CN109517890A - A kind of method of microorganism in detection timber - Google Patents
A kind of method of microorganism in detection timber Download PDFInfo
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Abstract
The invention discloses a kind of methods of microorganism in detection timber.The method of microorganism makes moisture content keep 20% or so firstly the need of timber is pre-processed in climatic chamber in advance in detection timber of the invention, then after surface sterilization, sawdust is drilled through by quantitative and depthkeeping degree, dodecyl trimethyl ammonium bromide (DTAB) the solution cracking of beta -mercaptoethanol is added, microbial total inhereditary material in wood tissue cell is extracted, and then the microbial information in timber is obtained by amplification and sequencing, determine the microbe species in timber.Method of the invention ruins microorganism and can obtain more full timber microbial information than traditional separation method separation wood tissue, conducive to foxiness effectively prevent or the degradation of timber, effectively compensate for current timber microorganism detects the deficiency for be unableing to do without the conventional method of separation, purifying and tissue cultures, provides completely new test method for the biodegrade and prevention and treatment of timber.
Description
Technical field
The invention belongs to wood protection field, a method of microorganism in detection timber.
Background technique
Timber microorganism ruins the main reason for being nature wood degradation and recycling, is that nature maintains carbon nitrogen cycle
Indispensable a part, but timber process with using being above to need in face of one of with the problem of solution.Microorganism is ruined
Type mainly has mouldy, discoloration, etches, is soft rotten and rotten.Anti-microbial agent is primarily referred to as bacterium and fungal agents.Detect timber
Harms of microbe judges the extent of injury of timber, understands the anti-microbial agent for causing timber, and prevention and treatment is made to shoot the arrow at the target.Bacterium danger
Evil, mould harm, stain fungus harm, rot fungi harm are needed using different control methods.Since foxiness is first from interior
Portion is difficult to touch region such as underground and starts, so being difficult to discover；Microorganism invades the early stage of timber, since the variation of timber is non-
It is often faint, it is also difficult to discover.Structure material establishes periodic detection and monitoring mechanism in early stage, can be by timber corruption
Harm and accident caused by rotten are preferably minimized.The microorganism detection and monitoring for carrying out timber accomplish early to discover the early prevention and treatment of early prediction.
All the time, the detection of timber microorganism be unable to do without separation, purifying and culture technique, to timber microorganism step-by-step movement
Phagocytic process take microexamination or inoculation method to be studied, to timber biological ruin mechanism study mainly for separate it is pure
Change wood chemistry and physical change caused by obtained single wood deterioration microorganism and carries out.However many microorganisms be unable to do without
The particular surroundings of own existence, it is difficult to which isolated, only 1%~5% microorganism can carry out pure culture, and due to tissue
The speed of growth of microorganism is different during being separately cultured, grow too fast microorganism may cover grow slow microorganism and
It is difficult to purify bacterial strain, brings the biased of result, be difficult to identify by morphology separating there are also many microorganisms,
This largely limits the research of microorganism.
Summary of the invention
The object of the present invention is to provide a kind of methods of microorganism in detection timber.
The method of microorganism in detection timber provided by the invention, comprising:
1) moisture content for adjusting timber to be measured, obtains pretreated timber, the moisture content of the pretreated timber
For 6%-16%；
2) it is crushed the pretreated timber, obtains sawdust；
3) DNA for extracting the sawdust, obtains DNA to be measured；
4) the microorganism sequence information in the DNA to be measured is analyzed, determines the type of microorganism, i.e., in the described timber to be measured
The type of microorganism.
The moisture content of the pretreated timber concretely 20%.
The moisture content for adjusting timber to be measured can carry out in climatic chamber.
Step 1), which may also include, carries out surface sterilization to the pretreated timber.The surface sterilization can utilize 75%
Ethanol water carry out.
In step 2), the DNA for being crushed the pretreated timber can be using drilling row.The brill can bore for six ribs.Institute
The diameter for stating brill can be 5mm.
