CN102277430A - Rape stem base botryosphaeria dothidea molecule standard sample and preparation method thereof - Google Patents
Rape stem base botryosphaeria dothidea molecule standard sample and preparation method thereof Download PDFInfo
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- CN102277430A CN102277430A CN2011102197078A CN201110219707A CN102277430A CN 102277430 A CN102277430 A CN 102277430A CN 2011102197078 A CN2011102197078 A CN 2011102197078A CN 201110219707 A CN201110219707 A CN 201110219707A CN 102277430 A CN102277430 A CN 102277430A
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Abstract
The invention relates to the technical search field of plant quarantine, in particular to a rape stem base botryosphaeria dothidea molecule standard sample. The rape stem base botryosphaeria dothidea molecule standard sample is prepared from raw material high-purity DNA extracted from a rape stem base botryosphaeria dothidea strain, the standard sample has the following physical and chemical properties: (1) the standard sample is pure white powdery; (2) content of each tube is 5 minus or plus 0.5 Mug; (3) DNA purity OD260/280 is equal to 1.8-2; and (4) the standard sample is soluble in water or a TE (Tris-EDTA) buffer solution. The rape stem base botryosphaeria dothidea strain self-separated in a laboratory is taken as raw material, mass culture under specific condition is carried out, high-purity DNA is extracted, and split charging and freeze drying are carried out to obtain the rape stem base botryosphaeria dothidea molecule standard sample, and the physical and chemical properties of the obtained rape stem base botryosphaeria dothidea molecule standard sample are excellent and can be detected by virtue of a PCR method by adopting a specific primer, and flexibility can reach 10-4 times. By virtue of a detection test, DNA completeness, uniformity and stability of the standard sample are good, the standard sample can be used as a positive reference substance during import and export detection, and accuracy of detection result is improved.
Description
Technical field
The present invention relates to rape stem foot ulcer bacteria molecular criteria sample, the invention still further relates to its preparation method in addition, belong to Plant Quarantine technical study field.
Background technology
A lot of about the report of animal virus and food microorganisms reference material both at home and abroad, the reference material research of phytopathogen is then at the early-stage, and a lot of problems all need to be resolved hurrily.The bacterial strains of American Type Culture Collecti (ATCC) of buying as positive control more when phytopathogen detected both at home and abroad, its price is very expensive, domesticly almost can't buy, and the rarely seen correlative study report of the molecular dna reference material of phytopathogen Molecular Detection, so deep comprehensively technology of preparing and the stable assurance technology of researching and solving phytopathogen detection DNA standard model, actively develop the development of China Plant Quarantine pathogenic bacteria detection molecules DNA standard model, replenish the blank of this fields of measurement, have very important practical sense.
Summary of the invention
The purpose of this invention is to provide rape stem foot ulcer bacteria molecular criteria sample and its production and application explanation.
Rape stem foot ulcer bacteria molecular criteria sample of the present invention, extracting high purity DNA with rape stem foot ulcer bacteria bacterial strain is that raw material makes, standard model has following physico-chemical property: (1) shape: pure white is Powdered; (2) the content 5 μ g ± 0.5 μ g of every pipe; (3) DNA purity: OD260/280=1.8～2; (4) soluble in water, or TE(Tris-EDTA) in the damping fluid.
Described DNA purity: OD260/280=1.85.
The preparation method of rape stem foot ulcer bacteria molecular criteria sample of the present invention comprises the steps:
(1) rape stem foot ulcer bacteria bacterial strain adopts potato sucrose (PD) liquid nutrient medium, places under 20～22 ℃ of conditions and cultivates 5～7 days;
(2) extract rape stem foot ulcer bacteria DNA, adopt the CTAB method to extract DNA;
(3) preparation of nucleic acid standard model: the DNA that will measure after the concentration carries out packing by 5 μ g ± 0.5 μ g/pipe, every kind of nucleic acid molecule standard specimen packing 100-400 pipe, and freeze-drying then is the purpose standard substance.
