CN106480202B - A method of seed Carried bacteria is detected based on silica solution density gradient centrifugation - Google Patents
A method of seed Carried bacteria is detected based on silica solution density gradient centrifugation Download PDFInfo
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Abstract
The present invention discloses a kind of method based on silica solution density gradient centrifugation detection seed Carried bacteria.This method comprises: the preparation of rice paddy seed to be checked, the isotonic mother liquor of Ludox HS40, discontinuous density Ludox HS40 mask work liquid prepare, are laid in centrifuge tube discontinuous density Ludox HS40 mask work liquid, centrifugation, the precipitating that suspended with 1 × TE buffer, seed Carried bacteria PCR amplification, the building of 16S rDNA clone library, the evaluation of 16S rDNA clone library, bacterial species analysis.The discontinuous density Ludox HS40 mask work liquid and parameter that the present invention utilizes silica solution to be laid in centrifuge tube, bacterium as much as possible can be isolated and purified, improve the accuracy of detection, it can reflect that the diversity of detected batch seed Carried bacteria is horizontal, it overcomes that separation of bacterial type in existing detection technique is few, the defect of bacterium entrained by seed cannot be reacted completely.
Description
Technical field
The invention belongs to seed quality detection technique fields, and in particular to the method for detection seed Carried bacteria.
Background technique
Currently, seed health degree detects during many countries have become seed " quality evaluation ", " plant quarantine "
Customary measure, it has also become indispensable component part in seed quality control system, some countries establish special seed
Healthy measurement experiment room.Seed health measurement is whether measurement seed carries pathogen (such as fungi, bacterium and virus), nocuousness
Animal (such as nematode and pest) health status, i.e., pest species entrained by seed and quantity are detected, wherein planting
The detection of sub- Carried bacteria is the important content in the detection of seed health degree.As International Rice institute sets up seed health from nineteen eighty-three
Portion (SEED HEALTH UNIT) and since building up seed health laboratory in 1987, has started to grind the system of seed health
Study carefully.
The purpose of seed health detection is: detection quarantine sexually transmitted disease evil；Determine the use value of seed lot；Evaluation kind
Whether son batch needs by seed treatment；Certain popularity for infecting disease is assessed for investigation or research purpose；Verify kind
The low reason of sub- germination percentage, makes up the deficiency of germination test.
The method of traditional detection seed Carried bacteria has washing method, agar ware method.
Washing method: 40, seed are extracted from seed lot at random, seed is put into sterilized 250mL triangular flask, so
Aqua sterilisa 25mL is added afterwards, it is 150rpm that triangle bottle closure, which is placed on revolving speed, and 1h fullys shake in 25 DEG C of shaking table, is collected outstanding
Supernatant liquid is centrifuged 10min in sterilized 10mL centrifuge tube, with the revolving speed of 3000rpm, and 200 μ L sterile waters are added after removing supernatant
Suspend precipitating.Suspension is subjected to 10 times of series of concentrations gradient dilutions with aqua sterilisa, LB solid plate is then applied and is detected.It washes
The method of washing is only capable of separating the part bacterium that the surface of the seed carries, and can not separate to the bacterium of Interior Seed, and need dilute
Cleaning solution is released, test of many times is needed more could completely to count the bacterial species and quantity of the surface of the seed carrying.Meanwhile by
In the not Culturability of part bacterium, the germy pure culture of institute cannot be isolated using washing method, it is difficult to carry to seed
Bacterial species effectively identified.
Agar ware method: being right specified in standard GB/T/T3543.7-1995 crop seeds-other item-tests
The bacterium that seed carries carries out separation inspection method.At present to the detection of seed Carried bacteria, substantially using standard GB/T/
Agar ware method in T3543.7-1995.40, seed are extracted from seed lot at random, 5mL first cleans rice paddy seed with aqua sterilisa
1 time, 3min successively is impregnated with 70% ethyl alcohol 2.5mL, 2.5% sodium hypochlorite 2.5mL impregnates 5min, and 70% ethyl alcohol 2.5mL impregnates
30s is then rinsed seed 3 times with aqua sterilisa, after seed is dried, is placed on LB agar medium, every ware puts 10, each
4 repetitions are handled, use the LB plate of blank as environmental control under same operation, then 28 DEG C of constant temperature incubation 72h detect rice
The bacterial species and quantity that seed carries.It is identical as washing method, due to the not Culturability of part bacterium, not using agar ware method
The germy pure culture of institute can be kept completely separate out, it is difficult to which the bacterial species carried to seed are effectively identified.
Silica solution category colloidal solution is dispersion liquid of the nanoscale silica dioxide granule in water or in solvent, odorless, nothing
Poison.Ludox HS40 silica solution is a kind of synthesis amorphous silica that partial size is 12nm, and dioxide-containing silica 40% is close
Degree is 1.312g/mL.
