CN107805658A - A kind of screening technique of the eucaryon miniature organism T RFLP fragment sensitive to water quality - Google Patents
A kind of screening technique of the eucaryon miniature organism T RFLP fragment sensitive to water quality Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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Abstract
The present invention relates to a kind of screening technique of the eucaryon miniature organism T RFLP fragment sensitive to water quality, belong to miniature organism ecological technique field.The screening technique of the present invention comprises the following steps：(1) water sample is gathered, and determines the physical and chemical factor of each water sample sample；(2) STb gene is extracted；(3) performing PCR amplification is entered to the v4 areas of the 18SrRNA genes of eukaryotic microorganisms, reclaims, purifies amplified production and carry out digestion, determine digestion products, obtain T RFLP data and collection of illustrative plates；(4) the T RFLP data obtained according to step (3), the percentage that same clip peak area sum in each water sample sample accounts for total peak area is calculated；(5) correlation analysis is carried out to physical and chemical factor and purpose fragment, filters out the fragment sensitive to physical and chemical factor change.The present invention utilizes eucaryon miniature organism, filters out to specific physical and chemical factor sensitivity and be present in the monoid of specific physical and chemical factor scope.
Description
Technical field
The invention belongs to miniature organism ecological technique field, and in particular to a kind of eucaryon miniature organism T- sensitive to water quality
The screening technique of RFLP (terminal restriction Length Polymorphism technique) fragment.
Background technology
In water quality monitoring, physics and chemistry monitoring has the advantages that speed is fast, simple and convenient, but still has certain limitation,
Be mainly manifested in can not reflect pollute it is comprehensive and its to biology toxic effect.Physics and chemistry monitoring can only represent the dirt during sampling
Dye situation, and for a long time, continuous biological monitoring than physics and chemistry monitoring can more reflect environmental quality change overall picture.Biological monitoring has
Ageing, comprehensive and sensitiveness, the situation that whole period environmental factor changes, and the association of each environmental factor change can be reflected
Same and antagonism result.Therefore in monitoring of environmental quality, biology is a preferable monitoring instrument.
In the prior art,《Using T-RFLP technologies to Wenyu River research on microbial community structure》One text is reported to difference
The water body of time is sampled to investigate the spatial variations of biological community structure, take 5,6,7, August part is in Shahe Reservoir in Liyang City sampled point
Water sample, extract microbiologic population's STb gene in water body, expand the 16S rDNA of bacterium, and analyze by T-RFLP, obtain four
The T-RFLP collection of illustrative plates of different time bacterial community, the bacterium kind that T-RFLP fragments are 135b, 151bp, 440bp is observed according to collection of illustrative plates
Group's change is bigger, most sensitive for external world's reflection, possibly as the indicative biology of environmental change.But the research does not have
The physical and chemical factor in water body environment is determined, the change at peak in the T-RFLP peak figures for the environmental samples taken according only to different months
To judge environmentally sensitive fragment so that the result of study lacks persuasion, and can not understand and some specific physical and chemical factor
Sensitiveness.
In addition,《Ammonia oxidation bacteria and bacterial quorum sensing and functional study in municipal sewage plant》One text report：Selection
Sewage disposal system in the sewage treatment plant in three cities is as survey region, using ammonia oxidation bacteria as research object, determine into
Water BOD, water outlet BOD, influent ammonia nitrogen, water outlet ammonia nitrogen, water inlet nitrite nitrogen, water outlet nitrite nitrogen, water temperature, DO, SRT and dirt
The physical and chemical factors such as mud concentration, carry out the Pearson correlation analyses of different length T-RFLP fragments and ambient parameter, obtain with it is specific
The related fragment of envirment factor, in conjunction with cloning and sequencing technology, obtains corresponding monoid.Different length T-RFLP fragments are joined with environment
Several Pearson correlation coefficient is as shown in table 1, from table 1：283bp fragments and water outlet BOD, temperature are significantly correlated, to this two
Individual physical and chemical factor change is sensitive；354bp fragments and water inlet BOD and water outlet ammonia nitrogen are significantly correlated, and the two physical and chemical factors are changed
It is sensitive；491bp fragments and influent ammonia nitrogen, water outlet ammonia nitrogen, temperature are significantly correlated, these three physical and chemical factors are changed sensitive.The skill
Art utilizes Pearson relative coefficients, quantifies the correlation of fragment relative abundance and physical and chemical factor value, so as to draw to specific reason
Change the sensitive monoid of the factor.But Pearson coefficients are only applicable to the data for meeting normal distribution；Furthermore obtained monoid is
The monoid sensitive to some physical and chemical factor, as the sensitive monoid resonableization factor which in the range of with indicative function not yet
Solution；Moreover, used biological group is the parent of economic animal and people's (being eucaryote) in prokaryotes, with water body
Edge relation is farther out, it is impossible to reacts chemical factors well to Eukaryotic influence.
