CN106520969A - Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample - Google Patents
Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample Download PDFInfo
- Publication number
- CN106520969A CN106520969A CN201611052428.6A CN201611052428A CN106520969A CN 106520969 A CN106520969 A CN 106520969A CN 201611052428 A CN201611052428 A CN 201611052428A CN 106520969 A CN106520969 A CN 106520969A
- Authority
- CN
- China
- Prior art keywords
- soil sample
- soil
- pseudomonass
- standard
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a quantitative detection method for pseudomonadaceae in soil on the basis of a standard soil sample. The method specifically comprises steps as follows; firstly, the standard soil sample with the pseudomonadaceae concentration known is prepared, and DNA of the standard soil sample and a to-be-detected soil sample is extracted; a fluorescent quantitative PCR procedure and a reaction system are established, and finally, the concentration of pseudomonadaceae in the to-be-detected soil sample is detected. A conventional plate coating counting method for the pseudomonadaceae is improved, a standard curve is drawn according to the standard soil sample with the pseudomonadaceae concentration known, the pseudomonadaceae in the to-be-detected soil sample is detected quantitatively, CFU/g is taken as the unit in the result, the operation steps are efficient and convenient, the concentration of the pseudomonadaceae in the soil can be detected accurately, and the method is suitable for large-scale popularization and application.
Description
Technical field
The present invention relates to biological technical field, the method for pseudomonass in particularly a kind of detection by quantitative soil.
Background technology
Pseudomonass (Pseudomonadaceae) belong on taxonomy antibacterial domain, Proteobacteria, γ-deformation Gammaproteobacteria,
The Rhodopseudomonass of Pseudomonadales, pseudomonadaceae.Pseudomonass are widely present in soil, are to study most deep soil
One of microbial population.Pseudomonass are the important component parts of soil micro-ecosystem system and soil carbon-nitrogen cyclings.It is many false single
Born of the same parents bacterium can improve plant microenvironment, and Induction of Systemic Resistance of Plant, suppression pathogenic microorganism infect root system, have promotion plant life
The effect of long and controlling plant diseases.Some pseudomonass also have potentiality of the degraded using various artificial-synthetic compounds.This
Outward, some of pseudomonass plant heavy metal and organic pollution has resistance and can be enriched with which in vivo.To false in soil
The detection by quantitative of Zymomonas mobiliss can preferably analyze the micro- of Ecological Distribution of Soil Microorganisms function, soil Soil pathogen ability and contaminated soil
Biological restoration ability etc..
The current quantitative detecting method to pseudomonass in soil mainly flat board coating counting method (Elad and Baker,
1985；Garbeva etc., 2006).Flat board coating counting method is coated on selective medium Jing after gradient dilution for soil extraction
On, then pseudomonass colony counting is carried out, and then pseudomonass concentration in soil is calculated, there is larger error in this method
And difficulty, accuracy is low, time-consuming, and is easily affected by miscellaneous bacteria, and testing crew pseudomonass identification ability is had high demands.
As Protocols in Molecular Biology is increasingly mature, in adopting quantitative PCR technique to soil on DNA level now, vacation is single
Born of the same parents bacterium carries out detection by quantitative, and (Kaare etc., 1999), but general measure PCR quantifies to treat test sample with copy number or genome concentration
Product, acquired results unit are that per gram of per gram of soil of copy number or ng genomic DNAs are native, rather than per gram of bacterium number is native, it is impossible to intuitively
Understand the concentration of pseudomonass in soil.And different batches testing result systematic error is larger, the different batches of same sample are fixed
Amount result there may be more than thousand times of difference.Therefore, general measure PCR also has during pseudomonass concentration in soil is detected
Limitation.