Summary of the invention
Separation of bacterial exists in the prior art the technical problem to be solved by the present invention is to overcome the detection of seed Carried bacteria
Type is few, cannot react that bacterium situation and disengaging time entrained by seed are long completely, is not able to satisfy lacking of quickly detecting
It falls into.
It is carried carefully in order to solve the above technical problems, the present invention provides one kind based on silica solution density gradient centrifugation detection seed
The method of bacterium, the following steps are included:
(1) preparation of rice paddy seed to be checked
Rice paddy seed 50 to be checked are taken, after first cleaning rice paddy seed 1~2 time to be checked with 200mL aqua sterilisa, successively with 70%
Ethyl alcohol 2.5mL impregnates 2~3min, and 2.5% sodium hypochlorite 2.5mL impregnates 3~5min, and 70% ethyl alcohol 2.5mL impregnates 20s~30s,
Then it is rinsed seed 3~4 times with aqua sterilisa, is ground in sterilizing mortar after seed is dried, filtered with 50 mesh sieve, take 50 mesh
The rice paddy seed that grinds below sieve uses 100 mesh sieve to filter again, and the rice paddy seed that grinds below 100 mesh sieve is taken to be placed in diameter
It is spare in the sterilizes culture dish of 9cm；
(2) preparation of the isotonic mother liquor of Ludox HS40
Ludox is mixed and made into according to the ratio of 1 ︰, 9 volume ratio with 10 × PBS buffer solution and Ludox HS40 silica solution
The isotonic mother liquor of HS40, the isotonic mother liquor density of Ludox HS40 are 1.308g/mL；
(3) discontinuous density Ludox HS40 mask work liquid is prepared
1#Ludox HS40 mask work liquid, 2#Ludox HS40 mask work liquid, 3# are prepared as follows
Ludox HS40 mask work liquid；
The preparation of 1#Ludox HS40 mask work liquid: isotonic with 10 × PBS buffer solution 1mL and 9mL Ludox HS40
The solution that mother liquor is hybridly prepared into is 1#Ludox HS40 mask work liquid, and 1#Ludox HS40 mask work liquid density is
1.108g/mL；
The preparation of 2#Ludox HS40 mask work liquid: isotonic with 10 × PBS buffer solution 4mL and 6mL Ludox HS40
The solution that mother liquor is hybridly prepared into is 2#Ludox HS40 mask work liquid, and 2#Ludox HS40 mask work liquid density is
1.082g/mL；
The preparation of 3#Ludox HS40 mask work liquid: isotonic with 10 × PBS buffer solution 6mL and 4mL Ludox HS40
The solution that mother liquor is hybridly prepared into is 3#Ludox HS40 mask work liquid, and 3#Ludox HS40 mask work liquid density is
1.042g/mL；
(4) discontinuous density Ludox HS40 mask work liquid is laid in centrifuge tube
In 10mL sterile centrifugation tube, it is first laid with 1#Ludox HS40 mask work liquid 2mL with pipettor, is then laid with
2#Ludox HS40 mask work liquid 2mL is finally laid with 3#Ludox HS40 mask work liquid 2mL, will be from after laying
Heart pipe is placed on rack for test tube, cannot be shaken；
(5) it is centrifuged
1. taking what step (1) laid in sterilizes culture dish to grind rice paddy seed 1.00g, it is placed in 10mL sterile centrifugation tube
In, 1 × HEPES buffer solution 8mL is added, 5min is mixed on turbula shaker, after being placed at room temperature for 5h, takes supernatant in Hunan instrument
20min, centrifugal condition are centrifuged on H1850R centrifuge are as follows: No. 3 rotors, 4 DEG C, 10000rpm, acceleration value is set as 5, brake value
It is set as 5；
2. after centrifugation, abandoning supernatant, precipitating is suspended to obtain suspension with 1 × HEPES buffer solution 1mL, takes 300 μ L of suspension laying
Solution surface in the centrifuge tube that step (4) is equipped with discontinuous density Ludox HS40 mask work liquid, each sample inspection
8 repetitions are done in survey, after laying, are centrifuged 15min, centrifugal condition on the instrument H1850R centrifuge of Hunan are as follows: No. 3 rotors, 4 DEG C,
8500rpm, acceleration value are set as 2, and brake value is set as 0, after forming density gradient separation layer, centrifuge tube is taken out, pipettor is used
Draw the cotton-shaped boundary layer solution between 1#Ludox HS40 mask work liquid and 2#Ludox HS40 mask work liquid；
(6) it is suspended and is precipitated with 1 × TE buffer
The cotton-shaped boundary layer solution of absorption is fitted into new centrifuge tube, 500 μ 10 × PBS buffer solution of L are added, is mixed
Afterwards, 3000rpm is centrifuged 2min, abandons supernatant and removes Ludox HS40, precipitating is suspended to obtain 1 × TE suspension with 1 × TE buffer；
(7) seed Carried bacteria PCR amplification
Using step (6) obtain 1 × TE suspension as total DNA template, with upstream primer 799F and downstream primer 1492R into