Table 1
| Variable | 219bp | 283bp | 354bp | 491bp |
| Water inlet BOD | 0.118 | -0.266 | -0.464* | 0.536 |
| Water outlet BOD | -0.113 | -0.475* | -0.025 | 0.262 |
| Water inlet NH4-N | 0.137 | 0.038 | 0.229 | -0.588* |
| Water outlet NH4-N | -0.144 | -0.220 | -0.633* | 0.645* |
| Water outlet NO3-N | 0.149 | -0.221 | -0.320 | 0.284 |
| Temperature | 0.189 | 0.665* | 0.152 | -0.587* |
| MLSS | 0.349 | 0.028 | -0.357 | -0.393 |
| DO | 0.181 | -0.028 | -0.011 | 0.312 |
| SRT | 0.109 | -0.175 | 0.193 | 0.053 |
* the P values for representing the coefficient correlation are：0.01 ＜ P ＜ 0.05
* represents that the P values of the coefficient correlation are：P ＜ 0.01
To sum up, the water quality monitoring method of T-RFLP technologies is utilized in forefathers' research, is existed in sensitive fragment screening technique
The defects of certain；And research object is prokaryotic micro-organisms such as bacterium, can not preferably illustrate change of water quality to eucaryote (such as
Fish, people) influence.
The content of the invention
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art part, there is provided a kind of eucaryon sensitive to water quality is micro-
The screening technique of type biology T-RFLP fragments, it can filter out sensitive to specific physicochemical factors using this method and be present in
The monoid of specific physicochemical factors scope.
To achieve the above object, the technical scheme taken of the present invention is：A kind of eucaryon miniature organism T- sensitive to water quality
The screening technique of RFLP fragments, it comprises the following steps：
(1) water sample is gathered, and determines the physical and chemical factor of each water sample sample；
(2) STb gene of each water sample sample is extracted；
(3) STb gene extracted using step (2) is template, using 18S rRNA gene primers as primer, to eucaryon miniature organism
18S rRNA genes v4 areas enter performing PCR amplification, reclaim, purify amplified production and carry out digestion, determine digestion products, obtain
T-RFLP data and collection of illustrative plates；Wherein, in the universal primer, 5 ' ends of a primer carry fluorescence labeling；
(4) the T-RFLP data that step (3) obtains are pre-processed, first deletes size and be less than 50 and the piece more than 500
Section, then the percentage that same clip peak area sum in each water sample sample accounts for total peak area is calculated, delete percentage and be less than 1%
Fragment, obtain purpose fragment；
(5) percentage that the physical and chemical factor of each water sample sample measured according to step (1) and step (4) calculate, to reason
Change the factor and carry out correlation analysis with purpose fragment, filter out the fragment sensitive to physical and chemical factor change.
In the water quality monitoring of prior art, the research object of biological monitoring is concentrated mainly on and the parent such as aquatic livestock, the mankind
The very remote prokaryotic micro-organisms of edge relation, or fish, invertebrate etc. catch that difficulty is big and distribution larger area to be present poor
Different monoid.Minority the study on monitoring method of water quality is substantially based on based on eucaryon miniature organism Morphological Identification, count and
The calculating of single index, take time and effort and need professional.The present invention breaks the normal procedure, and screens micro- to the sensitive eucaryon of water environment
Type biology T-RFLP fragments.
