CN101363055B - Molecule specificity making primer of mushroom Cr02 bacterial and detection method - Google Patents
Molecule specificity making primer of mushroom Cr02 bacterial and detection method Download PDFInfo
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Abstract
The invention provides a molecule particularity mark primer of mushroom Cr02 strain and a method for to distinguishing and detecting the mushroom Cr02 strain by using the primer. The sequence of the primer is: an upstream primer is 5'-TCTCGATGCAAGAAACTTGAA-3', a downstream primer is 5'-TCGGTCAAAATTAGTCAGGT-3'. The invention has the main advantages that compared with conventional morphological detection, antagonism and fruiting experiment, the method has short detection time and high accuracy; the method only needs 1-3 days for detection, while the conventional antagonism needs at least two weeds for detection, and the fruiting experiment needs at least 3 months for detection.
Description
(1) technical field
The present invention relates to a kind of molecular specificity labeled primers of mushroom Cr 02 bacterial, and the method for utilizing this primer that mushroom Cr 02 bacterial is differentiated and detected.
(2) background technology
China's mushroom production from 1.95 ten thousand tons of 2,420,000 tons of developing 2005 rapidly of nineteen eighty-three, accounts for more than 70% of global mushroom ultimate production.Scholarly forecast is arranged, because the fast development of Chinese mushroom industry, it will become the highest edible mushrooms of world wide production in nearly 10 years.China's mushroom is that world mushroom industry personage attractes attention with its alarming development speed, good quality and cheap cost, the own fashionable world of Chinese mushroom.Excellent species is very important to the contribution rate of mushroom production and quality, and this has determined the critical role of mushroom strain in the mushroom industry.China had signed " international new variety of plant protection method " in 1999, and this not only requires us to respect other national kind intellecture property, also will strengthen protecting the kind intellecture property of our country oneself simultaneously.Really protect the kind property right of China in order to set up edible mushrooms new variety resignation system, must at first set up sophisticated cultivar identification technology, for the new variety registration lays the foundation.Especially Japan on April 1st, 2004 came into effect " seedling method amendment ", to the export mixes of China edible mushrooms especially mushroom great threat.With regard to domestic, the phenomenon of cultivating champignon bacterial classification " different name of the same race, xenogenesis is of the same name " has brought great loss not only for the mushroom farming, has influenced their cultivation enthusiasm, has also greatly influenced the fast development of Chinese mushroom; And, more and more higher along with the appearance of large-scale factory culture mode to the requirement of cultivating champignon bacterial strain quality, need more easy, the identification of strains technology fast and accurately of development, with guarantee every batch with kind all accurate.
The foundation of the fast development of Protocols in Molecular Biology, particularly molecular marking technique is with ripe, and is easy for developing, the strain identification technology provides effective means fast and accurately.Be different from RAPD commonly used (Random Amplified Polymorphic DNA, randomly amplified polymorphic DNA) and AFLP (Amplified Fragment Length Polymorphism, amplified fragment length polymorphism) molecular marking technique, SCAR (Sequence CharacterizedAmplified Region, characteristic fragment amplification zone) mark is a kind of very stable molecule marker, has rapid, easy, characteristics cheaply on using.
(3) summary of the invention
The object of the invention provides a kind of molecular specificity labeled primers of mushroom Cr 02 bacterial, and a kind of method of utilizing this primer mushroom Cr 02 bacterial to be carried out Rapid identification.
The technical solution used in the present invention is:
A kind of molecular specificity labeled primers of mushroom Cr 02 bacterial, described primer sequence is:
Upstream primer 5 '-TCTCGATGCAAGAAACTTGAA-3 ';
Downstream primer 5 '-TCGGTCAAAATTAGTCAGGT-3 '.