It may also include before step 3) and the sawdust freezed at -20 DEG C.
The time of the freezing can be more than or equal to 2 hours.
Step 3) can include:
The sawdust 3a) is ground, wood powder is obtained；
Extracting solution 3b) is added in Xiang Suoshu wood powder, obtains extraction mixture；The extracting solution is into DTAB solution
The sterile solution that beta -mercaptoethanol obtains is added, beta -mercaptoethanol volume content is 2 μ l/mL extracting solutions in the extracting solution；Institute
DTAB solution is stated to be made of solvent and solute, the solvent be water, the solute be dodecyl trimethyl ammonium bromide (DTAB),
Tris, ethylenediamine tetra-acetic acid (EDTA), NaCl and the HCl for adjusting pH value, DTAB, Tris, EDTA and NaCl are described
Concentration in DTAB solution is respectively 0.02g/mL, 0.1M, 0.02M, 1.4M, and the pH value of the DTAB solution is 7.5；
Extract in the extraction mixture DNA 3c) to get the DNA to be measured is arrived.
It is 800 μ l:0.1g that the additive amount of the extracting solution, which can meet the extracting solution and the proportion of the sawdust,.
Step 3b) it may also include and be incubated for the extraction mixture at 65 DEG C.The time of the incubation can be greater than
Equal to 1 hour.
Step 3c) it extracts DNA in the extraction mixture and can be carried out using Promega wizard SV kit.
Step 4) can include:
4a) using the DNA to be measured as template, PCR amplification is carried out using the primer pair that ITS1 and ITS4 is formed, is expanded
Product；The ITS1 and ITS4 is respectively single stranded DNA shown in sequence 1 and sequence 2 in sequence table；
4b) amplified production is sequenced, the type of microorganism in the timber to be measured is determined according to sequencing result.
According to sequencing result determine microorganism in the timber to be measured type can by by sequencing result with containing micro- life
Sequence in the database of object sequence is compared, with the type of microorganism in the determination timber to be measured.
The database can be ncbi database.
The present invention also provides the methods that microbial DNA is extracted from timber or woodwork, which comprises according to institute
State detection timber in microorganism method in step 1) -3) extract obtain the DNA of timber to be measured, the DNA of the timber to be measured
In DNA i.e. containing microorganism in the timber to be measured.
Wherein, the timber to be measured can be for the timber for ruining symptom.
The extracting solution also belongs to protection scope of the present invention.
The method of microorganism or the method that microbial DNA is extracted from timber or woodwork in the detection timber,
Or following any applications of the extracting solution, also belong to protection scope of the present invention:
A1, the application in prevention and treatment wood deterioration；
A2, lead to the application in wood deterioration microorganism in detection；
A3, the application in wood degradation.
In the present invention, the timber can for have occurred it is rotten, mildew, discoloration and/or softening etc. biodeteriorations feature
Timber, can also be not have the timber of above-mentioned biodeterioration feature.
The timber concretely Gymnospermae plant or Angiospermae plant.The Gymnospermae plant can be
Song Shangang plant.The Song Shangang plant can be loose China fir mesh plant.The pine China fir mesh plant can be pinaceae plant, such as pinus sylvestris var. mongolica.
It is demonstrated experimentally that can be succeeded using method of the invention obtains microbial information from timber, determine micro- in timber
The type of biology, can to obtain more full timber micro- than the microorganism of ruining of traditional separation method separation wood tissue for this method
Biological information, conducive to the timber that effectively prevents or degrade of foxiness.Method of the invention compensates for current timber microorganism
Detection be unable to do without the deficiency of the conventional method of separation, purifying and tissue cultures, provides completely newly for the biodegrade and prevention and treatment of timber
Test method.
Detailed description of the invention
Fig. 1 is the timber that biodeterioration occurs in embodiment 1.A, B, C are that (biodeterioration has occurred for three kinds of timber ruined
Discoloration), Control is the timber (biodeterioration discoloration does not occur) that do not ruin.
