real-time fluorescence quantitative PCR quantitative approach, and then also set up bulk bacteria in golden flower black teas
The Rapid Quantification of number.
In summary, by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
The inventive method improves that traditional quantitative PCR labor intensity is big, quantitative inaccurate, poor repeatability shortcoming, also gram
The limitation of classic flat-plate cultivation and time-consuming is taken, it is scarce that the cumbersome and workloads of DGCE-PCR methods is big, expense is high etc.
Fall into, its application specific primer, by first order fluorescence quantitative PCR by the bacterial strain absolute quantitation of the corresponding kind in template, has
High specificity, it is time-consuming short, easy to operate, quantitative accurate the characteristics of, the quantitative determination of bacterial strain in tealeaves can be applied to well.Enter
And by the measure of bulk bacteria quantity in tealeaves, quickly determine whether the black tea is golden flower black teas, so as to set up to golden flower black teas
Quick false distinguishing method.
Brief description of the drawings
Fig. 1 gradient dilution plasmid standard amplification curve diagrams；
The canonical plotting of Fig. 2 bulk bacteria fluorescent quantitative PCR detection methods；
Fig. 3 fluorescent quantitation sensitivity technique figures；
Fig. 4 fluorescent quantitation specific detection figures；
The fort tea fluorescent quantitative pcr amplification curve maps of Fig. 5 six；
The initial bulk bacteria number figures of fort tea sample of Fig. 6 six.
Embodiment
For the objects, technical solutions and advantages of the present invention are more clearly understood, below with reference to accompanying drawing, and preferred reality is enumerated
Example is applied, the present invention is described in more detail.However, it is necessary to which many details listed in explanation, specification are only to be
Reader is set to have a thorough explanation to the one or more aspects of the present invention, even without these specific details can also
Realize the aspects of the invention.
Raw material selected by the present invention can bought on the market；Wherein：
Lysate is DNA extract solutions, and the kit is produced by precious bioengineering Co., Ltd, kit English full name：
The production of MightyPrep reagent for DNA treasured biotech firm, production code member：9182.
premiex TaqTMI.e. sonde method quantitative fluorescent PCR reaction special agent, purchased from precious bioengineering Co., Ltd, is produced
Product are numbered：RR390Q.
RNase, i.e. RNase.Purchased from Suo Laibao biotech firms, production code member：R1030.
Proteinase K, i.e. Proteinase K, purchased from Suo Laibao biotech firms, production code member：P1120.
Buffer Digestion (buffering digestive juice), Buffer PF (buffer solution PF), Buffer BD (buffer B D),
It is true that PW Solution (solution PW), Wash Solution (washing lotion) and TE Buffer (TE buffer solutions) are purchased from Ezup pillars
Bacterium genome DNA extraction kit, production code member：B518259-0050 manufacturers：Raw work bioengineering (Shanghai) share has
Limit company.
Solution I*, pMD19-T carrier, control Insert DNA*2It is all from the limited public affairs of the precious bioengineering of producer
Department, kit title：PMD19-T Simple Vector, production code member：D104A.
JM-109 competent cells, SOC culture mediums are purchased from precious organism Escherichia coli (JM109) competent cell reagent
Box, production code member：9052, producer：Precious bioengineering Co., Ltd.
X-Gal, instant solution, producer：Sangon Biotech (Shanghai) Co., Ltd., specification number：
B541006-0001。
IPTG instant solution, producer：Sangon Biotech (Shanghai) Co., Ltd., specification number：B541007-
0001。
Amp sterilizing ampicillin sodium solutions, production code member：B540722-0010 producers：Raw work bioengineering (Shanghai)
Limited company.
Solution I, solution II, Solution III, Spin Column：Nucleic acid purification post, Buffer WA,
Buffer WB are purchased from takara DNA small scale purifications reagent kit product numbering：9760 producers：The precious limited public affairs of bioengineering
Department.
