CN101042370A - AFLP rapid detection method for mushroom 7402 bacterium - Google Patents
AFLP rapid detection method for mushroom 7402 bacterium Download PDFInfo
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- CN101042370A CN101042370A CN 200710040320 CN200710040320A CN101042370A CN 101042370 A CN101042370 A CN 101042370A CN 200710040320 CN200710040320 CN 200710040320 CN 200710040320 A CN200710040320 A CN 200710040320A CN 101042370 A CN101042370 A CN 101042370A
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Abstract
This invention discloses one easy and rapid AFLP rapid test method on 7402 bacterial of mushroom, which comprises the following steps: bacterial wire fosters and gene set DNA extraction; DNA MseI and EcoRI doule enzyme slice; DNA slice uses Msel adapter and EcoRI adapter for connection; DNA enzyme product pre-enlarging; AFLP selective extending; genetic analyzing testing.
Description
Technical field
The invention belongs to biotechnology detection method field, be specifically related to a kind of mushroom 7402 bacterial AFLP quick-detecting methods.
Background technology
China's mushroom production is from 1.95 ten thousand tons of 2,420,000 tons of developing 2005 rapidly of nineteen eighty-three, account for more than 70% of global mushroom total production, rewritten the ranking list of world's edible fungi output, and constantly dwindle and the agaricus bisporus poor distance of ranking first, scholarly forecast is arranged, because the fast development of Chinese mushroom industry, it will become the highest edible fungi of world wide production in nearly 10 years.China's mushroom is with its alarming development speed, and the quality of high-quality and cheap cost are that world mushroom industry personage attractes attention the fashionable world of Chinese mushroom.
The contribution rate of good quality strain in mushroom per unit area yield and quality is very important, and this has determined the critical role of mushroom strain in the mushroom industry.China had signed " international new variety of plant Protection Code " in 1999; this not only requires us to respect other national kind intellecture property; also to strengthen simultaneously protecting the kind intellecture property of our country oneself; really protect the kind property right of China in order to set up edible fungi new varieties resignation system; must at first set up ripe cultivar identification technology, for the new varieties registration lays the foundation.Especially Japan on April 1st, 2004 came into effect " seedling method amendment ", to the export mixes of China edible fungi 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.
In recent years, the dna fingerprint technology rapid development is for identifying that on the dna molecular level mushroom strain provides brand-new means.Wherein AFLP (Amplified FragmentLength Polymorphism) technology be new development get up be considered to the most effective molecule labelling method.The ultimate principle of aflp analysis is a selective amplification genomic DNA endonuclease bamhi.Because the restriction enzyme site of different genes group DNA there are differences, thereby has produced the polymorphism of expanding fragment length.The finger-print that obtains with the AFLP method has reliable and stable and advantage such as rich polymorphism, is very suitable for the application of aspects such as strain identification.
Summary of the invention
Technical matters to be solved by this invention provides a kind of energy AFLP method for quick easy, that quickly and accurately mushroom 7402 bacterial classifications are identified.
A kind of mushroom 7402 bacterial AFLP quick-detecting methods comprise the following steps:
(1) extraction of cultural hypha and genomic DNA
Adopt PDY fluid nutrient medium (potato 20%, glucose 2%, yeast extract 0.1%), 150rpm, 25 ℃ of 15 days shake-flask culture mycelia.Membrane filtration is collected mycelia, and with aseptic water washing twice, it is standby that filter paper blots ℃ refrigerator preservation of back labelled putting into-20.
DNA extraction adopts the CTAB method, and the agarose gel electrophoresis with 0.8% detects the quality of DNA, measures the concentration and relative purity of DNA with uv-spectrophotometric instrument BECKMAN DU 640, and adjusts all samples DNA concentration to 100ng/ul.
(2) the dna double enzyme is cut
Sample DNA is carried out the double digestion of MseI and EcoRI.The DNA sample enzyme system of cutting comprises (cumulative volume 20 μ l):
ddH2O 14.8μl
10×Buffer 2.0μl
10U/μl?MseI 0.25μl
10U/μl?EcoRI 0.25μl
100×BSA 0.2μl
100ng/ μ l template DNA 2.5 μ l
37 ℃ of enzymes were cut 3 hours, handled 10min for 65 ℃.