Blazoning property of eucaryon miniature organism, is present in all parts of the world substantially.Single celled eucaryon miniature organism is more closely
Directly contacted with the environment that they are survived, thus compared to fish, invertebrate etc. to environmental change (such as water pollution)
With shorter, the more rapid reaction time, the change to survival environmental conditions is very sensitive.And eucaryon miniature organism growth and breeding
Speed is fast, and poisonous substance can be tested out in the shorter time and it is grown in several generation levels, bred, is metabolized and other life
The influence of physiological-biochemical characteristic, and this result then needs the time of a couple of days, the several months even more than several years for high experimental animal
Could obtain, and the latter on experimental facilities and sampling tool and step than the former complex and expensive much.It is in addition, most
Eucaryon miniature organism species is worldwide distribution, is not limited by season and regional disparity, therefore eucaryon miniature organism can conduct
The target monoid of monitoring water environment.Different groups are different with the sensitiveness of environmental pressure to pollutant in aquatic ecosystem,
Sensitive monoid is easily influenceed by environmental pressure and causes fluctuating for group and the ecosystem, therefore, screens to change of water quality
Sensitive eucaryon miniature organism monoid is most important for protection water environment safety.
However, T-RFLP is typically applied to environment eukaryotic microorganisms Study on Diversity by existing research, and not by T-
RFLP is applied to the screening for being related to the eucaryon miniature organism sensitivity monoid of the water quality monitoring of rivers., can using the method for the present invention
Filter out to specific physical and chemical factor sensitivity and be present in the monoid of specific physical and chemical factor scope, and the T- that the present invention uses
RFLP reduces cost compared with prior art.
As the preferred embodiment of screening technique of the present invention, the screening technique also includes step (6)：According to step
Suddenly the physical and chemical factor that (1) measures is classified to water sample sample, the percentage calculated according to classification results and step (4), is entered
Indicator species are analyzed between row group, filter out the sensitive fragment under different quality situation.
As the preferred embodiment of screening technique of the present invention, in the step (6), according to GB 3838-2002
(《Water environment quality standard》) regulation water sample is classified.According to the standard, water sample sample can be divided into one
Class water (a), two class water (b), three class water (c), four class water (d), five class water (e) and bad five class water (f).
As the preferred embodiment of screening technique of the present invention, in the step (5), to physical and chemical factor and purpose piece
Duan Jinhang Spearman rank correlation analysises.It is related to monoid relative abundance to quantify physical and chemical factor using Spearman rank correlation coefficients
Property, the instruction monoid of different range physical and chemical factor can be found, improves data reliability, and Spearman rank correlation analysises make total
Correlation analysis can be also carried out according to the situation for not meeting normal distribution.
As the preferred embodiment of screening technique of the present invention, the physical and chemical factor includes water temperature, pH value, dissolving
At least one of oxygen, total phosphorus, ammonia nitrogen, total nitrogen, permanganate index or COD.
As the more preferably embodiment of screening technique of the present invention, the physical and chemical factor be total phosphorus, ammonia nitrogen, total nitrogen,
Permanganate index and COD.Different physical and chemical factors influence to have differences on Zhujiang River eucaryon miniature organism structure of community,
The physical and chemical factor is the physical and chemical factor being had a great influence to eucaryon miniature organism structure of community.
As the preferred embodiment of screening technique of the present invention, the physical and chemical factor is screened by following methods
Arrive：Using R language, study physical and chemical factor using typical correspondence analysis (CCA) influences on eucaryon miniature organism structure of community, screening
It is more than the physical and chemical factor of 0.6 and extremely notable (P ＜ 0.01) with the coefficient correlation of eucaryon miniature organism structure of community.
As the preferred embodiment of screening technique of the present invention, in the step (2), the total of water sample sample is extracted
Before DNA, biological concentration processing first is carried out to water sample sample.
As the more preferably embodiment of screening technique of the present invention, the method for the biological concentration is：Filter water sample
Sample, collect the material on filter membrane.
As the preferred embodiment of screening technique of the present invention, in the step (3), 18S rRNA gene primers are
CCAGCA (G/C) C (C/T) GCGGTAATTCC and ACTTTCGTTCTTGAT (C/T) (A/G) A.