Described extraction rape stem foot ulcer bacteria DNA comprises the steps:
1. get the dried mycelia of 0.1 g and place the mortar of precooling, in liquid nitrogen, be ground into powder rapidly, powder is changed over to rapidly in the centrifuge tube of 1.5 ml (making multitube simultaneously);
2. the CTAB that adds 65 ℃ of preheatings of 800 μ l in the centrifuge tube of dried mycelia powder is housed in 1. extracts damping fluid, the vortex abundant mixing that vibrates, 65 ℃ of water-bath 50 min put upside down mixing once gently every 10 min;
3. be cooled to room temperature, centrifuge tube adds the chloroform of 800 μ l in 2. again: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down mixing gently, and leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
4. in 3., add and the isopyknic chloroform of this supernatant liquor in the gained supernatant liquor: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down mixing gently, leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
5. in 4., add in the gained supernatant liquor and the isopyknic Virahol of this supernatant liquor or 2 times of volume ice dehydrated alcohols, put upside down mixing gently, the DNA flocks appears, be placed on and place 30 min deposit D NA on ice, 4 ℃ of 12000 centrifugal 10 min of rpm abandons supernatant, and centrifuge tube is inverted on the filter paper dry, with 800 μ l concentration, 70 % ice ethanol washing and precipitating twice, to remove salt ion etc.After adding 800 μ l concentration, 70 % ice ethanol, put upside down centrifuge tube gently several times, the DNA that is deposited in the bottom is upspring, place centrifuge tube 10 min, 4 ℃ of 10000 centrifugal 1 min of rpm abandons supernatant; Wash the DNA precipitation with 800 μ l concentration, 70 % ice ethanol again, method is the same, and 4 ℃ of 15000 centrifugal 3 min of rpm abandons supernatant, air-dry removal ethanol;
6. in 5., add 200 μ l TE dissolving DNAs precipitation in the gained DNA precipitation.After treating fully dissolving, add 3 μ l RNase (10 mg/ml), 37 ℃ of water-bath 30 min; Handle by 3.-5. going on foot the same treatment method then;
7. with the 6. dry DNA precipitation of step after handling, dissolve with 100 μ l TE ,-20 ℃ of preservations are standby.
Described to the qualitative evaluation of standard model, carry out pcr amplification or qualitative analysis is carried out in order-checking by Auele Specific Primer, confirm that prepared nucleic acid is target DNA:
Get the nucleic acid standard model of preparation, add 50 μ lTE, use as template the dissolving back, carries out PCR and order-checking through adopting Auele Specific Primer Phom I and Phom II, carries out qualitative analysis, confirms that prepared nucleic acid standard model is the purpose nucleic acid samples,
Forward primer: 5'-CACCAATTGGATCCCCTA-3'
Reverse primer: 5'-AGGCGAGTCCCAAGTGGAACA-3'
Reaction system:
Template 1～2 μ l
Forward primer 1 μ l
Reverse primer 1 μ l
Taq enzyme 0.2U
dNTP 4?μl
10×Buffer 3?μl
Total 30?μl
Reaction conditions:
94℃?3min
94℃?30s
53℃?30s
72 ℃ of 40s circulate 35 times
72℃?5min。
Rape stem foot ulcer bacteria molecular criteria sample of the present invention is a raw material with laboratory isolating voluntarily rape stem foot ulcer bacteria bacterial strain, cultivate in a large number by specified conditions, extract high purity DNA, carry out packing, freeze-drying, obtain the molecular criteria product of rape stem foot ulcer bacteria, the physico-chemical property excellence can adopt specific primer to detect by PCR method, and sensitivity reaches 10
-4Doubly.Test after testing, these standard substance DNA integrity, homogeneity, good stability can be used for importing and exporting in the detection, as positive reference substance, improve the accuracy of detected result.
Four, description of drawings
Fig. 1 is the total DNA electrophorogram of rape stem foot ulcer bacteria.
Fig. 2 is rape stem foot ulcer bacteria primer amplified electrophoresis result figure, and wherein 1,2,3 is target DNA.
Fig. 3 is standard model sensitivity detected result figure of the present invention, and wherein by left-to-right, per two lattice are one group, are 10 successively
-1, 10
-2, 10
-3, 10
-4, 10
-5
Five, embodiment
Below in conjunction with specific embodiment the present invention is described in further detail, but the present invention is not limited to specific embodiment.
Embodiment 1
Rape stem foot ulcer bacteria molecular criteria sample preparation methods carries out as follows:
(1) rape stem foot ulcer bacteria bacterial strain adopts potato sucrose (PD) liquid nutrient medium, places under 20～22 ℃ of conditions and cultivates 5～7 days.