Row PCR amplification, total volume are the PCR reaction system of 50 μ L are as follows: 2 μ L, 1 × Taq reaction of DNA profiling, 5 μ L, 10nM/ μ L
22 μ L, 2.5M/ μ L dNTPs of μ L, 10nM/ μ L downstream primer 2 μ L, 1U/ μ L Taq enzyme of 1492R of upstream primer 799F, 4 μ L,
ddH2O 33μL；Response procedures: 94 DEG C of initial denaturation 5min, from 94 DEG C of denaturation 1min, 1min to 72 DEG C of extension 1min of 52 DEG C of annealing,
After 30 circulations, 72 DEG C of extension 10min, 4 DEG C of preservations；PCR product size is 750bp；The base sequence of the upstream primer 799F
Column are as shown in SEQ ID NO:1, and the base sequence of downstream primer 1492R is as shown in SEQ ID NO:2；
(8) building of 16S rDNA clone library
Will the obtained PCR product of amplification electrophoresis in 1.% Ago-Gel, after electrophoresis, cut in the UV lamp
700~800bp band recycles 750bp band, and JM109 competent cell is converted after being attached with carrier T and obtains transformant,
SOC culture medium culture is added, the SOC culture medium after culture is coated onto the LB plating medium containing ammonia benzyl, IPTG and X-Gal
On, after bacterium colony is grown, the white colony of random picking 160 or more is sequenced；
(9) 16S rDNA clone library is evaluated
By calculation formula: C=(1-n1/N) × 100%, wherein n1 is the number for containing only the OTU of a sequence；N is random
The total sequence number occurred in all white colonies of picking, and using QIIME software carry out the calculating of C value, C value be 95% with
On, (10) carry out bacterial species analysis as follows；
(10) bacterial species are analyzed
The 16S rDNA sequence that the sequencing result of step (8) is issued on NCBI is subjected to sequence alignment and determines bacterium kind
Class, through comparing, sequence similarity is 97% or more to be classified as same bacterial species.
Compared with prior art, the beneficial effects of the present invention are:
1, inventive process avoids traditional detection methods to need the step of cultivating, and can detect letter as comprehensive as possible
Breath can reflect that the diversity of this batch of seed Carried bacteria is horizontal.
In nature a large amount of microorganisms be can not cultivate (Wang Fengchao, Liu Weifeng, Chen Guanjun wait environmental microorganism
Genomics research and application [J] Chinese biological engineering magazine independent of culture, 2006,26(9): 76~83), therefore,
Although the method based on pure culture can obtain bacterial strain entity, since condition of culture limits, in the seed-borne fungi of a batch
It in detection, is only capable of being separated to a limited number of bacteriums, it is difficult to the integral status of reflection seed Carried bacteria completely.And use this hair
The method of bright physical separation, the bacterium that avoiding cannot cultivate are difficult to the drawbacks of obtaining, and can detecte letter as comprehensive as possible
Breath, to reflect that the diversity of this batch of seed Carried bacteria is horizontal.
2, time and cost are greatlyd save, and easy to operate, one sample time of separation detection is 48h, is able to satisfy quickly
The demand of detection.
16S rDNA is the most useful and most common molecular clock in the genealogical classification research of bacterium, and type is few, content
(account for about bacteria RNA content 80%) greatly, molecular size is moderate, is present in all biologies, and evolving has good clock
Property is known as the title of " bacterial fossil " with the conservative of height in structure and function.The rDNA in most of prokaryotes
All there are multiple copies, 5S, the copy number of 16S, 23S rDNA are identical.16S rDNA is due to being of moderate size, about 1.5Kb or so,
The difference between different Pseudomonas can be embodied and relatively easily obtain its sequence using sequencing technologies, compare conventional bacteria
Detection method, the step of eliminating microexamination, Determination of Physiological And Biochemical Indices, detecting a sample time is 48h, can be significantly
It saves time and cost, and easy to operate.And traditional washing method separates with agar ware method one sample time of separation detection and is
1-2 weeks, detection disengaging time was long, was not able to satisfy quick detection.
3, discontinuous density Ludox HS40 mask work liquid is laid in centrifuge tube using silica solution, energy will be as more as possible
Bacterium isolate and purify, improve the accuracy of detection, overcome in existing seed Carried bacteria detection technique that there is separation thin
Strain class is few, cannot react the defect of bacterium situation entrained by seed completely.