Compared with prior art, the beneficial effects of the present invention are：Prior art is faced with target monoid and aquatic products economy
Farther out (prokaryotic micro-organisms such as bacterium), or the cycle in epoch is long, distribution has obvious region (fish by animal, human relation
Deng)；Research method is time-consuming, it is laborious, to personnel requirement height；And sensitive monoid in the range of specific physical and chemical factor can not be filtered out
The problem of.The present invention to change of water quality to be quick on the draw and micro- with the nearer eucaryon of affiliation such as aquatic economic animals/mankind
Type biology is target monoid, goes out sensitive to specific physical and chemical factor based on T-RFLP technology screenings and is present in specific physical and chemical factor
Monoid/fragment of scope.
Embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment to the present invention
It is described further.It should be understood that these embodiments are merely to illustrate the present invention rather than limited the scope of the invention, under
NM specific experiment method in row embodiment, generally carried out according to normal experiment method.
The universal primer used in following embodiments is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 1
A kind of embodiment of the screening technique of the present invention eucaryon miniature organism T-RFLP fragment sensitive to water quality, this reality
The screening technique for applying the example eucaryon miniature organism T-RFLP fragment sensitive to water quality is as follows：
First, sample collection and physical and chemical factor measure
In Guangzhou creek and Xijiang River (Human impact point, 32), Guangzhou drinking water source area (reference point, 7),
43 sampled points such as Xijiang River estuary (estuary reference, 4) are investigated, using the method for sampling of direct water acquisition, reference《Ground
Table quality of water environment monitoring standard (GB3838-2002)》, it is measure water temperature, pH, dissolved oxygen (DO, dossolved oxygen), total
Phosphorus (TP, total phosphorus), ammonia nitrogen (Ammonia nitrogen), total nitrogen (TN, total nitrogen), permanganic acid
The physical and chemical factors such as salt index (Mn), COD (COD, Chemical Oxygen Demand).
2nd, biological concentration and DNA extractions
Get out Suction filtration device (suction filter pump, safety flack, suction filtering tube), filter membrane, tweezers, alcolhol burner, disposable glove, EP
Pipe, water sample；After assembling Suction filtration device, filter membrane is put with tweezers, respectively toward filter cup in pour into water sample, start to filter；Filter
Power supply is closed to water is dry, takes out filter membrane with tweezers, folding is put into EP and manages and carry out mark；Sample is stored in -80 DEG C of refrigerators.
With reference to kit PowerSoilIsolation kit specifications, carry out Genome DNA extraction.
3rd, T-RFLP
1st, PCR is expanded
Enter performing PCR amplification to the v4 areas of the 18SrRNA genes of eukaryotic microorganisms using the STb gene of extraction as template, under use
State primer：
TAReuk454FWD1 (5 '-CCAGCA (G/C) C (C/T) GCGGTAATTCC-3 ') and TAReukREV3 (5 '-
ACTTTCGTTCTTGAT (C/T) (A/G) A-3 '), and add chlordene fluorescein mark on primer TAReuk454FWD1
(Hexachlorofluoroscein) performing PCR amplification, is entered.Sequentially added in PCR pipe 21.4 μ LddH2O, 2 μ L samples DNA,
1.6 μ L primers (each 0.8 μ L of 18S-F-Hex, Euk516r-GC) and the μ L of enzyme (Ex Taq) 25, reaction system total amount are 50 μ L.
PCR response procedures are：95 DEG C of pre-degeneration 5min；94 DEG C are denatured 30 seconds, and 53 DEG C are annealed 45 seconds, and 72 DEG C extend 2 minutes, and totally 35 are followed
Ring；Last 72 DEG C extend 10 minutes, terminate at 4 DEG C.
2nd, product recovery and restricted digestion
Using kit TIANquick Mini Purification Kit (Tiangeng), recovery purifying is carried out；It will return afterwards
Receive product and carry out digestion (TAKARA, Japan), digestion system is as follows：10 μ L recovery products；2μLQC buffer；0.4μL Hae
III enzymes；7.6 μ L distilled waters, reaction system cumulative volume are 20 μ L；System is finally put into 37 DEG C water baths and carries out digestion.
3rd, capillary electrophoresis detection
Digestion products are subjected to Capillary Electrophoresis, and carry out the detection of fluorescence and fluorescence intensity, obtain T-RFLP data and
Collection of illustrative plates.