The content of the invention
For the problems referred to above, the present invention provides one kind and adopts quantitative PCR technique with standard soil known to pseudomonass concentration
Sample draws mark song so as to the pseudomonass in detection by quantitative soil sample to be measured, can be used for the tracing detection of pseudomonass, is that research is false
Zymomonas mobiliss provide a kind of easy, efficient method to the annidation of soil and pseudomonass related soil ecological functions,
What the present invention was realized in：
The quantitative detecting method of pseudomonass in a kind of soil based on standard sample, which comprises the following steps that：
A) pseudomonass concentration is prepared respectively be followed successively by 5 × 107、5×106、5×105、5×104、5×103、5×102With
The graded series standard soil sample of 50CFU/mL (CFU/mL refers to every milliliter of bacterium solution of viable count, similarly hereinafter)；
B) graded series standard soil sample DNA and soil sample to be measured is extracted respectively using soil microbial DNA extracts kit
DNA, extraction step are strictly carried out according to description；
C) (Widmer is invented the specific primer of selected amplifiable pseudomonass, and 1998), synthesis specificity upstream and downstream is drawn
Thing SEQ ID NO.1 (Psf) and SEQ ID NO.2 (Psr)；
Graded series standard soil sample DNA and soil sample DNA to be measured are obtained as template with step b) respectively, SEQ ID NO.1
It is upstream and downstream primer with SEQ ID NO.2, carries out quantitative fluorescent PCR reaction；
D) logarithm value and the mapping of corresponding threshold cycle number according to graded series standard soil sample pseudomonass concentration, goes forward side by side
Row linear fit obtains standard curve：Y=40.765-4.755x, R2=0.991；
Soil sample quantitative fluorescent PCR reaction result to be measured (threshold cycle number and linear equation) is substituted into into standard curve, you can
Obtain the concentration of pseudomonass in soil sample to be measured.
Further, in the soil based on standard soil sample of the present invention in the quantitative detecting method of pseudomonass, step c)
The quantitative fluorescent PCR reaction system is：2 × SYBR Premix Ex Taq, 10 μ L, concentration are 10 μm of ol/L upstream and downstream and draw
0.4 μ L of each 0.4 μ L of thing, ROX Reference Dye II, concentration are 2 μ L of 40-60ng/ μ L DNA profilings, and the ultra-pure water that sterilizes is mended
Enough to 20 μ L；
Response procedures are：95 DEG C of denaturations 30s；95 DEG C of denaturations 5s, 65 DEG C of annealing 34s, totally 40 circulations.
Further, in the soil based on standard soil sample of the present invention in the quantitative detecting method of pseudomonass, the system
Row gradient standard soil sample is obtained by：
A) 126 DEG C of sterilizing 120min of arable soil are taken, soil sample after air-drying, is obtained；
B) pseudomonass are inoculated in beef extract-peptone fluid medium and are activated, the pseudomonass after activation are inoculated into
In 250mL triangular flasks equipped with 100mL beef extract-peptone fluid mediums, 37 DEG C, 160r/min shaking table cultures are after 2 days, put down
Plate is standby after counting；Bacterium solution is diluted to 5 × 10 successively with sterilized water7、5×106、5×105、5×104、5×103、5×102
And 50CFU/mL；
The bacterium solution after 20mL dilutions is taken respectively is added in the soil sample that 100g step a) are obtained that (i.e. volume mass compares 1:5,
ML/g) and Quick-air-drying (wind desiceted soil earth in 12h under the conditions of forced ventilation darkness), that is, obtain pseudomonass concentration be followed successively by 5 ×
107、5×106、5×105、5×104、5×103、5×102With the graded series standard soil sample of 50CFU/mL.
In the present invention, Psf and Psr can go out the product of 990bp to pseudomonass specific amplification.
Colony counting method involved in the present invention is reference literature [Shen Ping, Fan Xiurong, Li Guangwu.Microbiology Experiment
[M].Beijing：Higher Education Publishing House, 1999,92-94] carry out, which concretely comprises the following steps：1mL aseptic straws are used, 1mL bacterium are drawn
Liquid is moved in the test tube equipped with 9mL sterilized water, the mixing bacterium solutions of pressure-vaccum 3 times, 10-1 diluents；Change an aseptic straw again to inhale
Take 1mL10-1 diluents to move in the test tube equipped with 9mL sterilized water, the mixing bacterium solutions of pressure-vaccum 3 times, 10-2 diluents；With this
Analogize, a series of diluents such as 10-3,10-4,10-5,10-6,10-7,10-8 are made in serial dilution.Each diluent is drawn
0.1mL is smoothened to dry with aseptic spatula on beef extract-peptone agar culture medium flat board.37 DEG C culture three days after count, bacterium
Bacterium colony average × 10 × extension rate that liquid concentration=same dilution factor repeats several times.Know the dense of pseudomonass in bacteria suspension
Degree.
Provided by the present invention for the method for pseudomonass concentration in detection by quantitative soil, pseudomonass in soil can be entered
Row quick detection is quantitative, compared with prior art, the invention has the beneficial effects as follows：
(1) it is practical, the true pseudomonass concentration of reaction soil.The inventive method, can substitute continue to use always flat
Plate coating is counted and general measure round pcr, by improving general measure round pcr, with standard known to pseudomonass concentration
Soil sample DNA as quantitative PCR mark song DNA, it is countable go out soil sample to be measured in pseudomonass concentration, the data obtained unit is bacterium number
Per gram native, can so avoid classic flat-plate from being coated with the unreliability of counting method result, can avoid general measure PCR results again
Not intuitive.