Ludox HS40 silica solution category colloidal solution is odorless, nontoxic.Silica solution is nanoscale silica dioxide granule in water
In or solvent in dispersion liquid, the silica gel particle of dispersion liquid is not of uniform size, after high speed centrifugation, can form a continuous density
Gradient, variety classes bacterium specific gravity, sedimentation coefficient are different, and the bacterium specific gravity, sedimentation coefficient under different conditions are also variant, can incite somebody to action
The different bacterium of sedimentation coefficient isolates and purifies.Living can not cultivate (viable but non-culturable state, VBNC)
Bacterium is a kind of self-protection form that bacterium resists severe living environment.For bacterium in poor environment, cell shortens spherical shape into
Or cell elongation is shown as, and volume increases, and it is more difficult or can not be separately cultured with conventional culture methods, it can not be examined
It surveys, but still there is metabolic activity, still can induce recovery under certain condition, have potential pathogenic.Use traditional agar ware
Method is detected after can not being cultivated when bacterium is in VBNC state, and uses Ludox HS40 silica solution density gradient
Centrifugation, is to be separated bacterium according to sedimentation coefficient, and different bacterium and bacterium sedimentation coefficient in different states are different, Ke Yitong
It crosses density gradient centrifugation method and obtains thallus, and detected by the method for PCR, therefore, the method for the present invention uses Ludox
HS40 silica solution density gradient and its suitable parameter compare existing agar ware method, energy separation detection to more bacteriums, tool
There is the advantage that traditional separation method is incomparable.
Detailed description of the invention
Fig. 1: the sub- Carried bacteria sample of the moon seed-grain of the method for the present invention density gradient centrifugation.It is 1# at arrow meaning
Cotton-shaped boundary layer solution between Ludox HS40 mask work liquid and 2#Ludox HS40 mask work liquid, as bacterium point
Absciss layer.
Fig. 2: the sub- Carried bacteria 16S rDNA clone library dilution curve of moon seed-grain, curve have reached plateau, because
This, it is believed that storage capacity meets analysis needs enough, and wherein abscissa is picked clones number, and ordinate is OTU(sorting of operation
Unit) number.
It is the base sequence of upstream primer 799F shown in SEQ ID NO:1 in sequence table.
It is the base sequence of downstream primer 1492R shown in SEQ ID NO:2 in sequence table.
Specific embodiment
The reagent that following embodiment is related to be it is commercially available, each embodiment without specified otherwise be conventional method, rice seed to be checked
Son is moon seed-grain, is provided by Yunnan Province Yuanyang County seed control station, moon seed-grain is also commercial product.
Ludox HS40 silica solution is commercial product.Ludox HS40 silica solution is a kind of synthesis that partial size is 12nm without fixed
Shape silica, dioxide-containing silica 40%, density 1.312g/mL.
Embodiment 1
The method of the present invention, steps are as follows:
(1) preparation of rice paddy seed to be checked
Moon seed-grain 50 is taken, is first cleaned the moon seed-grain son 1 time with 200mL aqua sterilisa, is successively 70% with volume fraction
Ethyl alcohol 2.5mL impregnates 3min, and the sodium hypochlorite 2.5mL that mass fraction is 2.5% impregnates 5min, and volume fraction is 70% ethyl alcohol
2.5mL impregnates 30s, then rinses moon seed-grain 3 times with aqua sterilisa, grinds in sterilizing mortar after seed is dried, first use
The screen filtration of 50 mesh takes and grinds moon seed-grain below 50 mesh sieve, then filtered with 100 mesh sieve, takes under 100 mesh sieve
Face grind moon seed-grain be placed in diameter be 9cm sterilizes culture dish in it is spare.
(2) preparation of the isotonic mother liquor of Ludox HS40
Ludox is mixed and made into according to the ratio of 1 ︰, 9 volume ratio with 10 × PBS buffer solution and Ludox HS40 silica solution
The isotonic mother liquor of HS40, the isotonic mother liquor density of Ludox HS40 are 1.308g/mL.
10 × the PBS buffer solution is prepared: acid dihydride potassium (KH2PO4): 0.27g；Disodium hydrogen phosphate (Na2HPO4):
1.42g；Sodium chloride (NaCl): 8g；Potassium chloride (KCl) 0.2g；800mL deionized water is added, dissolution is sufficiently stirred, is then added
Concentrated hydrochloric acid tune pH value is 7.4, last constant volume to 1L.