(3) connection of dna fragmentation joint
Each DNA sample that enzyme cuts adds the following reactant liquor of 5 μ L, and 16 ℃ of connections are spent the night.
ddH2O 0.85μl
Buffer 2.5μl
25uM?MseI?adapter 1.0μl
5uM?EcoRI?adapter 0.5μl
3U/ μ l T4-DNA ligase 0.15 μ l
(4) the DNA enzyme is cut the pre-amplification of product
Pcr amplification system (cumulative volume 20 μ l):
ddH，O 14μl
10×PCR Buffer 2.0μl
25mmol/L MgCl2 1.2μl
10mmol/L?dNTP 0.4μl
50ng/L?M00 0.6μl
50ng/μL?E00 0.6μl
5U/ μ L Taq DNA enzyme 0.2 μ l
Enzyme connects product 1 μ l
M00 and E00 are primer special, design according to Mse and EcoR I restriction enzyme site, and sequence is respectively:
E00：GAC?TGC?GTA?CCA?ATT?C
M00：GAT?GAG?TCC?TGA?GTA?A
PCR reaction conditions: 72 ℃ of 5min; 94 ℃ of 1min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, 20cycles; 72 ℃ of 5min.The PCR product, is observed and the record result with the VDS camera chain after the EB dyeing through 1.5% agarose gel electrophoresis.4 ℃ of preservations are standby.
(5) selective amplification of AFLP
20 times of pre-expansion volume increase thing dilutions are as the template of selective amplification.Pcr amplification system (cumulative volume 10 μ l):
ddH2O 4.7μl
10×PCR?Buffer 1.0μl
25mmol/L?MgCl2 0.8μl
10mmol/L?dNTP 0.2μl
50ng/L Mse11 primer 0.3 μ l
50ng/L EcoRI 14 primers 0.3 μ l
5U/ μ L Taq DNA enzyme 0.2 μ l
Pre-expansion volume increase thing dilution 2.5 μ l
Mse11 and EcoRI 14 primers design on primer M00 and E00 basis, and sequence is as follows:
EcoRI?14：GAC?TGC?GTA?CCA?ATT?CAT
Mse11：GAT?GAG?TCC?TGA?GTA?ACA
The PCR reaction conditions: 94 ℃ of 2min, 95 ℃ of 20s, 66 ℃ of 30s (each circulation reduces by 1 ℃) 30s, 72 ℃ of 2min, 10cycles; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 2min, 25cycles; 72 ℃ of 10min.The PCR product, with VDS camera chain observations, is used for Capillary Electrophoresis and detects after the EB dyeing through 1.5% agarose gel electrophoresis.
(6) genetic analyzer detects
The AFLP product of selective amplification carries out Capillary Electrophoresis at CEQTM 8000 genetic analysis instruments and detects, and each reacting hole adds 0.5 μ l amplified production, 30 μ l sample-loading buffers and 0.3 μ l standard molecular weight, last machine testing.
To 114 (for produce with kind) individual collection from mushroom production in all parts of the country with bacterial classification and totally 164 the bacterial strains experiments of minority wild species, have only mushroom 7402 bacterial strains augmentation detection to go out the special DNA band (Fig. 1) that molecular weight is 214bp, sample detects repeatability at 0.5bp (Fig. 2) at different time and different swimming lanes
This detection method is compared with conventional morphologic detection, antagonistic effect, fruiting experiment, has weak point detection time, advantage of high accuracy.This detection required time only needs 4-5 days, and conventional antagonistic effect required time needs two time-of-weeks at least, and fruiting experiment then needs at least 3 months time; The separating and combining fluorescence signal that this method adopts capillary electrophoresis technique to carry out amplified band detects this method and compares with the traditional detection technology, has the characteristics of accuracy, good reproducibility.
Description of drawings
Figure 1A FLP detects collection of illustrative plates 1
What arrow marked is the special band of 214bp.
Fig. 2 AFLP detects collection of illustrative plates 2
What arrow marked is the special band of 214bp, and sample detects repeatability at 0.5bp at different time and different swimming lanes.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
The extraction of cultural hypha and genomic DNA
Adopt PDY fluid nutrient medium (potato 20%, glucose 2%, yeast extract 0.1%), 150rpm, 25 ℃ of 15 days shake-flask culture mushrooms, 7402 bacterial strain mycelia.Membrane filtration is collected mycelia, and with aseptic water washing twice, it is standby that filter paper blots ℃ refrigerator preservation of back labelled putting into-20.
DNA extraction adopts the CTAB method, and the agarose gel electrophoresis with 0.8% detects the quality of DNA, measures the concentration and relative purity of DNA with uv-spectrophotometric instrument BECKMAN DU 640, and adjusts all samples DNA concentration to 100ng/ul.
The dna double enzyme is cut
Sample DNA is carried out the double digestion of MseI and EcoRI.The DNA sample enzyme system of cutting comprises (cumulative volume 20 μ l):
ddH2O 14.8μl
10×Buffer 2.0μl
10U/μl?MseI 0.25μl
10U/μl?EcoRI 0.25μl
100×BSA 0.2μl
100ng/ μ l template DNA 2.5 μ l
37 ℃ of enzymes were cut 3 hours, handled 10min for 65 ℃.