(2) repeatability is high.Standard soil sample DNA of known pseudomonass concentration carries out quantitative simultaneously with soil sample DNA to be measured
The pseudomonass concentration of PCR, the period (Ct) experienced at fluorescence thresholds using each sample and standard sample sets up mark
Directrix curve, calculates corresponding pseudomonass concentration on standard curve according to testing sample Ct values.So can effectively eliminate same
Soil sample carries out the systematic error of different batches detection.I.e. same soil sample different batches testing result difference is less.
(3) it is simple and efficient to handle, using the inventive method, soil microbial DNA is carried out to standard soil sample and soil sample to be measured
Extract, then obtain a result by carrying out quantitative PCR, it is easy to operate, it is adapted to detect in soil pseudomonass concentration and which is carried out
Real-time detection.
Description of the drawings
Fig. 1 is specific detection primer to pseudomonass and the amplification of other common soil earth antibacterials；
In figure：Swimming lane 1-2 positions pseudomonass, 3-4 is bacillus cereuss, and 5-6 is streptomycete, and 7-8 is escherichia coli, and 9-10 is
Agrobacterium, 11-12 are negative control.
Fig. 2 be the present invention in soil pseudomonass sensitivity technique test amplification curve, sample threshold cycle number from
Height is on earth successively to 107、106、105、104、103、102With 10 pseudomonass/g wind desiceted soils.
Fig. 3 is the pseudomonass fluorescent quantitative PCR solubility curve figure of standard sample and testing sample, and solution temperature is 88.5
℃。
Fig. 4 is the pseudomonass fluorescent quantitative PCR curve chart of standard sample and testing sample, and vertical coordinate is circulation
Number, abscissa are soil Pseudomonas concentration.
Fig. 5 is standard sample and the pseudomonass quantitative fluorescent PCR canonical plotting of testing sample, y=40.765-
4.755x, R2=0.991.
Specific embodiment
Detection method according to the present invention is described further below in conjunction with specific embodiment.
Strain and culture medium that embodiment is related to：
The pseudomonass being related in the present embodiment are that (referring to document, " Pseudomonas aeruginosa combines life to Pseudomonas aeruginosa P1
The effect of the stifling prevention and control capsicum epidemic disease of thing ", Wang Qiujun etc., Jiangsu's agriculture journal, 2015)；Bacillus cereuss are bacillus subtilises
BS211 is (referring to document " antagonistic activity of Citrullus vulgariss endophytic Bacillus subtilis BS211 and potted plant preventive effect ", Ma Yan etc., Jiangsu's agriculture
Journal, 2006)；Streptomycete is Lou Che Shi streptomycete (referring to document " wheat straw different parts biodegradation rate difference ", Wang Jiajia
Deng, agricultural resource and environment journal, 2015)；Agrobacterium is Agrobacterium tumefaciems SK1044 (referring to document " agrobacterium mediation converted
The system optimization of verticillium dahliae ", Chen emperor etc., Cotton Science, 2011)；Escherichia coli are DH5a, purchase most valuable treasure biological engineering
(Dalian) company limited.
Beef extract-peptone agar culture medium：Carnis Bovis seu Bubali cream 5g, peptone 10g, NaCl 5g, agar 20g, water 1L, pH
7.2-7.4。
Beef extract-peptone fluid medium：Carnis Bovis seu Bubali cream 5g, peptone 10g, NaCl 5g, water 1L, pH 7.2-7.4.
The primer sequence that embodiment is related to：
SEQ ID NO.1(Psf)：5‘-GGTCTGAGAGGATGATCAGT-3’；
SEQ ID NO.2(Psr)：5‘-TTAGCTCCACCTCGCGGC-3’.