(3) discontinuous density Ludox HS40 mask work liquid is prepared
Divided with the Ludox HS40 that 10 × PBS buffer solution dilutes the isotonic mother liquor of Ludox HS40 and obtains following 3 kinds of density
From the separation that working solution is used for bacterium.The density gradient of the Ludox HS40 mask work liquid of 3 kinds of density be 1.108g/mL,
1.082g/m,1.042g/mL.1#Ludox HS40 mask work liquid, 2#Ludox HS40 point are specifically prepared as follows
From working solution, 3#Ludox HS40 mask work liquid；
The preparation of 1#Ludox HS40 mask work liquid: isotonic with 10 × PBS buffer solution 1mL and 9mL Ludox HS40
It is 1#Ludox HS40 mask work liquid that mother liquor, which is hybridly prepared into solution, and 1#Ludox HS40 mask work liquid density is
1.108g/mL；
The preparation of 2#Ludox HS40 mask work liquid: isotonic with 10 × PBS buffer solution 4mL and 6mL Ludox HS40
The solution that mother liquor is hybridly prepared into is 2#Ludox HS40 mask work liquid, and 2#Ludox HS40 mask work liquid density is
1.082g/mL；
The preparation of 3#Ludox HS40 mask work liquid: isotonic with 10 × PBS buffer solution 6mL and 4mL Ludox HS40
The solution that mother liquor is hybridly prepared into is 3#Ludox HS40 mask work liquid, and 3#Ludox HS40 mask work liquid density is
1.042g/mL；
Above 10 × PBS buffer solution is dilution, and the isotonic mother liquor of Ludox HS40 is to be diluted liquid.
The solution density calculation method of density gradient working solution is as follows:
①Vx=V0(ρ0-ρi)/(ρi-ρ10)
②ρi=(V0ρ0+Vxρ10)/(V0+Vx)
Wherein V0For diluted liquid product, ρ0To be diluted liquid density, VxFor dilution volume (mL), ρiIt is close for working solution
It spends (g/mL), acquisition, ρ is 2. calculated by above-mentioned formula10For dilution density (g/mL).
ρ0Product description by consulting purchase obtains, ρ10It is obtained by weighing technique.
Above 3 kinds of Ludox HS40 mask work liquid density calculates the value being related to and is shown in Table 1.
The density of 13 kinds of Ludox HS40 mask work liquid of table calculates the value being related to
(4) discontinuous density Ludox HS40 mask work liquid is laid in centrifuge tube
In 10mL sterile centrifugation tube, it is first laid with 1#Ludox HS40 mask work liquid 2mL with pipettor, is then laid with
2#Ludox HS40 mask work liquid 2mL is finally laid with 3#Ludox HS40 mask work liquid 2mL, will be from after laying
Heart pipe is placed on rack for test tube, cannot be shaken.
(5) it is centrifuged
1. taking what step (1) laid in sterilizes culture dish to grind rice paddy seed 1.00g, it is placed in 10mL sterile centrifugation tube
In, 1 × HEPES buffer solution 8mL is added, 5min is mixed on turbula shaker, after being placed at room temperature for 5h, takes supernatant in Hunan instrument
20min, centrifugal condition are centrifuged on H1850R centrifuge are as follows: No. 3 rotors, 4 DEG C, 10000rpm, acceleration value is set as 5, brake value
It is set as 5；
2. after centrifugation, abandoning supernatant, precipitating is suspended to obtain suspension with 1 × HEPES buffer solution 1mL, takes 300 μ L of suspension laying
1#Ludox HS40 mask work liquid 2mL, 2#Ludox HS40 mask work liquid 2mL and 3# are successively equipped in step (4)
Solution surface in the 10mL centrifuge tube of Ludox HS40 mask work liquid 2mL, each sample detection are done 8 repetitions, are laid
Afterwards, 15min, centrifugal condition are centrifuged on the instrument H1850R centrifuge of Hunan are as follows: No. 3 rotors, 4 DEG C, 8500rpm, acceleration value is set as
2, brake value is set as 0, after forming density gradient separation layer, centrifuge tube is taken out, 1#Ludox HS40 is carefully drawn with pipettor
Cotton-shaped boundary layer solution between mask work liquid and 2#Ludox HS40 mask work liquid；
1 × the HEPES buffer solution is prepared: taking 100 × HEPES storing liquid 10mL, 990mL ddH is added2O is configured to
For 1 × HEPES buffer solution.
The preparation of 100 × HEPES storing liquid: it takes 23.8g HEPES to be dissolved in 90mL distilled water, uses lmol/L
NaOH tune pH to 7.5~8.0, then uses ddH2O is settled to 100mL, filtration sterilization, 4 DEG C or -20 DEG C preservations.
(6) it is suspended and is precipitated with 1 × TE buffer
The cotton-shaped boundary layer solution of absorption is fitted into new centrifuge tube, 500 μ 10 × PBS buffer solution of L are added, is mixed
Afterwards, 3000rpm is centrifuged 2min, abandons supernatant and removes Ludox HS40, precipitating is suspended to obtain 1 × TE suspension with 1 × TE buffer；
The preparation of 1 × TE buffer: Tris alkali 6.06g is first weighed, 40mL ddH is added2O dissolution, is added dropwise dense HCl about 2.1mL
PH to 8.0 is adjusted, 50mL is settled to and obtains Tris solution；9.306g EDTA-Na is weighed again2·2H2O adds ddH2O 35mL, after stirring
With about 1g NaOH particle tune pH to 8.0, it is settled to 50mL and obtains EDTA solution.Take prepared Tris solution 1mL, EDTA solution
0.2mL adds ddH2It is 1 × TE buffer that O, which is settled to 100mL,.
