CN109852712A - A kind of simple and effective bacterial fungus bacterium colony PCR general program - Google Patents
A kind of simple and effective bacterial fungus bacterium colony PCR general program Download PDFInfo
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Abstract
The present invention relates to a kind of simple and effective bacterial fungus bacterium colony PCR general programs.The bacterial fungus bacterium colony PCR general program is that bacterium, fungi are inoculated in respectively on corresponding culture medium and cultivated, and culture medium, which grows obvious macroscopic bacterium colony, can use bacterium colony for experiment；By mechanical lapping disruption of microorganisms cell, DNA profiling is obtained after TE buffer is only added in clasmatosis sediment, gained DNA profiling can be directly used for PCR amplification；In addition to primer is different, bacterium, the reaction system of fungi subsequent PCR amplification, response procedures are identical.The method of the present invention is easy to operate safely, success rate is high, as a result reliable, it can be used for the amplification of gramnegative bacterium, gram-positive bacterium, yeast and filamentous fungi specific fragment, the step of simplifying bacterium colony PCR, time cost and economic cost are reduced, high-volume, the Rapid identification of multi-class environmental microorganism are suitably applied.
Description
Technical field
The present invention relates to a kind of simple and effective bacterial fungus bacterium colony PCR general programs.
Background technique
Round pcr is widely used to the gene studies and analysis of the every field such as biology, medicine, archaeology.PCR is fast
Speed identification microorganism specific dna sequence is the basic demand of conventional molecular biological research, and the successful extraction of strain template DNA
It is the basis of round pcr.
Strain template DNA preparation before standard PCR amplification is needed by broken wall, extracting, precipitating, purifying, operation
It is cumbersome, time-consuming, operation will cause the loss of DNA repeatedly, and the reagents such as phenol, chloroform for DNA extracting are unfavorable for operating
The health of person.Bacterium colony PCR is a kind of method of single bacterium colony using round pcr screening specific dna sequence, due to being not required to
Genomic DNA is isolated and purified from a large amount of culture of microorganism, bacterium colony PCR can quickly, simply determine that a small amount of bacterium is wrapped
Containing specific DNA sequence dna, other than microorganism classification identification, bacterium colony PCR is also frequently used for the mirror of gene magnification, recombination and integration
The fields such as fixed and plasmid construction, medical diagnosis.
Currently, Gram-negative and gram-positive bacterium, yeast, filamentous fungi colony polymerase chain reaction (PCR) method have been reported
[Woodman,Michael E.Direct PCR of intact bacteria(colony PCR).[J].Current
Protocols in Microbiology, 2008, Appendix 3:Appendix 3D.], [Amberg D C, Burke D
J, Strathern J N.Yeast colony PCR. [J] .Csh Protocols, 2006,2006 (1)], [Zhang Xiaoli, Lyu
Saussurea involucrata, Shen Yongnian wait bacterium colony round pcr quickly to identify experimental study [J] Chinese journal of dermatology of filamentous fungi, and 2011,
44 (8)], these methods mainly suspension microorganism cell again in certain buffer, then carry out it is necessary boil,
Freezing step is to promote cell cracking released dna, but every kind of method, bacterium, yeast, silk effective to the microorganism of particular category
The bacterium colony PCR operating parameter of shape fungi is had nothing in common with each other, and does not have broad applicability, the bacterium that the current country there is no bacterial fungus general
Fall the report of PCR.Further, since fungal cell's outer layer has thicker and tough and tensile cell wall, simple thermodynamic activity is difficult to make thin
Cellular lysate, therefore fungi especially filamentous fungi bacterium colony PCR effect is generally undesirable.Actually most of laboratory uses at present
Liquid-nitrogen freeze drying grinds broken filamentous fungal cell wall, but this method and step is cumbersome, and liquid nitrogen grinding is time-consuming and laborious, is easy to freeze
Hurt operator, in addition to this, generally requires to use toxic organic reagent after cell liquid nitrogen grinding and further purify, be unfavorable for
Operator's health safety.