Specific primer and fluorescence probe sequence selected by the present invention, to refer to periodical《EPATechnology for
Mold Identification and Enumeration》, specific primer and probe sequence described in the 7-8 pages, content
For：
Eurotium(Aspergillus)amstelodami/chevalieri/herbariorum/rubrum/repens
(assay name：Eamst)
Forward Primer EamstF1：5'-GTGGCGGCACCATGTCT
Reverse Primer EamstR1：5'-CTGGTTAAAAAGATTGGTTGCGA
Probe EamstP1：5’-CAGCTGGACCTACGGGAGCGGG
First, implementation
(1) bacterial strain DNA is extracted
The extraction of Eurotium genomic DNA is carried out, DNA solution is obtained；Comprise the following steps that：
(a) purebred culture Eurotium microorganism, collects its mycelia；
(b) mortar, grinding rod and key clean and dry are wrapped up tight with masking foil, carries out high-temperature sterilization, grinding
Before, first appropriate liquid nitrogen carries out precooling in mortar to mortar and grinding rod；
(c) treat that liquid nitrogen volatilization is dry, then appropriate liquid nitrogen is in mortar, weighs 0.10g mycelial samples, adds in mortar, treats
Liquid nitrogen volatilizees completely, and quick ground sample obtains mycelia powder to powdered；
(d) mycelia powder is scraped in 1.5mL centrifuge tubes with key, 200 μ L are added directly over centrifugation bottom of the tube
Buffer Digestion and 2 μ L beta -mercaptoethanols, add 20 μ L Proteinase K solution, after concussion is mixed, 56 DEG C of perseverances
Tepidarium 1h is cracked completely to cell；During water-bath, a centrifuge tube is mixed per 10min, method is mixed：Played with finger from
Heart ttom of pipe end to liquid is homogeneous liquid；
(c) toward 100 μ L Buffer PF are added in the centrifuge tube after above-mentioned water-bath, mix quiet under conditions of -20 DEG C
Put 5min；Then room temperature 10000rpm centrifuges 5min, takes supernatant, is transferred into new 1.5mL centrifuge tubes；
(d) toward take out supernatant in add 200 μ L Buffer BD mix after, add 200 μ L absolute ethyl alcohol, fill
Divide after mixing, solution and translucent fibre shape suspension are all moved in the adsorption column of collecting pipe, stand 2min, then
10000rpm room temperatures centrifuge 1min, outwell the waste liquid in collecting pipe, and the waste liquid for collecting the mouth of pipe is dried with the clean scraps of paper；
(e) adsorption column is put back into collecting pipe, adds 500 μ L PW Solution, 10000rpm centrifugations 30s and outwell collecting pipe
In waste liquid, clean the mouth of pipe；Adsorption column is put back into collecting pipe, 500 μ L Wash Solution, 10000rpm centrifugations 30s are added
The waste liquid in collecting pipe is outwelled, the mouth of pipe is cleaned；Then adsorption column is placed back in collecting pipe, in room temperature 12000rpm centrifugations
2min, the Wash Solution for residual of leaving away, cleans the mouth of pipe；
(f) above-mentioned adsorption column is taken out, is put into new 1.5mL centrifuge tubes, 50 μ L TE Buffer is added and stands 3min,
12000rpm room temperatures centrifuge 2min, then collect DNA solution, are saved backup under the conditions of -20 DEG C.
(2) specific primer and fluorescence probe design
Specific primer and fluorescence probe sequence selected by the present invention, to refer to periodical《EPA Technology for
Mold Identification and Enumeration》, specific primer and probe sequence described in the 7-8 pages.
Forward primer Forward Primer EamstF1：5'-GTGGCGGCACCATGTCT
Reverse primer Reverse Primer EamstR1：5'-CTGGTTAAAAAGATTGGTTGCGA
The sequence Probe EamstP1 of fluorescence probe：5’-CAGCTGGACCTACGGGAGCGGG
Probe modification：6-FAM\Eclipse
(3) primer specificity is detected
The DNA solution obtained with step (1) extracting method, with ddH2O is negative control, the specificity of detecting step (2)
The specificity of primer；
Real-time fluorescence quantitative PCR reaction system：Carried out using Eppendorf quantitative fluorescent PCRs instrument, each sample takes 1 μ
L is used as template.Reaction system and reaction condition are as follows：
(a) reaction system：Carried out using Eppendorf quantitative fluorescent PCRs instrument, each sample takes 1 μ L as template；25
It is as follows that μ L quantitative fluorescent PCR reaction system contains composition：premiex TaqTM, 12.5 μ L；Primers F, 0.5 μ L；Primer R, 0.5
μL；DNA profiling, 2 μ L；6-FAM modifies probe, 0.1 μ L；dH2O, 25 μ L；
(b) reaction condition：95 DEG C of pre-degeneration 30s, 1 circulation：95 DEG C of denaturation 5s, 55 DEG C of annealing 10s, 72 DEG C of extension 20s,
45 circulations.