(7) seed Carried bacteria PCR amplification
Using step (6) obtain 1 × TE suspension as total DNA template, with upstream primer 799F and downstream primer 1492R into
Row PCR amplification, total volume are the PCR reaction system of 50 μ L are as follows: 2 μ L, 1 × Taq reaction of DNA profiling, 5 μ L, 10nM/ μ L
22 μ L, 2.5M/ μ L dNTPs of μ L, 10nM/ μ L downstream primer 2 μ L, 1U/ μ L Taq enzyme of 1492R of upstream primer 799F, 4 μ L,
ddH2O 33μL；Response procedures: 94 DEG C of initial denaturation 5min, from 94 DEG C of denaturation 1min, 1min to 72 DEG C of extension 1min of 52 DEG C of annealing,
After 30 circulations, 72 DEG C of extension 10min, 4 DEG C of preservations；PCR product size is about 750bp；The base of the upstream primer 799F
Sequence is as shown in SEQ ID NO:1, and the base sequence of downstream primer 1492R is as shown in SEQ ID NO:2.
(8) building of 16S rDNA clone library
Will the obtained PCR product of amplification electrophoresis in 1.% Ago-Gel, after electrophoresis, cut in the UV lamp
700~800bp band recycles 750bp band, and JM109 competent cell is converted after being attached with carrier T and obtains transformant,
SOC culture medium culture is added, the SOC culture medium after culture is coated onto the LB plating medium containing ammonia benzyl, IPTG and X-Gal
On, after bacterium colony is grown, the white colony of random picking 160 or more is sequenced.Specific step is as follows:
1. preparing the LB plating medium and SOC culture medium containing ammonia benzyl, IPTG and X-Gal, each connection reaction prepares 2
A LB plating medium containing ammonia benzyl, IPTG and X-Gal balances plate to room temperature before coated plate.
The preparation of LB plating medium containing ammonia benzyl, IPTG and X-Gal: taking yeast extract 5g, tryptone 10g,
DdH is added in sodium chloride 10g and agar powder 15g2After O 1L mixing, 121 DEG C, 0.1~0.15MPa, sterilize 20min, and sterilizing terminates
Afterwards, it is green that 40 μ L, 200mg/mL IPTG of 20mg/mL X-Gal, 4 μ L, 100mg/mL ammonia benzyl is added when being cooled to 55 DEG C~60 DEG C
10 μ L of mycin is the LB plating medium containing ammonia benzyl, IPTG and X-Gal.
The preparation of SOC culture medium: taking tryptone 20g, yeast extract 5g and sodium chloride 0.5g, and 950mLddH is added2O
After dissolution, 250mM Klorvess Liquid 10mL is added, adjusting pH is 7.0, then uses ddH2O is settled to 1L, and 121 DEG C, 0.1~
0.15MPa, sterilize 20min, and the glucose solution 20mL that 1M sterilizing is added after sterilizing, when being cooled to 55 DEG C~60 DEG C is
SOC culture medium.
2. being centrifuged 30s by the 0.5mL centrifuge tube of carrier T and the PCR product of recycling at 4 DEG C, 2000rpm, being allowed to be pooled to
0.5mL is centrifuged bottom of the tube.
3. establishing connection reaction in accordance with the following methods, total volume is that the coupled reaction system of 10 μ L is as follows:
4. being placed at room temperature for 1h after being allowed to mixing with pipettor piping and druming coupled reaction system.
5. centrifugation makes ligation reaction be pooled to 0.5mL centrifugation bottom of the tube, takes 2 μ L connection reaction products to be added to and be placed on ice
1.5mL centrifuge tube in.
6. the JM109 competent cell frozen is placed in and dissolves 5min on ice, gently vibrates centrifuge tube and be allowed to mix.
7. taking 50 μ L to be added in the 1.5mL centrifuge tube for having ligation reaction the competent cell of dissolution, ice bath after mixing
20min。
8. thermal shock 45s~50s(cannot vibrate in 42 DEG C of water-baths).
9. centrifuge tube is transferred on ice rapidly, cooling 2min.
10. 950 μ L of SOC culture medium is added, 37 DEG C, 150rpm shaken cultivation 1.5h.
SOC culture medium after culture is coated on the LB plating medium containing ammonia benzyl, IPTG and X-Gal, to bacterium colony
After growing, the bacterium colony (containing Insert Fragment) of picking white, blue bacterium colony is then given up.
JM109 competent cell is commercial product, is purchased from TakaRa company.