This primer is to being the employing round pcr, through a large amount of shaker tests, adopt RAPD to be labeled as the DNA fragment specific that primer obtains mushroom Cr 02 bacterial, with this fragment cloning order-checking, to obtain dna sequence dna, designed Auele Specific Primer carries out pcr amplification with this primer to mushroom Cr 02 bacterial, can obtain the specific fragment of 333bp size.
The invention still further relates to a kind of method that mushroom Cr 02 bacterial is identified and detected, described method is: extract mushroom strain genomic dna to be measured as template, with described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out electrophoresis detection, if the DNA band of 333bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom Cr 02 bacterial; Described molecular specificity labeled primers sequence is:
Upstream primer 5 '-TCTCGATGCAAGAAACTTGAA-3 ';
Downstream primer 5 '-TCGGTCAAAATTAGTCAGGT-3 '.
Described method key is the selection of amplimer, and DNA extraction, PCR reaction system and reaction conditions are determined, and electrophoresis detection, all can carry out according to this area ordinary method.
Preferably, PCR reaction system of the present invention is composed as follows:
PCR Buffer final concentration is 1 *
dNTPs 1mmol/L
MgCl
2 2.5mmol/L
Taq DNA enzyme 2.5U
Each 1.25 μ M of upstream and downstream primer
Template DNA 60ng
Surplus is ddH
2O;
The PCR reaction conditions is as follows:
Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 53 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
PCR Buffer final concentration is 1 *, be meant that the concentration of each component in reaction system is identical with 1 * PCR Buffer among the PCR Buffer, selecting volume usually for use is 10 * PCRBuffer of reaction system volume 1/10.10 * PCR Buffer composition is: 100mM Tris-HCl (pH8.5), 500mM KCl, 25mM MgCl
2And 1.0%Triton-X-100, solvent is ddH
2O.
Concrete, the method for the invention is as follows:
(1) get mushroom strain to be measured, be seeded to the PDA inclined-plane, cultivate 14d for 25 ℃, be forwarded in the PD liquid nutrient medium, 25 ℃ of following shaking culture 14d collect mycelia, extract mushroom strain genomic dna to be measured with the SDS-CTAB method;
(2) genomic dna that extracts with step (1) is a template, as amplimer, carries out pcr amplification with described molecular specificity labeled primers:
Per 20 μ L are composed as follows for the PCR reaction system:
10×PCR?Buffer 2μL
10mmol/L?dNTPs 2μL
25mmol/L?MgCl
2 2μL
5U/ μ L Taq DNA enzyme 0.5 μ L
Each 1 μ L of 25 μ M upstream and downstream primers
20ng/ μ L template DNA 3 μ L
DdH
2O complements to 20 μ L;
The PCR reaction conditions is as follows:
Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 53 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last;
(3) get step (2) amplified production 5 μ L, with 1 μ L0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, electrophoresis in 1 * TAB damping fluid, under the 5V/cm voltage, the EB dyeing of electrophoresis end back, EB dyeing was dyeed 30 minutes in the aqueous solution that contains 0.5 μ g/ml EB usually.Take a picture on automatic gel images analyser, if the DNA band of 333bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom Cr 02 bacterial.
Beneficial effect of the present invention is mainly reflected in: detection method of the present invention is compared with conventional morphologic detection, antagonistic effect, fruiting experiment, has weak point detection time, advantage of high accuracy; The inventive method detects required time and needed only 1～3 day, and conventional time-of-week of needed times at least two of antagonistic effect, fruiting experiment then needs at least 3 months time.