(4) foundation of standard curve
(a) preparation of plasmid standard
Above-mentioned Eurotium genome is used for template, using the specific primer in step (3), regular-PCR is carried out, obtains
Purifying recovery is carried out to product, purifying reclaims specific steps with reference to the universal DNA purifying QIAquick Gel Extraction Kit (centrifugation of kit Tiangeng
Column type) operating procedure；The target DNA fragment reclaimed will be purified by transduction conversion, plasmid standard is prepared into, and determine plasmid
Concentration, calculates plasmid standard copy number.
Comprise the following steps that：
1) pMD carriers are connected
Bulk bacteria genome is connected in pMD carriers, material shown in table 1 is added in microcentrifugal tube, full dose is 5 μ
L；Then 5ul Solution I* are added；16 DEG C of reaction 30min.
Table 1
| Reagent | Usage amount |
| PMD19-T carriers | 1ul |
| Control Insert DNA*2 | 1ul |
| dH2O | Up to 5ul |
2) JM-109 competent cells are imported
A) competent cell is melted using preceding on ice, slight mixing, take the above-mentioned JM109 competent cells of 100 μ L in
In 4mL centrifuge tubes；
B) the above-mentioned connection carriers of 10ul are added in 100ul JM109 competent cells, 30min are placed in ice, with nothing
Bacterium water sets negative control；
C) 42 DEG C are heated after 60s, then place 1min on ice, add vibration training under the conditions of 890ul SOC culture mediums, 37 DEG C
Support 60min, frequency of oscillation：180rpm；
D) and then in the LB- agar plate culture agent containing X-Gal, IPGT, Amp cultivate, form single bacterium colony, 37 DEG C of mistakes
Night cultivates；In superclean bench, from the positive bacterium colony of 3 whites of picking on the LB flat boards with bacterium colony and 1 blue negative bacterium
Fall, be inoculated in respectively in A, B, C and D test tube equipped with 4mL LB ammonia benzyl nutrient solutions, 15h is shaken in 37 DEG C；
E the reaction system of 25 μ L above-mentioned steps (3)) is prepared, enters performing PCR, its condition and system are consistent with regular-PCR, electricity
Swimming and gel imaging, then carry out positive recombinant plasmid sequencing.
3) plasmid extraction and Concentration Testing
A the bacterium colony nutrient solution of above-mentioned A, B, C and D test tube) is subjected to incubated overnight 15h, and takes out bacterium colony growing way preferable 2
Kind of bacterium colony nutrient solution, then take out from 2 kinds of bacterium colony nutrient solutions 1.5mL bacterium colony nutrient solution respectively, 12000rpm centrifugation 2min,
To precipitate strain, supernatant is abandoned；
B) precipitated with 250ul Solution I (A containing Rnase) fully suspended bacterials, add 250ul solution
II finger springing centrifuge tube bottoms, make thalline fully crack, and form clear solution, add 350ul 4 DEG C of precooling
Solution III, with finger springing centrifuge tube bottom, make thalline fully crack, up to forming consolidation aggegation block, then room temperature
Stand 2min；
C) room temperature 12000rpm centrifuges 10min, takes supernatant, the Spin Column in kit are placed in into centrifuge tube
In, then supernatant is transferred in Spin Column, 12000rpm centrifugation 1min abandon filtrate；
D) 500ul Buffer WA are added in Spin Column, 12000rpm centrifugation 30s abandon filtrate；
E) 700ul Buffer WB are added in Spin Culumn, 12000rpm centrifugation 30s abandon filtrate, repeat one
It is secondary；
F) Spin Culumn are placed on collecting pipe again, 12000rpm, centrifuge 1min.Residual washing lotion is eliminated, will
Spin Culumn are placed on new 1.5mLd centrifuge tube, and 30ul aseptic distillations are added in the centre of Spin Culumn films
Water, is stored at room temperature 1min, and 12000rpm centrifugation 1min eluted dnas, -20 DEG C of preservations are standby；
G the concentration and purity of plasmid stock) are detected with Nano Drop (ultramicron ultraviolet specrophotometer), it is calculated and copies
Shellfish number (copies/mL), calculation formula：
Copy number=6.02 × 1023× concentration × 10-9/ (amplification base number × 660)
4) Specification Curve of Increasing
It is respectively 2.0 × 10 that the plasmid standard of above-mentioned concentration known is carried out into 10 times of gradient dilutions, i.e. diluted concentration7,
2.0×106, 2.0 × 105, 2.0 × 104, 2.0 × 103, 2.0 × 102, 2.0 × 101copies/μL；Each dilution factor parallel three
It is secondary, while being used as negative control by template of sterilized water；Expanded by real-time fluorescence quantitative PCR, detection primer sensitivity, and with
Copy number logarithm value is x-axis, using Ct values as y-axis, builds real-time fluorescence quantitative PCR standard curve, and determine its sensitivity.