(2) extract rape stem foot ulcer bacteria DNA, adopt the CTAB method to extract DNA, concrete steps are as follows:
1. get the dried mycelia of 0.1 g and place the mortar of precooling, in liquid nitrogen, be ground into powder rapidly, powder is changed over to rapidly in the centrifuge tube of 1.5 ml (making multitube simultaneously);
2. the CTAB that adds 65 ℃ of preheatings of 800 μ l in the centrifuge tube of dried mycelia powder is housed in 1. extracts damping fluid, the vortex abundant mixing that vibrates, 65 ℃ of water-bath 50 min put upside down mixing once gently every 10 min;
3. be cooled to room temperature, centrifuge tube adds the chloroform of 800 μ l in 2. again: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down mixing gently, and leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
4. in 3., add and the isopyknic chloroform of this supernatant liquor in the gained supernatant liquor: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down mixing gently, leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
5. in 4., add in the gained supernatant liquor and the isopyknic Virahol of this supernatant liquor or 2 times of volume ice dehydrated alcohols, put upside down mixing gently, the DNA flocks appears, be placed on and place 30 min deposit D NA on ice, 4 ℃ of 12000 centrifugal 10 min of rpm abandons supernatant, and centrifuge tube is inverted on the filter paper dry, with 800 μ l, 70 % ice ethanol washing and precipitating twice, to remove salt ion etc.After adding 800 μ l, 70 % ice ethanol, put upside down centrifuge tube gently several times, the DNA that is deposited in the bottom is upspring, place centrifuge tube 10 min, 4 ℃ of 10000 centrifugal 1 min of rpm abandons supernatant; Wash the DNA precipitation with 800 μ l, 70 % ice ethanol again, method is the same, and 4 ℃ of 15000 centrifugal 3 min of rpm abandons supernatant, air-dry removal ethanol;
6. in 5., add 200 μ l TE dissolving DNAs precipitation in the gained DNA precipitation.After treating fully dissolving, add 3 μ l RNase (10 mg/ml), 37 ℃ of water-bath 30 min; Handle by 3.-5. going on foot the same treatment method then;
7. with the 6. dry DNA precipitation of step after handling, dissolve with 100 μ l TE ,-20 ℃ of preservations are standby.
(3) preparation of nucleic acid standard model: the DNA that will measure after the concentration carries out packing by 5 μ g/ pipes, every kind of nucleic acid molecule standard specimen packing 150 pipes, and freeze-drying then is the purpose standard substance, and have following physico-chemical property: shape: pure white is Powdered; The content 5 μ g ± 0.5 μ g of every pipe; DNA purity: OD260/280=1.8～2; Soluble in water, or TE(Tris-EDTA) in the damping fluid.
(4) get the nucleic acid standard model of preparation, add 50 μ lTE, use as template the dissolving back, carries out PCR and order-checking through adopting specificity, carries out qualitative analysis, confirms that prepared nucleic acid standard model is the purpose nucleic acid samples.Example:
Forward primer: 5'-CACCAATTGGATCCCCTA-3'
Reverse primer: 5'-AGGCGAGTCCCAAGTGGAACA-3'
Reaction system:
Template 1～2 μ l
Forward primer 1 μ l
Reverse primer 1 μ l
Taq enzyme 0.2U
dNTP 4?μl
10×Buffer 3?μl
Total 30?μl
Reaction conditions:
94℃?3min；?94℃?30s，53℃?30s，72℃?40s，35cycles；72℃?5min。
The standard model that embodiment 1 is made carries out DNA integrity, homogeneity, stability test:
1.DNA quality and integrity detection
Get the DNA of preparation, carry out gel electrophoresis, purity and concentration and detect.
(1) DNA integrity detection: the dna direct electrophoretic examinations that direct method-usefulness is extracted, 120V, 1.5% agarose gel electrophoresis, the integrity of observing band, detected result is seen Fig. 1, and band is complete, and no disperse shape illustrates that DNA band integrity is better.
(2) DNA concentration and purity detecting: ultraviolet spectrophotometer method-absorption 1 μ l DNA is with the absorption value of spectrophotometric determination 260nm and 280nm, then according to OD
260/ OD
280Value is judged DNA purity, and according to OD
260Calculate its concentration.Calculate as follows:
DNA concentration (ng/ μ l)=OD
260* 50
Work as OD
260/ OD
280＜1.8, the expression protein content is higher; OD
260/ OD
280＞2.0, the expression rna content is higher; Work as OD
260/ OD
280=1.8～2.0, DNA is purer in expression.
2. standard model analysis of Uniformity
For checking the homogeneity of the purpose nucleic acid standard model for preparing, adopt PicoGreen dna molecular fluorescent quantitation method and ultraviolet spectrophotometry, get and randomly draw 15 pipe samples, every pipe sample is divided into 2 one's share of expenses for a joint undertaking samples to be tested, and the results are shown in (table 1) data and carries out F check (table 2) confirmatory sample homogeneity.