Summary of the invention
The present invention provides a kind of simple and effective bacterial fungus bacterium colony PCR general program, easy to operate, is widely used in
The amplification of Gram-negative and gram-positive bacterium, yeast and filamentous fungi specific dna sequence, amplification positive rate are very high.
A kind of simple and effective bacterial fungus bacterium colony PCR general program is to be inoculated with bacterium, fungal cultures respectively
It on corresponding culture medium, is cultivated, when growing the visible single colonie of obvious naked eyes on culture medium, bacterium colony can be used as experiment,
DNA profiling is prepared, PCR amplification is carried out.
Above-mentioned culture medium and cultivation temperature are common knowledge, and general bacterium uses beef-protein medium, at 37 DEG C
Lower culture；General fungi uses PDA culture medium, cultivates at 28 DEG C.
DNA profiling the preparation method comprises the following steps:
Bacterium, the preparation method of fungal DNA template are identical, and operating procedure is as follows: with sterile pipette tip or going out
Bacterium toothpick picking directly from test dish tests bacterium colony, and moves into the centrifuge tube equipped with sterile water, adds into centrifuge tube
Enter to be put into beveller after a small amount of autoclaved stainless-steel grinding pearl and grind, takes lapping liquid to be centrifuged after grinding, remove in centrifugation
A small amount of TE buffer is added after clear liquid to be resuspended, made re-suspension liquid is the DNA mould that can be used as bacterial fungus bacterium colony PCR general program
Plate.
Picking experiment bacterium colony should as far as possible from experiment bacterium colony the suitable thallus of picking, if biomass is very few, meeting
PCR amplification efficiency is reduced, and excessive thallus may be such that PCR amplification is suppressed.According to applicant's research experience, work as lapping liquid
When centrifugation is 5~30mg, bacterium and yeast colony PCR result are ideal；When lapping liquid centrifugation is 10~50mg
When, filamentous fungi bacterium colony PCR result is ideal.
The volume of the sterile water is 400~1000 μ l；The volume of centrifuge tube is 1.5~2ml, stainless-steel grinding pearl diameter
For 1~3mm, dosage is 1.5~2.5g；Beveller is the quick beveller of automatic sample, and grinding frequency is 60~80Hz, time
For 5~10min；Centrifugal rotational speed is 8000~10000r/min, and the time is 1~2min；TE buffer volume is 20~60 μ l.
Preferably, bacterial fungus bacterium colony PCR general program DNA profiling the preparation method comprises the following steps: using sterile pipette
Suction nozzle appropriate thallus of picking from experiment bacterium colony, and move into the 1.5ml centrifuge tube equipped with 800 μ l sterile waters, into centrifuge tube
It after the autoclaved stainless-steel grinding pearl of 2.0g is added, covers tightly, is put into 70Hz in beveller and grinds 7min, then lapping liquid is taken to put
Enter in a new 1.5ml centrifuge tube, 8000r/min is centrifuged 2min, and the resuspension of 20 μ l TE buffers is added after abandoning supernatant, will weigh
Suspension 8000r/min is centrifuged 1min, and the supernatant after centrifugation is the DNA profiling that can be used as bacterial fungus bacterium colony PCR general program.
The general bacterium colony PCR reaction system of bacterial fungus are as follows: 1~5 μ l of DNA profiling, upstream and downstream primer each 1 μ l, PCR
Master mix12.5 μ l mends ddH2O to 25 μ l.
The upstream and downstream primer is common knowledge, for microbial identification, generally uses universal primer 27F (5'-
AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TCTGGACCTGGTGAGTTT CC-3') to bacterial 16 S rRNA into
Row PCR amplification uses universal primer ITS4 (5'-TCCTCCGCTT ATTGATATGC-3') and ITS1 (5'-
TCCGTAGGTGAACCTGCGG-3' PCR amplification) is carried out to the area fungi ITS.
As general knowledge known in this field, PCR Master Mix is comprising needed for Taq archaeal dna polymerase, dNTP and PCR
The all components other than DNA profiling and primer 2X concentrate solution.