(5) tea sample DNA is extracted
(a) tea sample is pre-processed, obtains mycelium, it is standby；What tea sample was pre-processed concretely comprises the following steps：Take 500mL conical flasks
Fill 100mL physiological saline (mass fraction is 0.85%NaCI) and a small amount of bead, high pressure steam sterilization；Weigh Tea Samples each
20g is poured into sterilized conical flask, and 150rpm vibrates 30min at room temperature, liquid is transferred in 50mI centrifuge tubes, at 4 DEG C
Under the conditions of, 10000r/min centrifugation 10min remove supernatant, collect precipitation, tea in conical flask is rinsed with sterile saline
Leaf；Lay equal stress on complex oscillation, centrifugation, collect precipitation and the above-mentioned steps 4 times of normal saline flushing tealeaves, obtained sediment is used
1.0mL sterile salines suspend, and obtain mycelium, standby；
(b) above-mentioned mycelium is added in the centrifuge tube equipped with 1mL lysates, stands 5min；Then concussion is mixed, 95
DEG C water bath with thermostatic control 10min；
(c) after water-bath, above-mentioned mixed liquor is centrifuged into 2min under conditions of 4 DEG C, 12000rpm；Then supernatant is taken, is obtained
To tea sample DNA extract solutions, saved backup under the conditions of -20 DEG C.
(6) number of bulk bacteria is detected in tea sample
Hypha,hyphae genomic DNA is template in the tea sample DNA extract solutions that step (5) is obtained, using in step (3)
Specific primer carries out fluorescent quantitative PCR, obtains the Ct values of different fluorescent quantitative PCRs, brings step (4) Plays into
In curvilinear equation, copy number is calculated, passes through conversion, you can obtain the number of Eurotium in tea sample.
2nd, test result
(1) standard curve
Use dH2O is by the plasmid standard of 10 times of gradient dilutions, and it is 2.0 × 10 to take copy number7-2.0×102copies/ul
6 gradients standard items carry out quantitative fluorescent PCR reaction, obtain the amplification curve of corresponding gradient, as shown in Figure 1 for gradient it is dilute
Release plasmid standard amplification curve diagram.System goes out standard curve according to the Ct value automatic Fittings of each gradient, using Ct values as vertical seat
Mark, starting copy number is the standard curve that abscissa does regression curve, as bulk bacteria fluorescent quantitative PCR detection method, such as Fig. 2
It is shown.Meaning system of the invention is to carry system inside the precious bioluminescence instruments of Takara TP800.
The standard curve of gained is：Y=-3.683 × log (X)+42.39.The slope of curve is -3.683, and ordinate is cut
Away from for 42.39, coefficient R2=0.999, amplification efficiency is that good linear pass is presented between 86.1%, Cq values and concentration
System.
(2) the sensitivity detection of primer and probe
With the standard plasmid (2.0 × 10 of 10 × times dilution1Copies/ul and 2.0 × 100Copies/ul it is) template, presses
Sensitivity detection is carried out according to above-mentioned fluorescent quantitation reaction condition.Fluorescent quantitation sensitivity technique figure is illustrated in figure 3, can by figure
Know that lowest detection is limited to 2.0 × 100Copies/ul, i.e., each reaction needs 2 copies, and the concentration of the detection above occurs good
Good amplified signal, blank control does not have amplified signal.