Reagent and instrument that embodiment is related to：
Soil microbial DNA extracts kit be MP soil DNA extracts kits, article No. 116560200；
SYBR premix Ex Taq test kits are (comprising 2 × SYBR Premix Ex Taq and ROX Reference Dye
II) purchased from precious biological engineering company limited, article No. is RR420A；
2 × Taq PCR Master Mix are purchased from Nanjing Ji Tian biotechnology Co., Ltd；
Psf/Psr primers are synthesized by Jin Sirui bio tech ltd；
PCR fluorescent quantitation instruments in embodiment are ABI7500 quantitative real time PCR Instruments, and data analysiss are determined by ABI7500 fluorescence
The analysis software that amount PCR instrument is carried is completed, and after the completion of quantitative fluorescent PCR reaction, it is glimmering that software is collected to each reaction automatically
Optical signal is analyzed, and selects suitable fluorescence thresholds, the period for then being experienced at fluorescence thresholds using each sample
(Ct) and the pseudomonass concentration of standard sample sets up standard curve, testing sample can be looked on standard curve according to its Ct value
To corresponding pseudomonass concentration.
Wind desiceted soil earth in 12h is referred under the conditions of i.e. forced ventilation darkness in embodiment to soil sample Quick-air-drying.
The special primer detection of 1 pseudomonass of embodiment
(1) prepared by each bacterial strain DNA
With all bacterial strains of beef extract-peptone fluid medium culture, then extract each with OMEGA DNA of bacteria extracts kit
Bacterial strain DNA.
(2) detect the synthesis of pseudomonass special primer
Psf：5’-GGTCTGAGAGGATGATCAGT-3’
Psr：5’-TTAGCTCCACCTCGCGGC-3’
Synthesized by Jin Sirui bio tech ltd.
(3) it is used for detecting the PCR reaction systems of pseudomonass
Respectively with each bacterial strain DNA as template, Psf and Psr is upstream and downstream primer, enters performing PCR amplification：
PCR reaction systems：
The each 0.5 μ L of primer of 2 × Taq PCR Master Mix, 12.5 μ L, 10 μm of ol/L, 1 μ L concentration are 40-60ng/ μ
The DNA profiling of L, sterilizing ultra-pure water complement to 25 μ L.
The PCR extents of reaction：
95 DEG C of denaturations 5min；92 DEG C of degeneration 1min, 64.1 DEG C of annealing 1min, 72 DEG C of extension 1min, totally 30 circulations；So
72 DEG C extend 10min afterwards.
(4) PCR primer identification
Taking 6 μ L PCR primers carries out 1% sepharose electrophoresis detection, observes under Jing SYBR-green dyeing uviol lamps, according to
The presence or absence of DNA bands and size are verified to the specificity of pseudomonass special primer.
(5) result of the test
PCR amplifications are as shown in figure 1, in Fig. 1, swimming lane 1-2 positions pseudomonass, 3-4 are bacillus cereuss, and 5-6 is strepto-
Bacterium, 7-8 are escherichia coli, and 9-10 is Agrobacterium, and 11-12 is negative control primer；As seen from Figure 1, Psf/Psr can only specificity
Amplify the band of 990bp from pseudomonass DNA (1-2), and bacillus cereuss (3-4), streptomycete (5-6), escherichia coli (7-
8), Agrobacterium (9-10) and negative control (11-12) are without amplified band (3-12).Therefore this pair of primer has well specifically
Property, only there is the amplified production of 990bp to pseudomonass, to other control strains without amplified production.
Embodiment 2 makes standard soil sample known to pseudomonass concentration
(1) culture of pseudomonass
The pseudomonas that test tube slant is preserved are inoculated in beef extract-peptone agar culture medium and are activated, then will be living
The pseudomonass for having changed are inoculated into the 250mL triangular flasks equipped with 100mL beef extract-peptone fluid mediums, are placed in shaking table, shake
Bed rotating speed is 160r/min, obtains pseudomonass liquid, carry out plate count to bacterium solution in 37 DEG C of environment after cultivating 2 days, cold
Hide standby.
Plate count method reference literature [Shen Ping, Fan Xiurong, Li Guangwu is carried out in embodiment to bacterium solution.Microorganism
Learn experiment [M].Beijing：Higher Education Publishing House, 1999,92-94] carry out, 1mL aseptic straws are used, 1mL bacterium solutions is drawn and is moved into dress
In having the test tube of 9mL sterilized water, the mixing bacterium solutions of pressure-vaccum 3 times, 10-1Diluent；An aseptic straw is changed again draws 1mL 10-1Diluent is moved in the test tube equipped with 9mL sterilized water, the mixing bacterium solutions of pressure-vaccum 3 times, 10-2Diluent；By that analogy, continuously
Dilution makes 10-3、10-4、10-5、10-6、10-7、10-8Etc. a series of diluents.Each diluent draws 0.1mL to Carnis Bovis seu Bubali cream egg
On white peptone agar culture medium flat board, smoothened to dry with aseptic spatula.37 DEG C of cultures were counted after 2 days, that is, know false unit cell in bacteria suspension
The concentration of bacterium.