PCR amplification program are as follows:
Bacterium, fungi PCR amplification program are identical, modify slightly on Standard PCR procedure basis, and operating procedure is such as
Under: 95 DEG C of 3~5min of initial denaturation；Recurring number (30~35 times) × (94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 50s)；
72 DEG C of extension 10min.
Microbial DNA template is prepared, DNA is needed to release from microbial cell, so clasmatosis is very heavy
The step wanted.Clasmatosis is broadly divided into chemical method and Mechanical Method two major classes, and compared to chemical method, mechanical milling method can be in short-term
Between shred cell, and there is versatility [Liang Ruifang, Xu Long, Yue Mingqiang cell breaking technology application study be in progress the Inner Mongol [J]
Agricultural science and technology, 2013 (1): 113-114.].Different classes of microbial cell breakage difficulty is different, usual gramnegative bacterium,
Gram-positive bacterium, yeast, filamentous fungi clasmatosis difficulty successively increase.If mechanical external force is too small, it is difficult to make micro-
Biological cell rupture；But if mechanical external force is excessive, it will lead to microbe genome DNA and destroyed, can not carry out subsequent
PCR amplification.Applicant it has been investigated that, be crushed various classification microbial cells using steel ball grinding, 70Hz grinds 7min or so,
Obtained DNA profiling can successfully carry out PCR amplification, milling time it is too long or it is too short be all unable to reach the above effect, so
The milling time of 7min or so is the key that bacterial fungus bacterium colony PCR general program of the present invention.
In gramnegative bacterium, gram-positive bacterium, yeast, the test of filamentous fungi bacterium colony PCR general program, Shen
It asks someone to be provided with regular colony PCR as method comparison, and sets up the positive control of Standard PCR and do not add the yin of DNA profiling
Property control, to reduce false positive or false negative result.
Bacterial fungus bacterium colony PCR general program of the present invention, PCR amplification positive rate are substantially better than regular colony PCR, have aobvious
The versatility of work not only solves the problems such as Standard PCR microbe genome DNA extraction step is cumbersome, broken wall is difficult, while also solving
Regular colony PCR positive rate of having determined is low, bacterial fungus processing method variability issues.The method of the present invention is easy to operate safely, success
Rate is high, as a result reliably, is generally applicable to gramnegative bacterium, gram-positive bacterium, yeast and filamentous fungi, passes through machinery
Disruption of microorganisms cell is ground, the preparation of pcr template can be completed in 15min, greatly reduces time cost and economic cost,
Especially suitable for high-volume, the Rapid identification of multi-class environmental microorganism, the mirror of transformant, recombination and integration simultaneously can be used for
It is fixed, plasmid construction and area of medical diagnostics.
Detailed description of the invention
Fig. 1 is the preparation flow of the DNA profiling of bacterial fungus bacterium colony PCR general program.1: adding into the centrifuge tube of sterilizing
Enter the 400 μ l sterile waters of μ l~1000；2: picking tests bacterium colony in right amount, moves into the centrifuge tube equipped with sterile water；3: to centrifuge tube
The middle stainless-steel grinding pearl being added after a small amount of high pressure sterilization；4: by centrifuge tube be put into beveller 60~80Hz grinding 5~
10min；5: the lapping liquid after taking grinding is put into the centrifuge tube of another sterilizing；6:8000~10000r/min is centrifuged 1~2min；
7: removing supernatant after centrifugation, 20~60 μ l TE buffers are added and are resuspended；8: using re-suspension liquid as DNA profiling, or with re-suspension liquid
Supernatant after micro- centrifugation is as DNA profiling.
Fig. 2 is the amplification of bacterial fungus bacterium colony PCR general program, including 16 kinds of bacteriums.1: Leuconostoc mesenteroides；2:
Lactococcus lactis；3: enterococcus faecium；4: class goldbeater's skin Wei Si Salmonella；5: Pediococcus acidilactici；6: citrobacter freundii；7: calcium acetate
Acinetobacter calcoaceticus；8: streptococcus pneumonia；9: staphylococcus saprophyticus；10: Sphingol single-cell；11: Siberia Exiguobacterium sp；12:
Spindle lysine bacillus；13: Bacillus anthracis；14: Bacillus cereus；15: bacillus subtilis；16: Xie Dian
Afnyloliquefaciens.