3. standard model stability analysis
Originally there is the card standard model to have the stability period limit gauge accepted argument of card standard model bright (as the production regulation explanation of U.S. SIGMA company with reference to the external card of band on an equal basis, this has the card standard model in vacuum, lucifuge ,-20 ℃ of storages down, stable validity period is 2 years) be foundation, biological nature according to microorganism, scheduled to last with 2 years, standard model to preparation is got two duplicate samples stage by stage at every turn, carries out qualitative test according to different preservation conditions, and every duplicate samples is its definite value result to measure 3 sub-values.In whole stability tests, used personnel, instrument, testing method and laboratory are all identical with uniformity test.
According to said determination method difference calculation result, this rape stem foot ulcer bacteria standard model is in vacuum, lucifuge ,-20 ℃ of storages down, and stable validity period is 2 years, and measurement result sees Table 3.
In the stability test, slope can calculate with following formula:
In the formula:
Intercept is calculated by following formula:
The standard deviation of the point on the straight line can be calculated by following formula:
Get its square root s=0.12857%, the uncertainty relevant with slope calculated with following formula:
Degree of freedom is n-2=5 and p=0.95(95% confidence level) student's t-factor that distributes equal 2.57.
Because
So slope is inapparent.Thereby do not observe unstable in the stability experiment.
4. sensitivity test
The molecular criteria product of this germ are diluted to 100ng/ μ l, as mother liquor, dilute by 10 times of concentration gradients then, four (4) methods are carried out the PCR detection in the by specification.Detected result show when this diluted sample to 10000(promptly 10
-4*) doubly, band is invisible, therefore judges that the sensitivity of these standard substance is 10
-4
Table 1 rape stem foot ulcer bacteria molecular criteria product homogeneity detected result
| Numbering | Increment 1 test result (μ g) | |
Mean value | Standard deviation |
| 1-1 | 5.3975 | 5.9195 | 5.659 | 0.36911 |
| 1-2 | 5.469 | 5.086 | 5.278 | 0.27082 |
| 1-3 | 5.364 | 5.47 | 5.417 | 0.07495 |
| 1-4 | 5.7105 | 5.901 | 5.806 | 0.13470 |
| 1-5 | 5.131 | 5.7865 | 5.459 | 0.46351 |
| 1-6 | 5.1415 | 5.608 | 5.375 | 0.32987 |
| 1-7 | 5.8975 | 5.9185 | 5.908 | 0.01485 |
| 1-8 | 5.6285 | 5.5045 | 5.567 | 0.08768 |
| 1-9 | 5.863 | 5.6815 | 5.772 | 0.12834 |
| 1-10 | 5.4235 | 5.317 | 5.370 | 0.07531 |
| 1-11 | 5.5945 | 5.4525 | 5.524 | 0.10041 |
| 1-12 | 5.057 | 5.036 | 5.047 | 0.01485 |
| 1-13 | 5.1665 | 5.9955 | 5.581 | 0.58619 |
| 1-14 | 5.398 | 5.9015 | 5.650 | 0.35603 |
| 1-15 | 5.332 | 5.044 | 5.188 | 0.20365 |
Table 2 rape stem foot ulcer bacteria molecular criteria product homogeneity F assay
Table 3 rape stem foot ulcer bacteria molecular criteria product Detection of Stability result
Claims (6)
1. rape stem foot ulcer bacteria molecular criteria sample is characterized in that: extracting high purity DNA with rape stem foot ulcer bacteria bacterial strain is that raw material makes, and standard model has following physico-chemical property: (1) shape: pure white is Powdered; (2) the content 5 μ g ± 0.5 μ g of every pipe; (3) DNA purity: OD260/280=1.8～2; (4) soluble in water, or TE(Tris-EDTA) in the damping fluid.
2. rape stem foot ulcer bacteria molecular criteria sample according to claim 1 is characterized in that: DNA purity: OD260/280=1.85.
3. the preparation method of rape stem foot ulcer bacteria molecular criteria sample is characterized in that: comprise the steps:
(1) rape stem foot ulcer bacteria bacterial strain adopts potato sucrose (PD) liquid nutrient medium, places under 20～22 ℃ of conditions and cultivates 5～7 days;
(2) extract rape stem foot ulcer bacteria DNA, adopt the CTAB method to extract DNA;
(3) preparation of nucleic acid standard model: the DNA that will measure after the concentration carries out packing by 5 μ g ± 0.5 μ g/pipe, every kind of nucleic acid molecule standard specimen packing 100-400 pipe, and freeze-drying then is the purpose standard substance.