(4) description of drawings
Fig. 1 is for carrying out the result of pcr amplification to mushroom strain; M is a dna molecular amount standard; The negative contrast of C; The arrow indication is for being the special DNA band of 333bp from being numbered the molecular weight that 17 mushroom Cr 02 bacterial amplifies; All the other are numbered other mushroom commonly used and produce bacterial classification.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
(1) extraction of mycelium culture and genomic dna: the mushroom strain of cryopreservation is transferred to PDA inclined-plane (PDA slant medium: remove skin potato 200 grams, be cut into small pieces, add 1000 milliliters in water and boiled 30 minutes, the elimination potato ball adds water with filtrate and complements to 1000 milliliters, add glucose 20 grams, agar 15 grams dissolve the back packing, sterilize 30 minutes for 121 ℃, cool off the inclined-plane) on, cultivate down for 25 ℃.Receive 100ml PD liquid nutrient medium (PD liquid nutrient medium: remove skin potato 200 grams behind the 14d, be cut into small pieces, add 1000 milliliters in water and boiled 30 minutes, the elimination potato ball adds water with filtrate and complements to 1000 milliliters, add glucose 20 grams, sterilized 30 minutes for 121 ℃, cooling promptly gets the PD liquid nutrient medium) in, 25 ℃ of following 100r/min shaking culture 14d, collect mycelia, it is standby to put into-20 ℃ of refrigerator preservations.The SDS-CTAB method is adopted in the extraction of genomic dna, and uses DNA purification kit (Hangzhou BIOER Technology Co., Ltd) that the DNA that extracts is carried out purifying.The genomic dna of purifying with 1.5% agarose gel electrophoresis qualitative detection, is used DNA/RNA ultraviolet spectrophotometer detection by quantitative earlier again.The DNA extraction thing is standby in-20 ℃ of refrigerator storages.
(2) design specific PCR amplimer, the right sequence of primer be upstream primer 5 '-TCTCGATGCAAGAAACTTGAA-3 ' and downstream primer 5 '-TCGGTCAAAATTAGTCAGGT-3 ', synthetic by Shanghai biotechnology company limited.
(3) pcr amplification of SCAR molecule marker: 10 * PCR Buffer2 μ l, 10mmol/L dNTPs2 μ l, 25mmol/L MgCl
22 μ l, 5U/ μ l Taq DNA enzyme 0.5 μ l, 25mM special primer are to each 1 μ l, 20ng/ μ l template DNA 3 μ l, ddH
2O complements to 20 μ l.Amplified reaction carries out on TC-XP type amplification instrument.Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 53 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
(4) electrophoresis detection: get step (3) pcr amplification product 5ul, with 1ul0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, in 1 * TAB damping fluid, electrophoresis under the 5V/cm voltage, after electrophoresis finished, dyeing was 30 minutes in the aqueous solution that contains 0.5 μ g/ml EB, took a picture on the automatic gel images analyser of the clear JS-380A of training then.
According to the method described above, (on behalf of mushroom strain, numbering 1～23 be followed successively by: 1: No. 2, Shen Xiang to multiple mushroom strain respectively; 2: No. 6, Shen Xiang No. 43: Shen Xiang; 4: No. 9, Shen Xiang; 5: No. 7, Shen Xiang; 6: No. 8, Shen Xiang; 7: No. 10, Shen Xiang; 8: Shen Xiang 12; 9: Su Xiang; 10: Wu Xiang; 11:L26; 12:L03; 13:L66; 14:241; 15:241-4; 16: celebrating section 20; 17:Cr02; 18:Cr04; 19: rich No. 1 of Fujian; 20: No. 1, Shanghai farming; 21: the fragrant 939-9 in Zhejiang; 22: the fragrant 939-6 in Zhejiang; 23: fragrant nine) detect, with the negative contrast of sterilized water, electrophoresis result is seen Fig. 1.Wherein number 17 and be mushroom Cr 02 bacterial, amplifying molecular weight is the special DNA band of 333bp, and all the other are numbered other mushrooms commonly used and produce bacterial classification, and not seeing has the special DNA band of 333bp to produce.
Sequence table _ ST25
SEQUENCE?LISTING
＜110〉Zhejiang Prov. Forest Science Inst
＜120〉a kind of molecular specificity labeled primers of mushroom Cr 02 bacterial and detection method
<130>
<160>2
<170>PatentIn?version3.4
<210>1
<211>21
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>1
<210>2
<211>20
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>2
Claims (5)
1. the molecular specificity labeled primers of mushroom Cr 02 bacterial, described primer sequence is:
Upstream primer 5 '-TCTCGATGCAAGAAACTTGAA-3 ';
Downstream primer 5 '-TCGGTCAAAATTAGTCAGGT-3 '.