(9) 16S rDNA clone library is evaluated
Clone library to be evaluated with C value (Coverage Value), C value is bigger, and the representativeness of clone library is stronger,
So as to show out the representative degree of sample Carried bacteria in constructed clone library.With dilution curve to clone library into
Row analysis and evaluation are then believed that storage capacity meets analysis needs, the moon enough when curve reaches plateau or the when of tending towards stability
The bright sub- Carried bacteria 16S rDNA clone library dilution curve of seed-grain is shown in Fig. 2, and wherein abscissa is picked clones number, and ordinate is
OTU(operational taxonomic unit) number, when curve tend to it is flat when, illustrate that sequencing data amount is reasonable, more data volumes only can
Generate a small amount of new OTU.Fig. 2 curve has reached plateau, thus, it is believed that storage capacity meets analysis needs enough, i.e.,
The clone library can represent the microbial diversity of " moon paddy " seed Carried bacteria group substantially.
By calculation formula: C=(1-n1/N) × 100%, wherein n1 is the number for containing only the OTU of a sequence；N is random
The total sequence number occurred in all white colonies of picking, and using QIIME software carry out the calculating of C value, C value be 95% with
On, show that the 16S rDNA clone library that step (8) is built can show out sample Carried bacteria in constructed clone library
Representative degree, in this example, C value is up to wherein n1=3.95 97.53%(；N=160), then bacterial species are carried out by step (10)
Analysis.If C value less than 95%, needs to re-establish library.
(10) bacterial species are analyzed
By step (8) from the survey of 160 clones of the random picking of LB plating medium containing ammonia benzyl, IPTG and X-Gal
Sequence result and the 16S rDNA sequence issued on NCBI carry out sequence alignment with BLAST, and it is higher close to receive Suo Yuqi homology
Edge species 16S rDNA sequence, and determine the affinity of known 16S rDNA sequence in sequence and NCBI be sequenced, warp
It compares, sequence similarity is 97% or more to be classified as same bacterial species, and see Table 2 for details for the bacterium situation that the present embodiment detects.
The website NCBI, that is, American National Bioinformatics Institute website (http；//www.ncbi.nlm.nih.gov).
Using the moon seed-grain of above-mentioned same batch with traditional agar ware method detection sub- Carried bacteria of moon seed-grain as control.
Table 1 uses the method for the present invention and traditional agar ware method separation detection bacterium situation
As can be seen from Table 2, for the moon paddy rice paddy seed of same batch, the method for the present invention can be examined more than traditional agar ware method
8 kinds of bacteriums out improve the accuracy of detection, overcome in existing seed Carried bacteria detection technique that there are separation of bacterial types
It is few, the defect of bacterium situation entrained by seed cannot be reacted completely.
SEQUENCE LISTING
<110>Cereal Crops Inst., Yunnan Prov. Agricultural Academy
<120>a kind of method based on silica solution density gradient centrifugation detection seed Carried bacteria
<130> /
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>bacillus megaterium (Bacillus megaterium)
<400> 1
aacaggatta gataccctg 19
<210> 2
<211> 18
<212> DNA
<213>Escherichia coli (Escherichia coli)
<400> 2
ggtaccttgt tacgactt 18
Claims (1)
1. a kind of method based on silica solution density gradient centrifugation detection seed Carried bacteria, it is characterised in that including following step
It is rapid:
(1) preparation of rice paddy seed to be checked
Rice paddy seed 50 to be checked are taken, after first cleaning rice paddy seed 1~2 time to be checked with 200mL aqua sterilisa, successively with 70% ethyl alcohol
2.5mL impregnates 2~3min, and 2.5% sodium hypochlorite 2.5mL impregnates 3~5min, and 70% ethyl alcohol 2.5mL impregnates 20s~30s, connects
With aqua sterilisa rinse seed 3~4 times, after seed is dried sterilizing mortar in grind, with 50 mesh sieve filter, take 50 meshes
The rice paddy seed that grinds below son uses 100 mesh sieve to filter again, and the rice paddy seed that grinds below 100 mesh sieve is taken to be placed in diameter and be
It is spare in the sterilizes culture dish of 9cm；
(2) preparation of the isotonic mother liquor of Ludox HS40
Ludox HS40 etc. is mixed and made into according to the ratio of 1 ︰, 9 volume ratio with 10 × PBS buffer solution and Ludox HS40 silica solution
Mother liquor is seeped, the isotonic mother liquor density of Ludox HS40 is 1.308g/mL；
(3) discontinuous density Ludox HS40 mask work liquid is prepared
1#Ludox HS40 mask work liquid, 2#Ludox HS40 mask work liquid, 3#Ludox HS40 are prepared as follows
Mask work liquid；