Fig. 2 is to be extracted in embodiment 1 by DNA and fungi universal primer amplifies the indicative band come, and compare wooden
Material does not amplify fungi characteristic bands.
Fig. 3 is the fungi information obtained in embodiment 1 by sequencing.
Fig. 4 is the wood-stain fungus separated by conventional method, collects sequencing through mycelia and learns, isolates on A
Nectria and Trichoderma have been isolated on Botryotina, B, and are also only separated to Diplodia on C wood component
Fungi.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is all from unless otherwise specified
Routine biochemistry reagent shop.Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
DTAB solution is made of solvent and solute, and solvent is water, solute be dodecyl trimethyl ammonium bromide (DTAB),
Tris, ethylenediamine tetra-acetic acid (EDTA), NaCl and the HCl for adjusting pH value, DTAB, Tris, EDTA and NaCl are in DTAB
Concentration in solution is respectively 0.02g/mL, 0.1M, 0.02M, 1.4M, pH value 7.5.
Extracting solution: the sterile solution that beta -mercaptoethanol obtains, beta -mercaptoethanol body in extracting solution are added into DTAB solution
Product content is 2 μ l/mL extracting solutions.
A kind of method of microorganism in embodiment 1, detection timber
Discoloration (i.e. wood deterioration caused by microorganism changes colour) camphor tree will be ruined from different colours three pieces of microorganisms of variation occur
Sub- pine material is denoted as A, B and C (Fig. 1) and is handled as follows respectively, these three timber contain the life of typical discoloration
Object ruins symptom:
One, the acquisition of timber sawdust
Timber is pre-processed in climatic chamber, the moisture content of timber is adjusted to 20% or so, is pre-processed
Timber afterwards；The ethanol water of pretreated wood utilization 75% is subjected to surface sterilization, the timber after being sterilized；?
The position that there is canonical biometric to ruin symptom is chosen in timber after disinfection, drills through three positions with six ribs that diameter is 5mm,
The sawdust of generation is collected, then every 0.1g sawdust is collected as portion and is placed in 1ml centrifuge tube, then puts centrifuge tube
Sawdust after being freezed at least 2 hours at -20 DEG C, carries out the extraction of further DNA.It is needed if temporarily not extracting DNA
Sawdust after will be cold is placed in the quick-frozen preservation of -80 DEG C of refrigerators.
Two, the extraction of DNA
Sawdust after freezing that step 1 obtains is ground in dismembyator, keeps the hereditary information of cell in wood tissue most
It measures and, obtain wood powder；800 μ l extracting solutions are added into wood powder, it is small to be then placed in incubation 1 in 65 DEG C of warm bath pots
When, obtain extraction mixture；
Extraction mixture is centrifuged 3 minutes under 15000g, collects supernatant；Supernatant is transferred to Promega wizard
In the purification column of SV kit, then Xiang Zhuzhong adds 650 μ l and washes column liquid, and then 13000g is centrifuged 1 minute, discards waste liquid,
Reuse and wash column liquid and cleaned three times, last time is added wash column liquid after, 13000g is centrifuged 2-3 minutes, discards waste liquid
It the water that 100 μ l are free of DNA enzymatic is added in backward column, places 2-3 minute at room temperature, purification column is then placed on one newly
In 1.5ml centrifuge tube, 13000g is centrifuged 1-2 minutes, is collected centrifuge tube liquid, as target DNA solution, is stored in -20 DEG C of ice
It is spare in case.
Three, sequencing is analyzed with microbial information
The target DNA that step 2 is obtained expands ITS1/ITS4 as template, using fungi universal primer, primer sequence
Column and amplification system are as shown in Tables 1 and 2.