(3) quantitative fluorescent PCR specificity analysis
Bulk bacteria and 6 kinds of non-bulk bacteria in 10 kinds of fungies, including 4 are chosen, with red bulk bacteria (Eurotium rubrum),
E.repens (Eurotium repens), the yellow bulk bacteria (Eurotium niveoglaucum) of snow, Amsterdam dissipates capsule
Bacterium (Eurotium amstelodami) DNA is template, with ddH2O is negative control, and ibid condition carries out real time fluorescent quantitative
PCR reacts, and analyzes the specificity of this method；Fluorescent quantitation specific detection figure is illustrated in figure 4, after testing, the yellow scattered capsule of snow
It is the positive that bacterium, Amsterdam, which dissipate capsule capsule bacterium, red scattered capsule capsule bacterium, E.repens by bacterial strain amplification, and black-koji mould, meter Qu
Mould, Tabin aspergillus bacterium, production Aspergillus flavus, penicillium citrinum, aspergillus tamarii bacterium do not occur fluorescence signal, and amplification is feminine gender,
Blank control NTC is feminine gender, illustrates that the detection of the primer and probe to Eurotium has good specificity.
(4) fluorescent quantitation reappearance is detected
Standard plasmid after taking that 2 μ L are above-mentioned respectively and being serially diluted carries out sonde method real-time fluorescence as template, ibid condition
Quantitative PCR reacts, and each dilution gradient standard plasmid does 3 multiple holes.It is TaqMan probe real time fluorescent quantitative as shown in table 2
PCR detects the reappearance testing result of bulk bacteria.
Table 2
X is average value in upper table, and SD is standard deviation, and CV is the coefficient of variation.As shown in Table 2, the CV values of this method exist
Between 0.10%~0.98%, less than 1%.
(5) bulk bacteria testing result in tealeaves
Tealeaves (by taking six fort tea as an example) is pre-processed with reference to the step (5) in implementation and DNA is extracted, back and forward
Part carries out sonde method real-time fluorescence quantitative PCR reaction, is illustrated in figure 5 six fort tea fluorescent quantitative pcr amplification curve maps, schemes
6 be the six initial bulk bacteria numbers of fort tea sample.Table 3 is six fort tea sample initial sample bulk bacteria numbers.
Table 3
| Tea sample title | Copy number copies/2l | Initial number (cfu/g) |
| Grey suitable six fort tea | 333174.50 | 9229210 |
| The fort tea of golden flower black teas six | 331741.50 | 9189515 |
| Wuzhou Liu Bao tea (1910#) | 231841.70 | 6422207 |
| The fort tea of three crane six (tinfoil paper dress) | 172813.40 | 4787074 |
| The fort tea 0712 of three crane six | 13652.61 | 378188 |
| Wuzhou Liu Bao tea (1916#) | 14263.07 | 395098 |
| The middle fort tea of tea six | 11972.82 | 331657 |
| The fort tea 1709 of three crane six | 151.46 | 4195 |
| Six fort tea sample teas (basket) | 108.06 | 2993 |
From Fig. 5, Fig. 6 and table 3, method of the invention can accurate, the quick number for determining bulk bacteria in tealeaves, this hair
Bright method detection high specificity, sensitivity are high, and experimental result can be drawn in the short period of time, greatly reduces workload and work
Time, it is worthy of popularization.
The fluorescence probe quantitative PCR quick determination method of the bulk bacteria of the present invention, can be applied to all black teas, including but
It is not limited to Hunan black tea (Fu tea, thousand tael tea, black brick tea, tricuspid), Hubei green brick tea, four Sichuan-Tibet tea (brick tea), ancient Yi County, Anhui black
Tea (peace tea), Yunnan black tea (Pu'er tea), Guangxi Liu Bao tea and Shaanxi black tea (Fu tea).