Fig. 3 is the amplification of bacterial fungus bacterium colony PCR general program, including 12 kinds of fungies.1: geotrichum candidum；2:
UnculturedGalactomyces；3: Kodak's yeast；4:Magnusiomyces capitatus；5: candida tropicalis；6:
Didymellakeratinophila；7: A Shi trichosporon cutaneum；8: long spore Lip river moral yeast；9: the basket bacterium of rope form；10: aspergillus niger；
11: Karl Jaspers；12: angstrom cutting candidiasis.
Fig. 4 is the amplification of Standard PCR, regular colony PCR and bacterial fungus bacterium colony PCR general program.M:100~
2000bp DNA Maker；1~3 is citric acid bacillus (gramnegative bacterium)；4~6 be bacillus subtilis (gram sun
Property bacterium)；7~9 be long spore Lip river moral yeast (yeast)；10~12 be Karl Jaspers (filamentous fungi)；1,4,7,10 be normal
Advise PCR amplification result；2,5,8,11 be regular colony PCR amplification result；3,6,9,12 be bacterial fungus bacterium colony PCR general program
Amplification；13 be negative control amplification.
Fig. 5 is the amplification of bacterial fungus bacterium colony PCR general program in 1 ufu pehtze of application example.M:100~
2000bp DNA Maker；1: spindle lysine bacillus；2: Lactococcus lactis；3: enterococcus faecium；4: class goldbeater's skin Wei Si
Salmonella；5:Klebsiella sp.；6: Young citric acid bacillus；7: Bacillus anthracis；8: staphylococcus saprophyticus；9: usually false
Silk yeast；10: aspergillus flavus；11: geotrichum candidum；12:Uncultured Galactomyces；13: yellow mucor.
Fig. 6 is the amplification of 2 workshop bacterial fungus bacterium colony PCR general program of application example.M:100~2000bp
DNA Maker；1:Klebsiella quasipneumoniae；2: bacillus amyloliquefaciens；3: Bacillus cereus；4: in plug
Jar (unit of capacitance) acetobacter；5: long spore Lip river moral yeast；6: Candida tropicalis；7: Tr. figueriae；8:Uncultured
fungus；9: Candida tropicalis.
Specific embodiment
Example 1: the broad applicability of bacterial fungus bacterium colony PCR general program
1. bacterium colony culture
Aseptically, it takes 16 plants of bacteriums and the 12 fungal strain bacteria suspensions of laboratory preservation a small amount of, bacterium is uniformly applied
It is distributed on the beef-protein medium prepared, 1~3d is cultivated at 37 DEG C；The PDA training that fungi even spread is prepared
It supports on base, 3~5d is cultivated at 28 DEG C.To grow the visible single colonie of obvious naked eyes on culture medium, bacterium colony can be used by being considered as experiment.
2. the preparation of the general DNA profiling of bacterial fungus bacterium colony PCR
1.5ml sterile centrifugation tube is taken, 800 μ l sterile waters are added, it is spare.It is chosen from experiment bacterium colony with sterile pipette tip
Appropriate thallus is taken, is moved into ready centrifuge tube, the stainless-steel grinding pearl after 2.0g high pressure sterilization, mark are added into centrifuge tube
Note, covers tightly, and is put into 70Hz in beveller and grinds 7min, then takes lapping liquid to be put into a new 1.5ml centrifuge tube, 8000r/
Min is centrifuged 2min, and the resuspension of 20 μ lTE buffers is added after abandoning supernatant, re-suspension liquid 8000r/min is centrifuged 1min, after centrifugation
Supernatant is used as the DNA profiling of bacterial fungus bacterium colony PCR general program, and operating process is shown in Fig. 1.
3.PCR amplification
200 μ l PCR pipes are taken, 12.5 μ l of PCR Master mix, each 1 μ l (bacterial universal primers of upstream and downstream primer are added
For 27F and 1492R, fungi universal primer is ITS1 and ITS4), 2.5 μ l of DNA profiling mends ddH2O to 25 μ l, label, covers tightly,
PCR pipe is put into PCR instrument and is expanded.PCR amplification program is as follows: 95 DEG C of initial denaturation 4min；33 circulations × (94 DEG C of denaturation
30s, 55 DEG C of annealing 30s, 72 DEG C of extension 50s)；72 DEG C of extension 10min.Reaction sets positive control and negative control, positive control
For the microbial DNA that conventional method is extracted, negative control replaces DNA profiling with deionized water.