(2) preparation of standard soil sample
In Jiangsu Province Agriculture Science Institute, six directions base installation Fructus Capsici booth takes topsoil soils and carries out 126 DEG C of sterilizing 120min,
Then air-dry and obtain soil sample；7 parts of 100g soil are taken, bacterium solution is diluted to into 5 × 107、5×106、5×105、5×104、5×
103、5×102And 50CFU/mL, taken during 20mL is added to 100g soil respectively and Quick-air-drying, obtain 107、106、105、104、
103、102With the graded series standard soil sample of 10CFU/g wind desiceted soils.
The sensitivity test of pseudomonass complete set technology in 3 detection by quantitative soil of embodiment
(1) preparation of standard soil sample DNA
10 are extracted using MP soil DNAs extracts kit7、106、105、104、103、102With the graded series mark of 10CFU/g
The DNA of quasi- soil sample.
(2) it is used for the PCR reaction systems of detection by quantitative pseudomonass
PCR reaction systems include 2 × SYBR Premix Ex Taq, 10 μ L, each 0.4 μ of Psf/Psr primers of 10 μm of ol/L
0.4 μ L of L, ROX Reference Dye II, 2 μ L of DNA profiling, sterilizing ultra-pure water complement to 20 μ L.
(3) it is used for the PCR response procedures of detection by quantitative pseudomonass
95 DEG C of denaturations 30s, 95 DEG C of denaturations 5s, 65 DEG C of annealing 34s, 40 circulations.
(4) quantitative pcr amplification
Standard soil sample DNA is carried out on ABI7500 instrument quantitative pcr amplification.
(5) result of the test
The present invention tests amplification curve as shown in Fig. 2 Fig. 2 to the sensitivity technique of variable concentrations pseudomonass in soil
In, sample threshold cycle number is corresponding in turn to 10 from high in the end7、106、105、104、103、102With 10 pseudomonass/g wind desiceted soils；Knot
Fruit is shown 10 under the method7、106、105、104、103、102Gradient amplification curve can be formed with the gradient standard specimen DNA of 10CFU/g,
Sensitivity is native up to 10CFU/g.10 pseudomonass be can detect that hence with pseudomonass concentration in the method detection soil
Per gram native.
The detection by quantitative of pseudomonass in 4 soil sample to be measured of embodiment
(1) preparation of soil sample DNA to be measured
Control soil：Take from Huai'an Qingpu District cultivated land；
Charcoal soil：Add in control soil 1.33wt% charcoal (by 20 500 DEG C of mesh wheat straw Jing Muffle furnaces or
Obtained by 600 DEG C of anaerobism cracking 1h)；
Phytophthora Capsici Zoospores liquid is uniformly added in above-mentioned two groups of soil so that Phytophthora capsici quantity is every gram of dry ground
500 zoospores, distinguish in above-mentioned two groups of soil and conventionally plant Fructus Capsici (Soviet Union green pepper No. 5), natural lighting, temperature
25 DEG C -35 DEG C, 32 days are planted (referring to document " control effect and study mechanism of the straw biological charcoal to capsicum epidemic disease ", Wang Guangfei
Deng, soil, 2015), then take respectively two groups of soil prepare according to the preparation process of embodiment 2 step (2) standard soil sample it is to be measured
Soil sample, by soil sample Quick-air-drying to be measured after, using soil microbial DNA extracts kit extract DNA.
(2) it is used for the PCR reaction systems of detection by quantitative pseudomonass
20 μ L PCR reaction systems：
The each 0.4 μ L of Psf/Psr primers of 2 × SYBR Premix Ex Taq, 10 μ L, 10 μm of ol/L, ROX Reference
Dye II0.4 μ L, concentration are that (in specific operation process, the concentration of DNA profiling can be in 40- for 2 μ L of 60ng/ μ L DNA profilings
Select in the range of 60ng/ μ L), sterilizing ultra-pure water complements to 20 μ L.
(3) it is used for the PCR response procedures of detection by quantitative pseudomonass
95 DEG C of denaturations 30s, 95 DEG C of denaturations 5s, 65 DEG C of annealing 34s, 40 circulations.
(4) quantitative pcr amplification
By 107、106、105、104With 103CFU/g standard soil samples and soil sample DNA to be measured carry out simultaneous quantitative PCR amplifications, and
Mapped with the logarithm value of standard soil sample pseudomonass concentration and corresponding threshold cycle number, and carry out linear fit and obtain standard song
Line.According to soil sample threshold cycle number to be measured and linear equation, the pseudomonass concentration in soil to be measured is calculated.