The preparation of 1#Ludox HS40 mask work liquid: mixed with 10 × PBS buffer solution 1mL and the isotonic mother liquor of 9mL Ludox HS40
Closing the solution being configured to is 1#Ludox HS40 mask work liquid, and 1#Ludox HS40 mask work liquid density is 1.108g/
mL；
The preparation of 2#Ludox HS40 mask work liquid: mixed with 10 × PBS buffer solution 4mL and the isotonic mother liquor of 6mL Ludox HS40
Closing the solution being configured to is 2#Ludox HS40 mask work liquid, and 2#Ludox HS40 mask work liquid density is 1.082g/
mL；
The preparation of 3#Ludox HS40 mask work liquid: mixed with 10 × PBS buffer solution 6mL and the isotonic mother liquor of 4mL Ludox HS40
Closing the solution being configured to is 3#Ludox HS40 mask work liquid, and 3#Ludox HS40 mask work liquid density is 1.042g/
mL；
(4) discontinuous density Ludox HS40 mask work liquid is laid in centrifuge tube
In 10mL sterile centrifugation tube, it is first laid with 1#Ludox HS40 mask work liquid 2mL with pipettor, is then laid with 2#
Ludox HS40 mask work liquid 2mL is finally laid with 3#Ludox HS40 mask work liquid 2mL, after laying, by centrifuge tube
It is placed on rack for test tube, cannot shake；
(5) it is centrifuged
1. taking what step (1) laid in sterilizes culture dish to grind rice paddy seed 1.00g, it is placed in 10mL sterile centrifugation tube, then
1 × HEPES buffer solution 8mL is added, 5min is mixed on turbula shaker, after being placed at room temperature for 5h, takes supernatant in Hunan instrument
20min, centrifugal condition are centrifuged on H1850R centrifuge are as follows: No. 3 rotors, 4 DEG C, 10000rpm, acceleration value is set as 5, brake value
It is set as 5；
2. after centrifugation, abandoning supernatant, precipitating is suspended to obtain suspension with 1 × HEPES buffer solution 1mL, and 300 μ L of suspension is taken to be layed in step
Suddenly (4) are equipped with the solution surface in the centrifuge tube of discontinuous density Ludox HS40 mask work liquid, and each sample detection does 8
Secondary repetition after laying, is centrifuged 15min, centrifugal condition on the instrument H1850R centrifuge of Hunan are as follows: No. 3 rotors, 4 DEG C, 8500rpm,
Acceleration value is set as 2, and brake value is set as 0, and after forming density gradient separation layer, centrifuge tube is taken out, and draws 1# with pipettor
Cotton-shaped boundary layer solution between Ludox HS40 mask work liquid and 2#Ludox HS40 mask work liquid；
(6) it is suspended and is precipitated with 1 × TE buffer
The cotton-shaped boundary layer solution of absorption is fitted into new centrifuge tube, 500 μ L10 × PBS buffer solution is added, after mixing,
3000rpm is centrifuged 2min, abandons supernatant and removes Ludox HS40, precipitating is suspended to obtain 1 × TE suspension with 1 × TE buffer；
(7) seed Carried bacteria PCR amplification
1 × TE the suspension obtained using step (6) is carried out as total DNA template with upstream primer 799F and downstream primer 1492R
PCR amplification, total volume are the PCR reaction system of 50 μ L are as follows: on 2 μ L, 1 × Taq reaction of DNA profiling, 5 μ L, 10nM/ μ L
22 μ L, 2.5M/ μ L dNTPs of μ L, 10nM/ μ L downstream primer 2 μ L, 1U/ μ L Taq enzyme of 1492R of primer 799F, 4 μ L is swum,
ddH2O 33μL；Response procedures: 94 DEG C of initial denaturation 5min, from 94 DEG C of denaturation 1min, 1min to 72 DEG C of extension 1min of 52 DEG C of annealing,
After 30 circulations, 72 DEG C of extension 10min, 4 DEG C of preservations；PCR product size is 750bp；The base sequence of the upstream primer 799F
Column are as shown in SEQ ID NO:1, and the base sequence of downstream primer 1492R is as shown in SEQ ID NO:2；
(8) building of 16S rDNA clone library
Will the obtained PCR product of amplification electrophoresis in 1% Ago-Gel, after electrophoresis, cut 700 in the UV lamp~
800bp band recycles 750bp band, and JM109 competent cell is converted after being attached with carrier T and obtains transformant, is added
SOC culture medium after culture is coated on the LB plating medium containing ammonia benzyl, IPTG and X-Gal by SOC culture medium culture, to
After bacterium colony is grown, the white colony of random picking 160 or more is sequenced；
(9) 16S rDNA clone library is evaluated
By calculation formula: C=(1-n1/N) × 100%, wherein n1 is the number for containing only the OTU of a sequence；N is to choose at random
The total sequence number occurred in all white colonies taken, and using QIIME software carry out the calculating of C value, C value be 95% with
On, (10) carry out bacterial species analysis as follows；
(10) bacterial species are analyzed
The 16S rDNA sequence that the sequencing result of step (8) is issued on NCBI is subjected to sequence alignment and determines bacterial species, is passed through
It compares, sequence similarity is 97% or more to be classified as same bacterial species.
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