4. agarose gel electrophoresis
Pcr amplification product carries out electrophoresis, each 5 μ l of well point sample, electrophoretic buffer 1 using 1% Ago-Gel
× TAE, constant pressure 121V, electrophoresis about 35min.After electrophoresis, Ago-Gel is taken to dye in the EB solution of 1 μ g/ml, dyed
Time about 15min.After dyeing, takes Ago-Gel to observe result under ultraviolet (uv) transmission in gel imager and take pictures.Bacterium
Electrophoresis result is shown in Fig. 2, and fungi electrophoresis result is shown in Fig. 3.
5. sequencing
PCR product serves Hai Shenggong bioengineering Co., Ltd and carries out base sequence measurement, and passes through ncbi database pair
Bacterial 16 S r DNA and fungi ITS area's base sequence carry out Blast comparison identification, and bacterium sequencing result is shown in Table 1, fungi sequencing knot
Fruit is shown in Table 2.
The general bacterium colony PCR sequencing result of 1:16 plants of bacteriums of table
The general bacterium colony PCR sequencing result of table 2:12 fungal strain
6. interpretation of result
In order to understand the success rate and popularity of bacterial fungus bacterium colony PCR general program of the present invention, applicant is arbitrarily chosen
16 plants of bacteriums of separated preservation and 12 fungal strains carry out the general bacterium colony PCR of bacterial fungus, and are surveyed to these microorganisms
Sequence identification.
According to fig. 2 and Fig. 3,16 plants of bacteriums and 12 fungal strains have amplified band after PCR, and wherein fungi amplified fragments are long
Degree is between 350~750bp, and bacterium expanding fragment length is in 1800bp or so.According to Tables 1 and 2,16 plants of bacteriums and 12 plants it is true
The PCR amplification sequence of bacterium can successfully be sequenced, and compare through Blast, and highest similitude is 97% or more.
In this test, bacterial fungus bacterium colony PCR general program is 100% for the success rate of microbial identification, although
Be not excluded for it is some be not used for this experiment, the method for the present invention can not successful identification peculiar microorganism, but just it is common, deposit extensively
Bacterial fungus sequence amplification for, the method for the present invention have very high broad applicability.
Example 2: bacterial fungus bacterium colony PCR general program is compared with regular colony PCR
1. bacterium colony culture
Choose identified gramnegative bacterium (citric acid bacillus), gram-positive bacterium (withered grass gemma in example 1
Bacillus), yeast (long spore Lip river moral yeast), filamentous fungi (Karl Jaspers) etc. cultivated, cultural method is the same as example 1.
2. the preparation of the general DNA profiling of bacterial fungus bacterium colony PCR
With example 1.
3. the preparation of Standard PCR template
Genomic DNA (kit operation instructions are shown in concrete operations) is extracted using DNA extraction kit, kit is purchased from
Shanghai Sheng Gong bioengineering Co., Ltd.
4. the preparation of regular colony pcr template
The preparation of regular colony pcr template, the suspension microorganism cell again mainly in certain buffer, then into
Row it is necessary boil, freezing step is to promote cell cracking released dna, articles of reference " Direct PCR of intact
bacteria(colony PCR)》[Woodman,Michael E.Direct PCR of intact bacteria(colony
PCR) [J] .Current Protocols in Microbiology, 2008, Appendix 3:Appendix 3D.] and specially
[Liu Weida, Zhang Xiaoli, Wang Miaomiao wait the bacterium of fungi to benefit " colony polymerase chain reaction (PCR) method of fungi and the method for identifying pathogenic epiphyte "
Fall PCR method and identify the method for pathogenic epiphyte: CN.] DNA profiling of bacterium and fungi is prepared respectively.