(5) result of the test
Addition charcoal soil and control soil Pseudomonas concentration are determined using said method.Solubility curve is shown in Fig. 3, molten
Solution temperature is 88.5 DEG C, and standard sample and testing sample solubility curve are unimodal.
Amplification curve is shown in Fig. 4 (vertical coordinate is period, and abscissa is soil Pseudomonas concentration), and amplification efficiency is
102.1。
Standard curve is shown in Fig. 5, the logarithm value of standard soil sample DNA and the mapping of corresponding threshold cycle number, and carries out Linear Quasi
Conjunction obtains standard curve, and mark song is y=40.765-4.755x, coefficient R2=0.991.
Amplification curve, solubility curve and standard curve meet quantitative PCR requirement, reliable results.According to soil sample threshold to be measured
Value period and linear equation, can calculate the pseudomonass concentration in soil to be measured, test repetitive operation twice, twice technology
As a result it is as shown in table 1：
1 same sample different batches testing result of table
| Sample | Pseudomonass concentration |
| Control soil 1 | 1.40×106CFU/g |
| Charcoal soil 1 | 1.63×106CFU/g |
| Control soil 2 | 1.37×106CFU/g |
| Charcoal soil 2 | 1.54×106CFU/g |
As shown in Table 1, add charcoal soil Pseudomonas and be significantly higher than control soil, this and relevant report research (are joined
See document " Effect of biochar amendments on mycorrhizal associations and Fusarium
Crown and root rot of asparagus in replant soils ", Elmer etc., Plant Disease, 2011；
“Induction of systemic resistance in plants by biochar,a soil applied carbon
Sequestering agent ", Elad etc., Phytopathology are 2010) consistent.
The repeatability detection test of 5 testing sample of embodiment
Fixed quantity detection is carried out to same soil sample using 4 methods described of embodiment, is sentenced with the testing result of this sample
Determine the repeatability of the method.
Soil sample to be measured：Huai'an Qingpu District cultivated land soil is taken from, Fructus Capsici (Soviet Union green pepper 5 is conventionally manually planted
Number), natural lighting, 25 DEG C -35 DEG C of temperature, 32 days (i.e. document " control effects and machine of the straw biological charcoal to capsicum epidemic disease of plantation
Reason research ", Wang Guangfei etc., soil, 1.2 section, CK1 blanks in 2015), using method same as Example 4, to above-mentioned
Soil sample carries out repeated detection, and testing result is as shown in table 2：
2 same sample different batches testing result of table
| Sample | Pseudomonass concentration |
| First time testing result | 1.32×106CFU/g |
| Second testing result | 1.37×106CFU/g |
| Third time testing result | 1.26×106CFU/g |
| 4th testing result | 1.23×106CFU/g |
From data in table 2 can be seen that the sample to be repeated several times testing result error less, examine hence with the method
When surveying pseudomonass concentration in soil, its testing result is affected less by systematic error, can accurately react false unit cell in soil
Bacteria concentration.
The 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 (3)
1. in a kind of soil based on standard soil sample pseudomonass quantitative detecting method, it is characterised in that comprise the following steps that：
a）Pseudomonass concentration is prepared respectively is followed successively by 5 × 107、5×106、5×105、5×104、5×103、5×102With 50
The graded series standard soil sample of CFU/mL；
b）Graded series standard soil sample DNA and soil sample DNA to be measured is extracted respectively；
c）Respectively with step b）Middle acquisition graded series standard soil sample DNA and soil sample DNA to be measured are template, with SEQ ID NO.1
It is upstream and downstream primer with SEQ ID NO.2, carries out quantitative fluorescent PCR reaction；
d）According to logarithm value and the mapping of corresponding threshold cycle number of graded series standard soil sample pseudomonass concentration, standard is obtained
Curve：Soil sample quantitative fluorescent PCR reaction result to be measured is substituted into into standard curve, you can pseudomonass is dense in acquisition soil sample to be measured
Degree.
2. quantitative detecting method according to claim 1 based on pseudomonass in the soil of standard soil sample, it is characterised in that
Step c）The quantitative fluorescent PCR reaction system is：2 × SYBR Premix Ex Taq, 10 μ L, concentration are 10 μm of ol/L's
0.4 μ L of each 0.4 μ L of upstream and downstream primer, ROX Reference Dye II, template 2 μ L of the concentration for 40-60ng/ μ L, sterilizing are super
Pure water complements to 20 μ L；
Response procedures are：95 DEG C of denaturations 30s；95 DEG C of degeneration 5s, 65 DEG C of annealing 34s, totally 40 circulations.
