CN101041859A - Mushroom 507 bacterial AFLP quick-detecting method - Google Patents

Abstract

The invention discloses a rapid AFLP detecting method of mushroom 507 bacterium, which comprises the following steps: extracting bacterial culture and genome DNA; cutting DNA through Msel and EcoR1; connecting DNA segment through MseI adapter and EcoRI adapter; preaugumenting DNA enzyme-cut product; augumenting through AFLP selectively; detecting through genetic analyzer.

Description

A kind of mushroom 507 bacterial AFLP quick-detecting methods

Technical field

The invention belongs to the detection method field, be specifically related to a kind of mushroom 507 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 ultimate production, rewritten the ranking list of world's edible mushrooms output, and constantly dwindle and the Twospore Mushroom poor distance of ranking first, 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 with its alarming development speed, and fine 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 method " 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 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.

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 marking 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 printing 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 problem to be solved by this invention provides a kind of energy AFLP method for quick easy, that quickly and accurately mushroom 507 bacterial classifications are identified.

A kind of mushroom 507 bacterial AFLP quick-detecting methods comprise the following steps:

(1) extraction of mycelium culture and genomic dna

Adopt PDY liquid nutrient medium shake-flask culture mushroom mycelium, membrane filtration is collected mycelia.Adopt the CTAB method to extract 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.Pcr amplification system (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 diluent 2.5 μ l

Mse1 17 and EcoRI 11 primers design on primer M00 and E00 basis, and sequence is as follows:

EcoRI?11：GAT?GAG?TCC?TGA?GTAACG

Mse1?17：GAC?TGC?GTA?CCAATT?CAC

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 is at CEQ ^TM8000 genetic analysis instruments carry out capillary electrophoresis and detect, 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 with bacterial classification and totally 164 the bacterial strains experiments of minority wild species, has only mushroom 507 bacterial strains augmentation detection to go out the special DNA band (Fig. 1) that molecular weight is 171bp from mushroom production in all parts of the country

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 separation and combination fluorescent 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

Fig. 1 adopts special-purpose 507 AFLP to detect primer to be the special band of 171bp to what Xianggu mushroom strain carried out that AFLP detects gained collection of illustrative plates arrow mark.

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

The extraction of mycelium culture and genomic dna

Adopt PDY liquid nutrient medium (potato 20%, glucose 2%, yeast extract 0.1%), 150rpm, 25 ℃ of 15 days shake-flask culture mushrooms, 507 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 reaction solution 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 enzyme 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/μ?L?M00 0.6μl

50ng/μL?E00 0.6μl

5U/L Taq DNA enzyme 0.2 μ l

Enzyme connects product 1 μ l

M00 and E00 are primer special, design according to MseI 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.

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 MseI 17 primers 0.3 μ l

50ng/L EcoRI 11 primers 0.3 μ l

5U/ μ L Taq DNA enzyme 0.2 μ l

Pre-expansion volume increase thing diluent 2.5 μ l

Mse1 17 and EcoRI 11 primers design on primer M00 and E00 basis, and sequence is as follows:

EcoRI?11：GAT?GAG?TCC?TGA?GTA?ACG

Mse1?17：GAC?TGC?GTA?CCA?ATT?CAC

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, 25 cycles; 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 171bp.

Claims (7)

1. mushroom 507 bacterial AFLP quick-detecting methods comprise the steps:

(1) extraction of mycelium culture 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 17 of selective amplification and EcoRI11 are that the sequence that designs on primer M00 and E00 basis is as follows:

EcoRI?11：GAT?GAG?TCC?TGA?GTAACG

Mse1?17：GAC?TGC?GTA?CCAATT?CAC

(6) genetic analyzer detects

The AFLP product of selective amplification is at CEQ ^TM8000 genetic analysis instruments carry out capillary electrophoresis and detect, 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.

2. a kind of mushroom 507 bacterial AFLP quick-detecting methods as claimed in claim 1, it is characterized in that: the extraction of described mycelium culture of step (1) and genomic dna comprises the following steps: to adopt PDY liquid 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.

3. a kind of mushroom 507 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 507 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 reaction solution 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 507 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，O 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 507 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 diluent 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 507 bacterial AFLP quick-detecting methods as claimed in claim 1 is characterized in that: mushroom 507 bacterial classifications can augmentation detection go out the special DNA band that molecular weight is 17lbp.

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