5.PCR amplification
PCR amplification system and program are the same as example 1.Reaction sets positive control and negative control, and positive control is conventional method
The microbial DNA of extraction, negative control replace DNA profiling with deionized water.
6. agarose gel electrophoresis
For method with example 1, electrophoresis result is shown in Fig. 4.
7. interpretation of result
In order to identify reliability and validity of the invention, under the same conditions to gramnegative bacterium, gram sun
Property the 4 seed type microorganisms such as bacterium, yeast, filamentous fungi, carried out Standard PCR positive control and regular colony PCR control
Test, and replace DNA profiling to carry out negative control experiments with deionized water.
According to Fig. 4, four kinds of microorganism Standard PCR electrophoresis results have obvious amplified band；Regular colony PCR electrophoresis result
Middle gramnegative bacterium, gram-positive bacterium, yeast have amplified band, and filamentous fungi can not show amplified band, and
And saccharomycete band is darker；The electrophoresis result of bacterial fungus bacterium colony PCR general program has obvious amplified band, and fragment length
Consistent with positive control, band brightness is darker relative to positive control, but brighter compared with regular colony PCR.
Compared to Standard PCR, bacterial fungus bacterium colony PCR general program has saved a large amount of labour cost and time cost,
And there is reliability；Compared to regular colony PCR, bacterial fungus bacterium colony PCR general program not only increases PCR amplification result
Validity, and it is common to gramnegative bacterium, gram-positive bacterium, yeast and filamentous fungi.Analysis is the reason is that, originally
Inventive method is ground by high speed machine, and 7min or so is ground at 70Hz, it is sufficient to be crushed various microbial cells, and basic
Microbial DNA will not be caused by mechanical damage and damage.
Application example 1: the application in ufu pehtze in microbial identification
1.1 background information
Certain fermented bean curd producer produces fermented bean curd by spontaneous fermentation, because being open production, bacterium, yeast in environment and mould
Bacterium is possible to invade, therefore fermented bean curd quality is unstable, easily occurs during prior fermentation putrid and deteriorated, and applicant utilizes bacterium
Fungus colony PCR general program has carried out separation identification to the microorganism in producer's blank.
1.2 sample treatment
Sample packaging is aseptically opened, takes ufu pehtze 25g in sterile homogenizing bag from fermented bean curd tank centre,
And 225ml sterile saline is added, homogeneous is carried out using homogenizer.
1.3 bacterium colony cultures
After sufficiently being patted fermented bean curd sample uniformly by homogenizer, it is immediately placed in aseptic operating platform and carries out gradient dilution
(10-1To 10-7), it then takes each gradient dilution liquid 1ml in sterilized petri dishes respectively, topples over melted and be cooled to 50 DEG C immediately
Culture medium (beef-protein medium, PDA culture medium), agar plate is made after shaking up, is then placed in incubator culture,
Cultural method is the same as example 1.
The preparation of the 1.4 general DNA profilings of bacterial fungus bacterium colony PCR
With example 1.
1.5PCR amplification:
With example 1.
1.6 agarose gel electrophoresis
For method with example 1, electrophoresis result is shown in Fig. 5.
The analysis of 1.7 sequencing results
The measuring method of sequence is the same as example 1.
13 kinds of microorganisms have been isolated from ufu pehtze, through sequencing identification be respectively spindle lysine bacillus,
Lactococcus lactis, enterococcus faecium, class goldbeater's skin Wei Si Salmonella, Klebsiella sp., Young citric acid bacillus, anthrax spore bar
Bacterium, staphylococcus saprophyticus, candida inconspicua, aspergillus flavus, geotrichum candidum, Uncultured Galactomyces, yellow mucor.
Wherein yellow mucor, it is mould be dominant fungi, Lactococcus lactis, Young citric acid bacillus and Bacillus anthracis be advantage it is thin
Bacterium.
Bacillus anthracis, staphylococcus saprophyticus are conditioned pathogen, and citric acid bacillus, enterococcus are enteric bacteria, yellow
Aspergillus can produce mycotoxin, this illustrates that the sanitary condition of production of preserved beancurd is poor, and ufu pehtze is contaminated, it is proposed that is stopped immediately
Using Pollution Field, tool, timely thoroughly sterilizing are prevented something from spreading, and suggest using the Karl Jaspers separated from blank in one's power
Artificial infection produces fermented bean curd, to reduce or inhibit varied bacteria growing.