3. in the soil based on standard soil sample according to claim 1 or claim 2 pseudomonass quantitative detecting method, its feature exists
In what the graded series standard soil sample was obtained by：
A) arable soil is taken, and sterilize 120min in 126 DEG C, soil sample is obtained after air-drying；
B) pseudomonass after activation are inoculated in beef extract-peptone fluid medium, 37 DEG C, 160rpm shaking table cultures 2 days
Afterwards, bacterium solution is diluted to 5 × 10 successively with sterilized water7、5×106、5×105、5×104、5×103、5×102With 50 CFU/
ML, then according to volume mass compares 1：5 are added separately to step a）In the soil sample of acquisition and air-dry, that is, obtain pseudomonass concentration
It is followed successively by 5 × 107、5×106、5×105、5×104、5×103、5×102With the graded series standard soil sample of 50 CFU/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611052428.6A CN106520969A (en) | 2016-11-24 | 2016-11-24 | Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611052428.6A CN106520969A (en) | 2016-11-24 | 2016-11-24 | Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106520969A true CN106520969A (en) | 2017-03-22 |
Family
ID=58357191
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611052428.6A Pending CN106520969A (en) | 2016-11-24 | 2016-11-24 | Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106520969A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164462A (en) * | 2017-04-24 | 2017-09-15 | 江苏省农业科学院 | The quantitative detecting method of withered germ of water-melon in a kind of soil |
| CN107190055A (en) * | 2017-04-28 | 2017-09-22 | 浙江大学 | Soil bacteria high flux absolute quantification method |
| CN111057782A (en) * | 2020-01-19 | 2020-04-24 | 南京林业大学 | Method for detecting number of soil bacteria based on real-time fluorescent quantitative PCR |
| CN112129732A (en) * | 2020-08-13 | 2020-12-25 | 江苏大学 | Method for rapidly detecting bacillus cereus based on up-conversion magnetic separation |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613744A (en) * | 2009-06-04 | 2009-12-30 | 中国科学院南京土壤研究所 | A kind of soil Salmonellas fast quantitative measurement method for detecting that is used for |
| CN102337344A (en) * | 2011-11-04 | 2012-02-01 | 南京农业大学 | Quantitative detection method of escherichia coli in soil and assay kit thereof |
| CN102337343A (en) * | 2011-11-04 | 2012-02-01 | 南京农业大学 | Quantitative detection method of salmonella in soil and assay kit thereof |
| CN102936619A (en) * | 2012-06-15 | 2013-02-20 | 清华大学 | Method for quantitative determination of escherichia coli RNA, and specialized standard substance and application thereof |
| CN103397099A (en) * | 2013-08-16 | 2013-11-20 | 江苏省农业科学院 | Method for detecting quantity of pseudomonas fluorescens in rhizospheric soil during growth period of transgenic wheat by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction) |
| CN103477748A (en) * | 2013-09-06 | 2014-01-01 | 江苏省农业科学院 | Method for preventing and controlling soil-borne diseases of vegetables by aid of crop straws |
| CN106011256A (en) * | 2016-06-27 | 2016-10-12 | 江苏省农业科学院 | Method for detecting number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescence quantification PCR |
-
2016
- 2016-11-24 CN CN201611052428.6A patent/CN106520969A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613744A (en) * | 2009-06-04 | 2009-12-30 | 中国科学院南京土壤研究所 | A kind of soil Salmonellas fast quantitative measurement method for detecting that is used for |
| CN102337344A (en) * | 2011-11-04 | 2012-02-01 | 南京农业大学 | Quantitative detection method of escherichia coli in soil and assay kit thereof |
| CN102337343A (en) * | 2011-11-04 | 2012-02-01 | 南京农业大学 | Quantitative detection method of salmonella in soil and assay kit thereof |
| CN102936619A (en) * | 2012-06-15 | 2013-02-20 | 清华大学 | Method for quantitative determination of escherichia coli RNA, and specialized standard substance and application thereof |
| CN103397099A (en) * | 2013-08-16 | 2013-11-20 | 江苏省农业科学院 | Method for detecting quantity of pseudomonas fluorescens in rhizospheric soil during growth period of transgenic wheat by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction) |
| CN103477748A (en) * | 2013-09-06 | 2014-01-01 | 江苏省农业科学院 | Method for preventing and controlling soil-borne diseases of vegetables by aid of crop straws |