4th, data analysis
1st, T-RFLP data predictions
Resulting T-RFLP data are pre-processed, first, size is deleted and is less than 50 and the fragment more than 500, because
Purpose fragment magnitude range is not belonging to for it；Secondly, the peak area summation of identical purpose fragment is carried out, and seeks each water sample sample
In identical purpose fragment peak area sum account for the percentage of total peak area, then reject percentage less than 1% purpose fragment in order to avoid
Error interference；Finally, Data Integration, the data after being handled are carried out.T-RFLP pretreatment portions divided data (table 2 as shown in table 2
In, row represents sampling point, and row represent fragment).
Table 2
2nd, alternative physical and chemical factor is selected
Because different physical and chemical factors influence to have differences on Zhujiang River eucaryon miniature organism structure of community, and consider institute's rationalization
Factor pair its influence it is unrealistic, so we need to select to influence it significant physical and chemical factor and analyze.Use R languages
Speech, using typical correspondence analysis (CCA), show that COD (COD), Mn (permanganate index), AN (ammonia nitrogen), TN are (total
Nitrogen), TP (total phosphorus) has a great influence (correlation coefficient r to eucaryon miniature organism structure of community2It is all higher than 0.6 and extremely notable P ＜
0.001), therefore this 5 physical and chemical factors are used to carry out subsequent analysis.Physical and chemical factor influences such as table to eucaryon miniature organism structure of community
Shown in 3.
Table 3
Conspicuousness identifies：0‘***’0.001‘**’0.01‘*’0.05‘.’
Replace number：1000
3rd, physical and chemical factor and fragment correlation analysis
The percentage that the physical and chemical factor and step 4 measured according to step 1 calculates, using SPSS softwares, to what is picked out
Physical and chemical factor COD (COD), Mn (permanganate index), AN (ammonia nitrogen), TN (total nitrogen), TP (total phosphorus) and fragment are carried out
Spearman rank correlation analysises, filter out the fragment sensitive to the change of specific physical and chemical factor, T-RF fragments and physical and chemical factor
The result of Spearman rank correlations analysis is as shown in table 4, wherein first is classified as fragment length, P represents test value, each physics and chemistry
The factor represents coefficient correlation；Overstriking and the fragment marked with * represent small with three and above physical and chemical factor correlation test P values
Show that correlation test P values are less than 0.05 in 0.05 fragment, remaining overstriking and with * marks.Thus, we can pick out
Fragment (95,119,175,300,311,387,412,419) to permanganate index (Mn) concentration sensitive, to COD
The fragment (194,300,387,412,416) of concentration sensitive, the fragment (95,119,174,175,419) sensitive to ammonia nitrogen concentration,
The fragment (95,119,175,419) sensitive to total nitrogen concentration, to the sensitive fragment of total phosphorus concentration (95,119,175,300,412,
419)；And to three and the fragment (95,119,175,300,412,419) of above physical and chemical factor concentration sensitive, i.e. these pieces
Section is more sensitive for environmental change.
Table 4
* P ＜ 0.05 are represented
4th, indicator species are analyzed between group
The physical and chemical factor of each water sample sample measured according to step 1, reference《Water environment quality standard-(GB3838-
2002)》, water sample sample is divided into a kind of water (a), two class water (b), three class water (c), four class water (d), five class water (e) and bad
Five class water (f), the percentage calculated according to classification results and step 4, indicator species are analyzed between carrying out group by R language, screening
Sensitive fragment under different quality situation, the result of group difference analysis are as shown in table 5.