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (8)
1. a kind of fluorescence probe quantitative PCR quick determination method of bulk bacteria, it is characterised in that：Comprise the following steps：
(1) bacterial strain DNA is extracted
Purebred culture Eurotium microorganism, collects its mycelia, carries out the extraction of Eurotium genomic DNA, obtains DNA molten
Liquid；
(2) specific primer and fluorescence probe design
Forward primer Forward Primer EamstF1：5'-GTGGCGGCACCATGTCT
Reverse primer Reverse Primer EamstR1：5'-CTGGTTAAAAAGATTGGTTGCGA
The sequence Probe EamstP1 of fluorescence probe：5’-CAGCTGGACCTACGGGAGCGGG
Probe modification：6-FAM\Eclipse
(3) primer specificity is detected
Obtained DNA solution is extracted with step (1), with ddH2O is negative control, the specific primer of detecting step (2) it is special
Property；
(4) foundation of standard curve
It is respectively 2.0 × 10 that the plasmid standard of concentration known is carried out into 10 times of gradient dilutions, i.e. diluted concentration7, 2.0 × 106,
2.0×105, 2.0 × 104, 2.0 × 103, 2.0 × 102, 2.0 × 101copies/μL；Each dilution factor is parallel three times, while with
Sterilized water is that template is used as negative control；Expanded by real-time fluorescence quantitative PCR, detection primer sensitivity, and with copy number pair
Numerical value is x-axis, using Ct values as y-axis, builds real-time fluorescence quantitative PCR standard curve, and determine its sensitivity；
(5) tea sample DNA is extracted
(a) tea sample is pre-processed, obtains mycelium, it is standby；
(b) above-mentioned mycelium is added in the centrifuge tube equipped with 1mL lysates, stands 5min；Then concussion is mixed, 90~
100 DEG C of water bath with thermostatic control 8-15min；
(c) after water-bath, above-mentioned mixed liquor is centrifuged into 2min under conditions of 4 DEG C, 12000rpm；Then supernatant is taken, tea is obtained
Sample DNA extract solutions, are saved backup under the conditions of -20 DEG C；
(6) number of bulk bacteria is detected in tea sample
Hypha,hyphae genomic DNA is template in the tea sample DNA extract solutions that step (5) is obtained, using special in step (3)
Property primer carry out fluorescent quantitative PCR, obtain the Ct values of different fluorescent quantitative PCRs, bring step (4) standard curve into
In equation, copy number is calculated, passes through conversion, you can obtain the number of Eurotium in tea sample.
2. the fluorescence probe quantitative PCR quick determination method of bulk bacteria according to claim 1, it is characterised in that：In step
Suddenly (1), the bacterial strain DNA is extracted, and is comprised the following steps：
(a) liquid nitrogen is poured into through high-temperature sterilization, the mortar of precooling, adds 0.05~0.15g bulk bacteria mycelial samples, treat liquid
After nitrogen volatilization completely, quick ground sample obtains mycelia powder to powdered；
(b) mycelia powder is put in centrifuge tube, adds 150~250 μ L Buffer Digestion, 1~3 μ L β-sulfydryl second
Alcohol and 10~30 μ L Proteinase K solution, after concussion is mixed, 50~60 DEG C of 0.5~1.5h of water bath with thermostatic control；
(c) toward 80~120 μ L Buffer PF of addition in the centrifuge tube after above-mentioned water-bath, the condition after -15~-25 DEG C is mixed
3~6min of lower standing；Then room temperature 10000rpm centrifuges 5min, takes supernatant；
(d) toward after being mixed 150~250 μ L Buffer BD of addition in the supernatant taken out, 150~250 anhydrous second of μ L are added
Alcohol, after mixing, solution and translucent fibre shape suspension are all moved in the adsorption column of collecting pipe, stand 1~5min, then
10000rpm room temperatures centrifuge 5min, outwell the waste liquid in collecting pipe；
(e) adsorption column is put back into collecting pipe, adds 400~600 μ L PW Solution, 10000rpm centrifugations 30s and outwell collection
Waste liquid in pipe；400~600 μ L Wash Solution are added, 10000rpm centrifugations 30s outwell the waste liquid in collecting pipe,
Then 2min, the Wash Solution for residual of leaving away are centrifuged in room temperature 12000rpm, cleans the mouth of pipe；
(f) above-mentioned adsorption column is taken out, is put into new centrifuge tube, 40~60 μ l TE Buffer is added and stands 3min,
12000rpm room temperatures centrifuge 2min, then collect DNA solution, are saved backup under the conditions of -20 DEG C.