1 primer sequence of table
| Primer | Sequence |
| ITS1 | 5 '-CTTGGTCATTTAGAGGAAGTAA-3 ' (sequence 1 in sequence table) |
| ITS4 | 5 '-TCCTCCGCTTATTGATATGC-3 ' (sequence 2 in sequence table) |
2 amplification system of table
| Template DNA | 5-10ng |
| 5×buffer | 10μl |
| dNTPs | 1μl |
| MgCl2 | 3μl |
| ITS1 | 0.5μl |
| ITS4 | 0.5μl |
| Taq enzyme | 0.2μl |
| BSA | 2.5μl |
| ddH2O | It mends to 50 μ l |
In table 2,5 × buffer, dNTPs and Taq enzyme are Tiangeng biochemical technology Co., Ltd product.
It has configured after amplification system according to table 2 in being expanded on gene-amplificative instrament by the program of table 3, has obtained amplification and produce
Object.
3 amplification program of table
Finally, taking 2 μ l to carry out 2% detected through gel electrophoresis amplimer.Using same timber do not ruin part as
It compares (Control).The results show that not ruining timber does not obtain fungi band, and timber is ruined at three and has amplified typical case
Fungi band (Fig. 2).
By amplified production carry out sequencing and with sequence alignment is carried out in NCBI, as a result (Fig. 3) is shown, detects 230 in A
Kind timber microorganism, wherein most is Leptographium bacterium, accounts for 55.2%, followed by Clonostachys, accounts for
13.59%, other timber microorganism accountings are both less than 1%, and the another microorganism there are also 25.8% does not identify specific type；In B
Detect 173 kinds of timber microorganisms, it is Haematonectria bacterium that wherein accounting is biggish, accounts for 16.84%, Clonostachys
It accounts for 15.83%, Aspergillus and accounts for 9.08%, Xenoacremonium and account for 2.24%, other accountings are both less than 1%, separately also
49.65% microorganism does not identify specific type；206 kinds of timber microorganisms are detected in C, wherein Sporothrix is accounted for
38.92%, Trichoderma, which account for 23.63%, Leptographium and account for 15.2%, Xenoacremonium, accounts for 14.47%,
His accounting is all smaller；Only detect 4 kinds of microorganisms in control, respectively Botryotinia, Nectria, Trichoderma and
Diplodia.Using the isolated multiple-microorganism of the above method, it is significantly higher than the microorganism isolated using conventional method
Species number.
Wherein, the operating procedure for ruining microorganism by conventional method separation wood tissue is as follows:
Isolated method is organized according to tradition, and each stained wood surface A, B and C is stripped into a small amount of wood tissue and carries out group
Separation is knitted, then isolated group is woven in PDA culture medium and is cultivated, culture carries out at 28 DEG C, cultivates 5~7 days.Culture
After, it separates each bacterial strain and purifies, single bacterium after purification is cultivated respectively and utilizes ITS1 and ITS4 progress PCR above
Amplification and sequencing, carry out sequence alignment to sequencing result in NCBI, and as a result (Fig. 4) is shown, isolate on A
Nectria and Trichoderma have been isolated on Botryotina, B, and Diplodia fungi is also only separated on C.
Comparative example 1, when not adjusting moisture content timber microorganism detection
According to the method for embodiment 1, the timber of discoloration symptom will be ruined not containing representative microbial from pinus sylvestris var. mongolica
Adjust its moisture content, according to step 1 method acquire sawdust, later according to the method for step 2 and three extract respectively DNA and
Carry out analysis of biological information.
The results show that ten several microorganisms are detected in the timber altogether.
The detection of comparative example 2, the timber microorganism not freezed
According to the method for embodiment 1, the sawdust 1 step 1 of embodiment obtained is straight by obtained sawdust without freezing
It connects and extracts DNA according to the method for step 2, then carry out analysis of biological information according to the method for step 3.
The results show that detecting 53 kinds of timber microorganisms in A；41 kinds of timber microorganisms are detected in B；46 are detected in C
Kind timber microorganism.