2. method of mushroom Cr 02 bacterial being identified and being detected with molecular specificity labeled primers as claimed in claim 1, described method is: extract mushroom strain genomic dna to be measured as template, with described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out electrophoresis detection, if the DNA band of 333bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom Cr 02 bacterial; Described molecular specificity labeled primers sequence is:
Upstream primer 5 '-TCTCGATGCAAGAAACTTGAA-3 ';
Downstream primer 5 '-TCGGTCAAAATTAGTCAGGT-3 '.
3. method as claimed in claim 2 is characterized in that:
Described PCR reaction system is composed as follows:
PCR Buffer final concentration is 1 *
dNTPs 1mmol/L
MgCl
2 2.5mmol/L
Taq DNA enzyme 2.5U
Each 1.25 μ M of upstream and downstream primer
Template DNA 60ng
Surplus is ddH
2O;
The PCR reaction conditions is as follows:
Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 53 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
4. method as claimed in claim 3 is characterized in that described method is as follows:
(1) get mushroom strain to be measured, be seeded to the PDA inclined-plane, cultivate 14d for 25 ℃, be forwarded in the PD liquid nutrient medium, 25 ℃ of following shaking culture 14d collect mycelia, extract mushroom strain genomic dna to be measured with the SDS-CTAB method;
(2) genomic dna that extracts with step (1) is a template, as amplimer, carries out pcr amplification with described molecular specificity labeled primers:
Per 20 μ L are composed as follows for the PCR reaction system:
10×PCR?Buffer 2μL
10mmol/L?dNTPs 2μL
25mmol/L?MgCl
2 2μL
5U/ μ L Taq DNA enzyme 0.5 μ L
Each 1 μ L of 25 μ M upstream and downstream primers
20ng/ μ L template DNA 3 μ L
DdH
2O complements to 20 μ L;
The PCR reaction conditions is as follows:
Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 53 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last;
(3) get step (2) amplified production 5 μ L, with 1 μ L, 0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, electrophoresis in 1 * TAB damping fluid, under the 5V/cm voltage, electrophoresis finishes the back and dyes with EB, take a picture on automatic gel images analyser, if the DNA band of 333bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom CrO2 bacterial classification.
5. method as claimed in claim 4 is characterized in that described EB dyeing is dyeing in the aqueous solution that contains 0.5 μ g/ml EB 30 minutes.
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| CN101363055B true CN101363055B (en) | 2010-12-15 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005261228A (en) * | 2004-03-16 | 2005-09-29 | Hokuto Corp | Method for identifying strain of mushroom belonging to genus pleurotus, marker for identifying strain and method for producing marker |
| CN101086015A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 45 bacteria molecular specific mark and its obtaining method and uses |
-
2008
- 2008-08-25 CN CN 200810120315 patent/CN101363055B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005261228A (en) * | 2004-03-16 | 2005-09-29 | Hokuto Corp | Method for identifying strain of mushroom belonging to genus pleurotus, marker for identifying strain and method for producing marker |
| CN101086015A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 45 bacteria molecular specific mark and its obtaining method and uses |
Non-Patent Citations (3)
| Title |
|---|
| Su H,et al..Development of strain-specific SCAR markers for authentication of Ganoderma lucidum.《World J Microbiol Biotechnol》.2007,第24卷1223-1226. * |
| 吴学谦，等.SCAR分子标记技术在香菇菌株鉴定上的应用研究.《菌物学报》.2005,第24卷(第2期),259-266. |
| 吴学谦等.SCAR分子标记技术在香菇菌株鉴定上的应用研究.《菌物学报》.2005,第24卷(第2期),259-266. * |
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