Table 5
Conspicuousness marks：P ＜ 0.01, " * * "；P ＜ 0.05, " * "；P ＜ 0.1, " "
Therefore deduce that：The sensitive fragment of three class water has OTU411, OTU165, OTU84, OTU158, OTU370；Five classes
The sensitive fragment of water has OTU393；The sensitive fragment of bad five classes water has OTU117 and OTU406；The sensitive fragment of first three class water has
OTU119；The sensitive fragment of first five class water has OTU95, OTU175 and OTU412.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (10)
- A kind of 1. screening technique of the eucaryon miniature organism T-RFLP fragment sensitive to water quality, it is characterised in that：Including following step Suddenly：(1) water sample is gathered, and determines the physical and chemical factor of each water sample sample；(2) STb gene of each water sample sample is extracted；(3) STb gene extracted using step (2) is template, using 18SrRNA gene primers as primer, to eucaryon miniature organism Performing PCR amplification is entered in the v4 areas of 18SrRNA genes, is reclaimed, is purified amplified production and carries out digestion, is determined digestion products, is obtained T- RFLP data and collection of illustrative plates；Wherein, in the universal primer, 5 ' ends of a primer carry fluorescence labeling；(4) the T-RFLP data that step (3) obtains are pre-processed, first deletes size and be less than 50 and the fragment more than 500, then The percentage that same clip peak area sum in each water sample sample accounts for total peak area is calculated, deletes the fragment that percentage is less than 1%, Obtain purpose fragment；(5) percentage that the physical and chemical factor of each water sample sample measured according to step (1) and step (4) calculate, to physics and chemistry because Son carries out correlation analysis with purpose fragment, filters out the fragment sensitive to physical and chemical factor change.
- 2. screening technique as claimed in claim 1, it is characterised in that：The screening technique also includes step (6)：According to step (1) physical and chemical factor measured is classified to water sample sample, the percentage calculated according to classification results and step (4), is carried out Indicator species are analyzed between group, filter out the sensitive fragment under different quality situation.
- 3. screening technique as claimed in claim 2, it is characterised in that：In the step (6), according to GB 3838-2002 rule It is fixed that water sample is classified.
- 4. screening technique as claimed in claim 1, it is characterised in that：In the step (5), to physical and chemical factor and purpose fragment Carry out Spearman rank correlation analysises.
- 5. screening technique as claimed in claim 1, it is characterised in that：The physical and chemical factor include water temperature, pH value, dissolved oxygen, At least one of total phosphorus, ammonia nitrogen, total nitrogen, permanganate index or COD.
- 6. screening technique as claimed in claim 5, it is characterised in that：The physical and chemical factor is total phosphorus, ammonia nitrogen, total nitrogen, Gao Meng Hydrochlorate index and COD.
- 7. screening technique as claimed in claim 6, it is characterised in that：The physical and chemical factor screens to obtain by following methods： Using R language, study physical and chemical factor using typical correspondence analysis influences on eucaryon miniature organism structure of community, and screening is micro- with eucaryon The coefficient correlation of type biology community structure is more than the physical and chemical factor of 0.6 and extremely notable (P ＜ 0.01).
- 8. screening technique as claimed in claim 1, it is characterised in that：In the step (2), the STb gene of water sample sample is extracted Before, biological concentration processing first is carried out to water sample sample.
- 9. screening technique as claimed in claim 8, it is characterised in that：The method of the biological concentration is：Water sample sample is filtered, Collect the material on filter membrane.
- 10. screening technique as claimed in claim 1, it is characterised in that：In the step (3), 18SrRNA gene primers are CCAGCA (G/C) C (C/T) GCGGTAATTCC and ACTTTCGTTCTTGAT (C/T) (A/G) A.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109712673A (en) * | 2018-12-24 | 2019-05-03 | 江苏师范大学 | A kind of method of quick export Terminal restriction fragment length polymorphism data |
| CN115691670A (en) * | 2022-08-16 | 2023-02-03 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | River ecosystem health evaluation method based on microbial community specific response |
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| CN101792807A (en) * | 2010-03-25 | 2010-08-04 | 复旦大学 | Method for analyzing microbial community structures |
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| CN101792807A (en) * | 2010-03-25 | 2010-08-04 | 复旦大学 | Method for analyzing microbial community structures |
| CN104109717A (en) * | 2014-07-18 | 2014-10-22 | 新疆农业科学院微生物应用研究所 | Earthquake monitoring method based on bacterial community structure changes for Xinjiang region |
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| CN109712673A (en) * | 2018-12-24 | 2019-05-03 | 江苏师范大学 | A kind of method of quick export Terminal restriction fragment length polymorphism data |
| CN115691670A (en) * | 2022-08-16 | 2023-02-03 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | River ecosystem health evaluation method based on microbial community specific response |
| CN115691670B (en) * | 2022-08-16 | 2023-06-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | River ecosystem health evaluation method based on microbial community specific response |
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