The connection of dna fragmentation joint
Each DNA sample that enzyme cuts adds the following reactant liquor of 5 μ L, and 16 ℃ of connections are spent the night.
ddH2O 0.85μl
Buffer 2.5μl
25uM?MseI?adapter 1.0μl
5uM?EcoRI?adapter 0.5μl
3U/ μ l T4-DNA ligase 0.15 μ l
The DNA enzyme is cut the pre-amplification of product
Pcr amplification system (cumulative volume 20 μ l):
ddH，O 14μl
10×PCR?Buffer 2.0μl
25mmol/L?MgCl2 1.2μl
10mmol/L?dNTP 0.4μl
50ng/μL?M00 0.6μl
50ng/μL?E00 0.6μl
5U/ μ L Taq DNA enzyme 0.2 μ l
Enzyme connects product 1 μ l
M00 and E00 are primer special, design according to Mse and EcoRI restriction enzyme site, and sequence is respectively:
E00：GAC?TGC?GTA?CCA?ATT?C
M00：GAT?GAG?TCC?TGA?GTA?A
PCR reaction conditions: 72 ℃ of 5min; 94 ℃ of 1min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, 20cycles; 72 ℃ of 5min.The PCR product, is observed and the record result with the VDS camera chain after the EB dyeing through 1.5% agarose gel electrophoresis.4 ℃ of preservations are standby.
The selective amplification of AFLP
20 times of pre-expansion volume increase thing dilutions are as the template of selective amplification.Pcr amplification system (cumulative volume 10 μ l):
ddH2O 4.7μl
10×PCR?Buffer 1.0μl
25mmol/L?MgCl2 0.8μl
10mmol/L?dNTP 0.2μl
50ng/ μ L Mse11 primer 0.3 μ l
50ng/ μ L EcoRI 14 primers 0.3 μ l
5U/ μ L Taq DNA enzyme 0.2 μ l
Pre-expansion volume increase thing dilution 2.5 μ l
Mse11 and EcoRI 14 primers design on primer M00 and E00 basis, and sequence is as follows:
EcoRI?14：GAC?TGC?GTA?CCA?ATT?CAT
Mse11：GAT?GAG?TCC?TGA?GTA?ACA
The PCR reaction conditions: 94 ℃ of 2min, 95 ℃ of 20s, 66 ℃ of 30s (each circulation reduces by 1 ℃) 30s, 72 ℃ of 2min, 10cycles; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 2min, 25cycles; 72 ℃ of 10min.The PCR product, with VDS camera chain observations, is used for Capillary Electrophoresis and detects after the EB dyeing through 1.5% agarose gel electrophoresis.
Genetic analyzer detects
The AFLP product of selective amplification carries out Capillary Electrophoresis at CEQTM 8000 genetic analysis instruments and detects, and each reacting hole adds 0.5 μ l amplified production, 30 μ l sample-loading buffers and 0.3 μ l standard molecular weight, last machine testing.But augmentation detection goes out the special DNA band that molecular weight is 214bp.
Claims (8)
1. mushroom 7402 bacterial AFLP quick-detecting methods comprise the steps:
(1) extraction of cultural hypha and genomic DNA
(2) the dna double enzyme is cut
Sample DNA is carried out the double digestion of MseI and EcoRI;
(3) connection of dna segment joint
Joint uses MseI adapter and EcoRI adapter;
(4) the DNA enzyme is cut the pre-amplification of product
The primer special of pre-amplification designs according to MseI and EcoRI restriction enzyme site, and sequence is respectively:
E00：GAC?TGC?GTA?CCA?ATT?C
M00：GAT?GAG?TCC?TGA?GTA?A
(5) selective amplification of AFLP
20 times of pre-expansion volume increase thing dilutions are as the template of selective amplification.The primer Mse1 11 of selective amplification and EcoRI14 are that the sequence that designs on primer M00 and E00 basis is as follows:
EcoRI?14：GAC?TGC?GTA?CCA?ATT?CAT
Mse1?11：GAT?GAG?TCC?TGA?GTA?ACA
(6) genetic analyzer detects
The AFLP product of selective amplification carries out Capillary Electrophoresis at the genetic analysis instrument and detects.
2. a kind of mushroom 7402 bacterial AFLP quick-detecting methods as claimed in claim 1, it is characterized in that: the extraction of described cultural hypha of step (1) and genomic DNA comprises the following steps: to adopt PDY fluid nutrient medium (potato 20%, glucose 2%, yeast extract 0.1%), 150rpm, 25 ℃ of 15 days shake-flask culture mycelia; Membrane filtration is collected mycelia, and with twice of aseptic water washing, filter paper blots back labelled putting into-20 ℃ refrigerator and preserves standby, DNA extraction adopts the CTAB method, agarose gel electrophoresis with 0.8% detects the quality of DNA, measure the concentration and relative purity of DNA with uv-spectrophotometric instrument BECKMAN DU 640, and adjust all samples DNA concentration to 100ng/ul.