3. the fluorescence probe quantitative PCR quick determination method of bulk bacteria according to claim 1, it is characterised in that：In step
Suddenly (3), the reaction system and reaction condition of the primer specificity detection are：
(a) reaction system：Carried out using Eppendorf quantitative fluorescent PCRs instrument, each sample takes 1 μ L as template；25 μ L's
It is as follows that quantitative fluorescent PCR reaction system contains composition：premiex TaqTM, 12.5 μ L；Primers F, 0.5 μ L；Primer R, 0.5 μ L；
DNA profiling, 2 μ L；6-FAM modifies probe, 0.1 μ L；dH2O, 25 μ L；
(b) reaction condition：95 DEG C of pre-degeneration 30s, 1 circulation：95 DEG C denaturation 5s, 55 DEG C annealing 10s, 72 DEG C extension 20s, 45
Circulation.
4. the fluorescence probe quantitative PCR quick determination method of the bulk bacteria according to any one of claims 1 to 3, its feature
It is：In step (4), the preparation method of the plasmid standard is：
Above-mentioned Eurotium genome is used for template, using specific primer in step (3), regular-PCR is carried out, obtains product
Gel extraction is carried out, the target DNA fragment of gel extraction is converted by transduction, plasmid standard is prepared into, and determine plasmid
Concentration, calculates plasmid standard copy number.
5. the fluorescence probe quantitative PCR quick determination method of bulk bacteria according to claim 4, it is characterised in that：It is described
The calculation formula of plasmid standard copy number is：Copy number=6.02 × 1023× concentration × 10-9/ (amplification base number × 660).
6. the fluorescence probe quantitative PCR quick determination method of bulk bacteria according to claim 1, it is characterised in that：In step
Suddenly (5), the tea sample pretreatment is comprised the steps of：
Weigh each 20g of Tea Samples to pour into sterilized conical flask, 150rpm vibrates 30min at room temperature, and liquid is transferred to
In 50mI centrifuge tubes, under the conditions of 4 DEG C, 10000r/min centrifugation 10min remove supernatant, collect precipitation, use sterile physiological salt
Water rinses tealeaves in conical flask；Repeat the above steps 4 times, finally obtained sediment suspended with 1.0mL sterile salines,
Mycelium is obtained, it is standby.
7. the fluorescence probe quantitative PCR quick determination method of bulk bacteria according to claim 6, it is characterised in that：It is described
Sterilize to add after physiological saline and bead toward in conical flask, carry out high pressure steam sterilization.
8. the fluorescence probe quantitative PCR quick determination method of the bulk bacteria according to claim any one of 1-7, its feature exists
In：The tea sample is Fu tea, thousand tael tea, black brick tea, green brick tea, brick tea, peace tea, Pu'er tea or six fort tea.
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| CN109182464A (en) * | 2018-10-29 | 2019-01-11 | 漳州龙文琪睿生物科技有限公司 | Coronoid process dissipate capsule bacterium loop-mediated isothermal amplification detection kit and its detection method |
| CN113249448A (en) * | 2021-05-17 | 2021-08-13 | 河南农业大学 | Method for accurately quantifying mycelium biomass of bag-cultivated oyster mushrooms and application of method |
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| US6387652B1 (en) * | 1998-04-15 | 2002-05-14 | U.S. Environmental Protection Agency | Method of identifying and quantifying specific fungi and bacteria |
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| CN109182464A (en) * | 2018-10-29 | 2019-01-11 | 漳州龙文琪睿生物科技有限公司 | Coronoid process dissipate capsule bacterium loop-mediated isothermal amplification detection kit and its detection method |
| CN113249448A (en) * | 2021-05-17 | 2021-08-13 | 河南农业大学 | Method for accurately quantifying mycelium biomass of bag-cultivated oyster mushrooms and application of method |
| CN113249448B (en) * | 2021-05-17 | 2024-01-30 | 河南农业大学 | Method for accurately quantifying bag-cultivated oyster mushroom mycelium biomass and application thereof |
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