Comparative example 3, DNA extracting solution detect microorganism in timber after changing
According to the method for embodiment 1, the sawdust after freezing that 1 step 1 of embodiment obtains is ground in dismembyator, is obtained
To sawdust powder；800 μ l DTAB solution are added into sawdust powder, is then placed in 65 DEG C of warm bath pots and is incubated for 1 hour, obtain
Extraction mixture；The extraction mixture is extracted into DNA according to the method for step 2 and three respectively and carries out analysis of biological information.
The results show that detecting 2 kinds of timber microorganisms, respectively Leptographium bacterium and Clonostachys in A；B
In detect 4 kinds of timber microorganisms, respectively Penicillum, Haematonectri, Clonostachys and
Aspergillus；Detect 3 kinds of timber microorganisms in C, respectively Sporothrix, Trichoderma and
Xenoacremonium。
Sequence table
<110>INST OF WOOD INDUDTRY CHINESE, Forestry Institute of New Technology, Chinese Academy of Forestry
<120>a kind of method for detecting microorganism in timber
<130> WHOI180081
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
cttggtcatt tagaggaagt aa 22
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
tcctccgctt attgatatgc 20
Claims (10)
1. the method for detecting microorganism in timber, comprising:
1) moisture content for adjusting timber to be measured, obtains pretreated timber, the moisture content of the pretreated timber is
6%-16%；
2) it is crushed the pretreated timber, obtains sawdust；
3) DNA for extracting the sawdust, obtains DNA to be measured；
4) the microorganism sequence information in the DNA to be measured is analyzed, determines the type of microorganism in the timber to be measured.
2. according to the method described in claim 1, it is characterized by: the moisture content of the pretreated timber is 20%.
3. method according to claim 1 or 2, it is characterised in that: further include before step 3) to the sawdust at -20 DEG C
It is freezed.
4. according to the method described in claim 3, it is characterized by: the time of the freezing be more than or equal to 2 hours.
5. method according to any one of claims 1-4, it is characterised in that: step 3) includes:
The sawdust 3a) is ground, wood powder is obtained；
Extracting solution 3b) is added in Xiang Suoshu wood powder, obtains extraction mixture；The extracting solution is to add into DTAB solution
The solution that beta -mercaptoethanol obtains, beta -mercaptoethanol content is 2 μ l/mL extracting solutions in the extracting solution；The DTAB solution by
Solvent and solute composition, the solvent be water, the solute be dodecyl trimethyl ammonium bromide, Tris, ethylenediamine tetra-acetic acid,
NaCl and HCl for adjusting pH value, dodecyl trimethyl ammonium bromide, Tris, ethylenediamine tetra-acetic acid and NaCl are described
Concentration in DTAB solution is respectively 0.02g/mL, 0.1M, 0.02M, 1.4M, and the pH value of the DTAB solution is 7.5；
Extract in the extraction mixture DNA 3c) to get the DNA to be measured is arrived.
6. according to the method described in claim 5, it is characterized by: the additive amount of the extracting solution meets the extracting solution and institute
The proportion for stating sawdust is 800 μ l:0.1g.
7. any method in -6 according to claim 1, it is characterised in that: step 4) includes:
4a) using the DNA to be measured as template, PCR amplification is carried out using the primer pair that ITS1 and ITS4 is formed, amplification is obtained and produces
Object；The ITS1 and ITS4 is respectively single stranded DNA shown in sequence 1 and sequence 2 in sequence table；
4b) amplified production is sequenced, the type of microorganism in the timber to be measured is determined according to sequencing result.
8. extracting the method for microbial DNA from timber or woodwork, comprising: according to any the method in claim 1-7
In step 1) -3) extract and obtain the DNA of timber to be measured, i.e. containing micro- in the timber to be measured in the DNA of the timber to be measured
The DNA of biology.
9. extracting solution described in claim 5.
10. following any applications of extracting solution described in any the method or claim 5 in claim 1-8:
A1, the application in prevention and treatment wood deterioration；
A2, lead to the application in wood deterioration microorganism in detection；
A3, the application in wood degradation.
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