| CN106011256A (en) * | 2016-06-27 | 2016-10-12 | 江苏省农业科学院 | Method for detecting number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescence quantification PCR |
Non-Patent Citations (2)
| Title |
|---|
| FRANCO WIDMER等: "A Highly Selective PCR Protocol for Detecting 16S rRNA Genes of the Genus Pseudomonas (Sensu Stricto) in Environmental Samples", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 孙飞龙等: "铜绿假单胞菌荧光实时定量PCR标准品的构建", 《生物技术通报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164462A (en) * | 2017-04-24 | 2017-09-15 | 江苏省农业科学院 | The quantitative detecting method of withered germ of water-melon in a kind of soil |
| CN107190055A (en) * | 2017-04-28 | 2017-09-22 | 浙江大学 | Soil bacteria high flux absolute quantification method |
| CN107190055B (en) * | 2017-04-28 | 2018-10-02 | 浙江大学 | Soil bacteria high throughput absolute quantification method |
| CN111057782A (en) * | 2020-01-19 | 2020-04-24 | 南京林业大学 | Method for detecting number of soil bacteria based on real-time fluorescent quantitative PCR |
| CN112129732A (en) * | 2020-08-13 | 2020-12-25 | 江苏大学 | Method for rapidly detecting bacillus cereus based on up-conversion magnetic separation |
| CN112129732B (en) * | 2020-08-13 | 2023-10-10 | 江苏大学 | Method for rapidly detecting bacillus cereus based on up-conversion magnetic separation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103352078B (en) | Method and primer composition for detecting soybean fusarium oxysporum based on LAMP (loop-mediated isothermal amplification) technology | |
| Hadas et al. | Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds | |
| CN106520969A (en) | Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample | |
| Li et al. | Biosafety and colonization of Burkholderia multivorans WS-FJ9 and its growth-promoting effects on poplars | |
| CN105238876B (en) | LAMP primer group and its application method for tobacco ralstonia solanacearum detection | |
| CN105316265A (en) | Bacillus amyloliquefaciens used for biocontrol and application thereof | |
| CN108251545A (en) | A kind of method for quantitatively detecting VBNC state bacteriums | |
| CN107858316A (en) | Stenotrophomonas maltophilia, screening technique and the application of antagonism tobacco black shank bacterium | |
| CN107164497A (en) | Loop-mediated isothermal amplification technique detects the primer and kit of pseudomonas aeruginosa | |
| CN102634580B (en) | Primers and method for simultaneously detecting various pathogenic bacteria | |
| CN102586469A (en) | Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers | |
| CN104232782B (en) | A kind of detect tobacco soil-borne fungus pathogen PCR primer and application and method | |
| CN101812541B (en) | Lentinus edodes virus detection kit and method | |
| CN104946754A (en) | Method and detection kit for quantitatively detecting ralstonia solanacearum in soil | |
| CN103397099A (en) | Method for detecting quantity of pseudomonas fluorescens in rhizospheric soil during growth period of transgenic wheat by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction) | |
| CN105316427A (en) | Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum | |
| CN105018631B (en) | Method, its Primer composition and detection kit based on loop-mediated isothermal amplification technique detection carrot soft rot Pectinatus | |
| CN104195254B (en) | Method based on loop-mediated isothermal amplification technique detection Herba Equiseti Hiemalis's Fusariumsp and Primer composition | |
| CN103602727B (en) | Detection primer set, detection kit and detection method for rapidly detecting Ralstonia solanacearum | |
| CN111440890A (en) | Characteristic nucleotide sequence, identification primer and identification method of wild ganoderma lucidum W141201 | |
| CN102321548A (en) | Rhizobium sp. T3 and applications thereof in microbial degradation hydrogen sulfide | |
| CN102154462B (en) | Method and applications for fast detecting pathogenic bacteria molecules of bacterial soft rotting disease for banana | |
| JP2006238838A (en) | Method for assaying existence of bacterium | |
| CN113736679B (en) | Cold-resistant bacillus FJW2 and application thereof in preparation of plant pathogenic bacteria bacteriostatic agent | |
| Lingling et al. | Isolation and Identification ofthe Pathogen of Ginger Bacterial Wilt and Establishment of a PCR Detection System |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170322 |
|
| WD01 | Invention patent application deemed withdrawn after publication |