Application example 2: the application in workshop in microbial identification
2.1 background information
For food, drug, the sanitary condition of workshop directly affects the hygienic quality of product.Certain juice production
Producer's collecting sample from its workshop condition carries out microculture, the bacterium of as a result isolated a variety of different shapes and true
Bacterium in order to understand major microorganisms classification in workshop condition, and takes corresponding control measure, and applicant is entrusted by manufacturer
Support, identifies these microorganisms using bacterial fungus bacterium colony PCR general program.
2.2 bacterium colony cultures
Aseptically, the sample presentation plate for opening producer is drawn with a small amount of thallus of oese picking after sterilizing with plate
The mode of line is by microbionation in beef-protein medium, and fungi is inoculated in PDA culture medium, and cultural method is the same as example 1.
The preparation of the 2.3 general DNA profilings of bacterial fungus bacterium colony PCR
With example 1.
2.4PCR amplification
With example 1.
2.5 agarose gel electrophoresis
For method with example 1, electrophoresis result is shown in Fig. 6.
The analysis of 2.6 sequencing results
The measuring method of sequence is the same as example 1.
Producer sample presentation microorganism is respectively Sphingol single-cell, bacillus amyloliquefaciens, waxy brood cell's bar through sequencing identification
Bacterium, Siberia Exiguobacterium sp, long spore Lip river moral yeast, Candida tropicalis, A Shi trichosporon cutaneum, the basket bacterium of rope form,
Didymellakeratinophila.Wherein bacillus and yeast bacterial content are higher, and bacillus is widely present in nature ring
In border, vitality is strong, and Bacillus cereus can cause to poison by food, and the production environment of fruit juice is particularly suitable for yeast growth, yeast
It is rotten that pollution can easily cause fruit juice, therefore producer mainly should take corresponding control measure to these two types of microorganisms.
Claims (4)
1. a kind of bacterial fungus bacterium colony PCR general program, which is characterized in that bacterium, fungi are inoculated in corresponding culture respectively
It is cultivated on base, culture medium, which grows obvious macroscopic bacterium colony, can use bacterium colony for experiment；Micro- life is crushed by mechanical lapping
Object cell obtains DNA profiling after TE buffer is only added in clasmatosis sediment, and gained DNA profiling can be directly used for
PCR amplification；In addition to primer is different, bacterium, the reaction system of fungi subsequent PCR amplification, response procedures are identical.
2. bacterial fungus bacterium colony PCR general program as described in claim 1, which is characterized in that bacterium, fungal DNA template
Preparation method is identical, the specific steps are as follows: is directly chosen from test dish with sterile pipette tip or sterilizing toothpick
Experiment bacterium colony is taken, and is moved into the centrifuge tube equipped with sterile water, a small amount of autoclaved stainless-steel grinding is added into centrifuge tube
It is put into beveller and grinds after pearl, take lapping liquid to be centrifuged after grinding, a small amount of TE buffer resuspension is added after removing centrifuged supernatant,
Made re-suspension liquid is the DNA profiling that can be used as bacterial fungus bacterium colony PCR general program.
3. bacterial fungus bacterium colony PCR general program as described in claim 1, which is characterized in that be prepared through claim 2
DNA profiling, bacterium, fungi PCR amplification program are identical, the specific steps are as follows: 95 DEG C of 3~5min of initial denaturation；Recurring number
(30~35 times) × (94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 50s)；72 DEG C of extension 10min.
4. DNA profiling preparation method as claimed in claim 2, which is characterized in that the volume of sterile water is 400~1000 μ l；
The volume of centrifuge tube is 1.5~2ml；Stainless-steel grinding pearl diameter is 1~3mm, and dosage is 1.5~2.5g；Beveller grinding frequency
Rate is 60~80Hz, and the time is 5~10min；Centrifugal rotational speed is 8000~10000r/min, and the time is 1~2min；TE buffer
Volume is 20~60 μ l.
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