4. the preparation method of rape stem foot ulcer bacteria molecular criteria sample according to claim 1 is characterized in that: described extraction rape stem foot ulcer bacteria DNA comprises the steps:
1. get the dried mycelia of 0.1 g and place the mortar of precooling, in liquid nitrogen, be ground into powder rapidly, powder is changed over to rapidly in the centrifuge tube of 1.5 ml;
2. the CTAB that adds 65 ℃ of preheatings of 800 μ l in the centrifuge tube of dried mycelia powder is housed in 1. extracts damping fluid, the vortex abundant mixing that vibrates, 65 ℃ of water-bath 50 min put upside down mixing once gently every 10 min;
3. be cooled to room temperature, centrifuge tube adds the chloroform of 800 μ l in 2. again: the primary isoamyl alcohol mixed solution, put upside down mixing gently, and leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
4. in 3., add and the isopyknic chloroform of this supernatant liquor in the gained supernatant liquor: the primary isoamyl alcohol mixed solution, put upside down mixing gently, leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
5. in 4., add in the gained supernatant liquor and the isopyknic Virahol of this supernatant liquor or 2 times of volume ice dehydrated alcohols, put upside down mixing gently, the DNA flocks appears, be placed on and place 30 min deposit D NA on ice, 4 ℃ of 12000 centrifugal 10 min of rpm abandons supernatant, and centrifuge tube is inverted on the filter paper dry, with 800 μ l concentration, 70 % ice ethanol washing and precipitating twice, to remove salt ion etc.;
After adding 800 μ l concentration, 70 % ice ethanol, put upside down centrifuge tube gently several times, the DNA that is deposited in the bottom is upspring, place centrifuge tube 10 min, 4 ℃ of 10000 centrifugal 1 min of rpm abandons supernatant; Wash the DNA precipitation with 800 μ l concentration, 70 % ice ethanol again, method is the same, and 4 ℃ of 15000 centrifugal 3 min of rpm abandons supernatant, air-dry removal ethanol;
6. in 5., add 200 μ l TE dissolving DNAs precipitation in the gained DNA precipitation;
After treating fully dissolving, add 3 μ l RNase (10 mg/ml), 37 ℃ of water-bath 30 min; Handle by 3.-5. going on foot the same treatment method then;
7. with the 6. dry DNA precipitation of step after handling, dissolve with 100 μ l TE ,-20 ℃ of preservations are standby.
5. the preparation method of rape stem foot ulcer bacteria molecular criteria sample according to claim 3, it is characterized in that: to the qualitative evaluation of standard model, carry out pcr amplification or qualitative analysis is carried out in order-checking by Auele Specific Primer, confirm that prepared nucleic acid is target DNA:
Get the nucleic acid standard model of preparation, add 50 μ lTE, use as template the dissolving back, carries out PCR and order-checking through adopting Auele Specific Primer Phom I and Phom II, carries out qualitative analysis, confirms that prepared nucleic acid standard model is the purpose nucleic acid samples,
Forward primer: 5'-CACCAATTGGATCCCCTA-3'
Reverse primer: 5'-AGGCGAGTCCCAAGTGGAACA-3'
Reaction system:
Template 1～2 μ l
Forward primer 1 μ l
Reverse primer 1 μ l
Taq enzyme 0.2U
dNTP 4?μl
10×Buffer 3?μl
Total 30?μl
Reaction conditions:
94℃?3min
94℃?30s
53℃?30s
72 ℃ of 40s circulate 35 times
72℃?5min。
6. the preparation method of rape stem foot ulcer bacteria molecular criteria sample according to claim 3 is characterized in that: the chloroform that adopts among the described extraction rape stem foot ulcer bacteria DNA: the primary isoamyl alcohol mixeding liquid volume is than being 24:1.
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| CN103255207A (en) * | 2013-01-15 | 2013-08-21 | 内蒙古农牧业科学院 | Molecular detection method for brassica campestris seeds carrying brassica campestris leptosphaeria maculans pathogenic bacterial |
| CN107099613A (en) * | 2017-06-27 | 2017-08-29 | 中国检验检疫科学研究院 | A kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria |
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| CN107099613A (en) * | 2017-06-27 | 2017-08-29 | 中国检验检疫科学研究院 | A kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria |
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Application publication date: 20111214 |