3. a kind of mushroom 7402 bacterial AFLP quick-detecting methods as claimed in claim 1 is characterized in that: the described dna double enzyme of step (2) tangent condition is: the DNA sample enzyme system of cutting comprises (cumulative volume 20 μ l):
ddH2O 14.8μl
10×Buffer 2.0μl
10U/μl?MseI 0.25μl
10U/μl?EcoRI 0.25μl
100×BSA 0.2μl
100ng/ μ l template DNA 2.5 μ l
37 ℃ of enzymes were cut 3 hours, handled 10min for 65 ℃.
4. a kind of mushroom 7402 bacterial AFLP quick-detecting methods as claimed in claim 1 is characterized in that: being connected to of step (3) described dna segment joint: each DNA sample that enzyme cuts, add the following reactant liquor of 5 μ L, and 16 ℃ of connections are spent the night:
ddH
2O 0.85μl
Buffer 2.5μl
25uM?MseI?adapter 1.0μl
5uM?EcoRI?adapter 0.5μl
3U/ μ l T
4-dna ligase 0.15 μ l.
5. a kind of mushroom 7402 bacterial AFLP quick-detecting methods as claimed in claim 1 is characterized in that: the amplification system that the described DNA enzyme of step (4) is cut the pre-amplification of product is: (cumulative volume 20 μ l):
ddH
2O 14μl
10×PCR?Buffer 2.0μl
25mmol/L?MgCl
2 1.2μl
10mmol/L?dNTP 0.4μl
50ng/μL?M
00 0.6μl
50ng/μL?E
00 0.6μl
5U/ μ L Taq DNA enzyme 0.2 μ l
Enzyme connects product 1 μ l
PCR reaction conditions: 72 ℃ of 5min; 94 ℃ of 1min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, 20cycles; 72 ℃ of 5min.The PCR product, is observed and the record result with the VDS camera chain after the EB dyeing through 1.5% agarose gel electrophoresis.4 ℃ of preservations are standby.
6. a kind of mushroom 7402 bacterial AFLP quick-detecting methods as claimed in claim 1 is characterized in that: the selective amplification pcr amplification system of the described AFLP of step (5) (cumulative volume 10 μ l):
ddH
2O 4.7μl
10×PCR?Buffer 1.0μl
25mmol/L?MgCl
2 0.8μl
10mmol/L?dNTP 0.2μl
50ng/ μ L MseI 17 primers 0.3 μ l
50ng/ μ L EcoRI 11 primers 0.3 μ l
5U/ μ L Taq DNA enzyme 0.2 μ l
Pre-expansion volume increase thing dilution 2.5 μ l
The PCR reaction conditions: 94 ℃ of 2min, 95 ℃ of 20s, 66 ℃ of 30s (each circulation reduces by 1 ℃) 30s, 72 ℃ of 2min, 10cycles; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 2min, 25cycles; 72 ℃ of 10min.The PCR product, with VDS camera chain observations, is used for Capillary Electrophoresis and detects after the EB dyeing through 1.5% agarose gel electrophoresis.
7. a kind of mushroom 7402 bacterial AFLP quick-detecting methods as claimed in claim 1 is characterized in that: the described genetic analyzer of step (6) is CEQ
TM8000 genetic analyzers.
8. a kind of mushroom 7402 bacterial AFLP quick-detecting methods as claimed in claim 1 is characterized in that: mushroom 7402 bacterial classifications can augmentation detection go out the special DNA band that molecular weight is 214bp.
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| CN 200710040320 CN101042370A (en) | 2007-04-29 | 2007-04-29 | AFLP rapid detection method for mushroom 7402 bacterium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831422A (en) * | 2010-04-28 | 2010-09-15 | 北京市农林科学院 | Edible mushroom genomic DNA quick extraction method applicable to PCR reaction |
| CN106801094A (en) * | 2017-02-07 | 2017-06-06 | 上海市农业科学院 | A kind of SSR marker finger-print of the strain of mushroom 7402 and its construction method and application |
-
2007
- 2007-04-29 CN CN 200710040320 patent/CN101042370A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831422A (en) * | 2010-04-28 | 2010-09-15 | 北京市农林科学院 | Edible mushroom genomic DNA quick extraction method applicable to PCR reaction |
| CN101831422B (en) * | 2010-04-28 | 2012-07-11 | 北京市农林科学院 | Edible mushroom genomic DNA quick extraction method applicable to PCR reaction |
| CN106801094A (en) * | 2017-02-07 | 2017-06-06 | 上海市农业科学院 | A kind of SSR marker finger-print of the strain of mushroom 7402 and its construction method and application |
| CN106801094B (en) * | 2017-02-07 | 2020-05-29 | 上海市农业科学院 | SSR marker fingerprint spectrum of mushroom 7402 strain and construction method and application thereof |
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Open date: 20070926 |