CN105018637B - Application and method of the tetA genes in detecting soil antibiotic resistance level of pollution - Google Patents
Application and method of the tetA genes in detecting soil antibiotic resistance level of pollution Download PDFInfo
- Publication number
- CN105018637B CN105018637B CN201510526038.7A CN201510526038A CN105018637B CN 105018637 B CN105018637 B CN 105018637B CN 201510526038 A CN201510526038 A CN 201510526038A CN 105018637 B CN105018637 B CN 105018637B
- Authority
- CN
- China
- Prior art keywords
- teta
- soil
- pollution
- antibiotic resistance
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The application and method that the invention discloses a kind of tetA genes in detecting soil antibiotic resistance level of pollution.According between specific resistance gene tetA enrichment times and the total enrichment times of ARGs have extremely significant positive correlation (P<0.001), the level that soil antibiotic resistance pollutes is detected and is evaluated according to the concentration level of this gene in soil, has greatly saved human and material resources, and can preferably react soil antibiotic resistance level of pollution.
Description
Technical field
The invention belongs to the detection technique fields of Soil Environmental Pollution object, and in particular to tetA genes are in detection soil antibiosis
Application in plain resistance level of pollution and method.
Background technology
In physianthropy and cattle breeding, antibiotic is widely used in treating or preventing disease generation and promotes family dirty swine
It is long to improve yield.However, the antibiotic in intake human body or animal body can seldom be absorbed by humans and animals, most of antibiotic meeting
Discharge humans and animals it is external and with after processing the discharge of sewage and human and animal excreta be diffused into soil as Fertilizer application, make
Soil environment becomes a huge potential antibiotics resistance gene (Antibiotic Resistance Genes, ARGs)
Repository.More and more evidences show that antibiotic release in the environment and diffusion can apply edaphon selectivity pressure
Power significantly increases so as to cause the micro organism quantity for carrying ARGs.The research of the World Health Organization delivered recently is warned " anti-
Raw element tolerant bacteria is just popular in global many places especially China, this would potentially result in humans and animals and suffers from meningitis
With so that skin, blood, kidney and other organs is infected ".
After the microbial strains death for carrying ARGs, including the DNA molecular of ARGs is releasably to existing in environment for a long time, and
May by be in direct contact or food chain migration etc. number of ways enter human body, increase the antibiotic resistance of human body, make bacterium
The treatment of infectious diseases is more difficult.Further, since gene contamination is more special, it can be situated between by Mobile genetic elements
The Horizontal Gene Transfer mechanism led unlimitedly is propagated between phase bacteroid and pathogen and nonpathogenic bacteria, and has heredity
Property, it is difficult to control and eliminate, long-term, irreversible harm will be caused to human health and ecology erroneous zone once being formed.Cause
This, evaluation soil antibiotic resistance level of pollution has become the hot spot of international concern.
Since ARGs is mainly entrained by all kinds of drug-resistant microorganisms in soil, detection soil antibiotic resistance pollution
Level can be detected in terms of resistant microorganism quantity and resistant gene abundance two, and corresponding detection method is respectively：Resistance
Culture of microorganism and resistant gene quantitative pcr amplification method.Resistant microorganism cultivation evaluates soil antibiotic level of pollution master
The ratio of total Bacterial diversity is accounted for by resistant microorganism to indicate；Resistant gene quantitative pcr amplification method, mainly by anti-
Property gene accounts for the ratio of total bacterial 16 S rRNA gene abundances to indicate.Since in soil only 0.1~10% microorganism is
It is educable, therefore the accuracy of resistant microorganism cultivation evaluation soil antibiotic resistance level of pollution is under suspicion.Resistance
Gene quantification PCR amplification method can avoid the defect of culture of microorganism, by directly extracting DNA from soil, and to purpose
Genetic fragment carries out amplification and carries out accurate quantitative analysis to gene abundance according to the fluorescence intensity in amplification procedure, in antibiotic
It is received in resistance contamination analysis widely used.However the antibiotic resistance pollution in soil often refers to Multiple Classes of Antibiotics, because
This to accurate evaluation it is total soil antibiotic resistance pollution, need the method by high-throughput quantification PCR to tens of kinds or even
Hundreds of kinds of genes are expanded, by quasi- evaluation soil with compare the rich of all kinds of ARGs in soil after bacteria abundance corrects
Degree is compared analysis, calculates separately out the enrichment times of each resistant gene in quasi- evaluation soil, all ARGs enrichments times
Several adductions can react the total antibiotic resistance level of pollution of soil.But the resistant gene quantity that this detection method covers is huge
Greatly, therefore cost is of high cost, and takes time and effort, and is unfavorable for large-scale promotion.
Time saving, the cheap and accurate soil antibiotic resistance level of pollution detection method of one kind facing the evaluation mankind
Potential antibiotic resistance pollution risk and implement prevent antibiotic resistance propagate strategy be very crucial.
Invention content
The purpose of the present invention is exactly to solve the above-mentioned problems, to provide tetA genes in detection soil antibiotic resistance pollution
Application in level and method.
To achieve the goals above, the present invention adopts the following technical scheme that：
Applications of the gene tetA in detecting soil antibiotic resistance level of pollution.
Above-mentioned application is that the positive correlation between the total enrichment times of enrichment times and ARGs by gene tetA embodies.
Relationship between the enrichment times and the total enrichment times of ARGs of the gene tetA：Y=569.635+4.671x,
Middle x is tetA enrichment times, and y is the total enrichment times of ARGs.
A method of soil antibiotic resistance level of pollution being detected using gene tetA, specifically includes following step：
(1) forward primer sequence such as SEQIDNo.1 and reverse primer sequences such as SEQIDNo.2, tetA-F 5 '-are selected
CTCACCAGCCTGACCTCGAT-3 '/tetA-R 5 '-CACGTTGTTATAGAAGCCGCATAG-3 ' are to purpose resistant gene
TetA carries out PCR amplification；
(2) the purpose resistant gene after gel extraction purifying amplification, resistant gene is connected in carrier T, is then converted
Competent E.coli cell is selected positive clone molecule after applying LB/ ammonia benzyl/IPTG/X-Gal tablets, is determined by gene sequencing
The positive clone molecule for carrying target gene, is used in combination the LB culture solutions containing ammonia benzyl to be enlarged culture to it；
(3) concentration for the plasmid that detection is extracted from the LB culture bacterium solutions containing ammonia benzyl, converses unit volume plasmid institute
The copy number for carrying resistant gene, the plasmid containing target gene is serially diluted by 10 times of gradients, as resistant gene
Standard items；
(4) soil DNA is extracted, soil DNA and resistant gene standard items are carried out at the same time quantitative pcr amplification tetA genes, root
According to the enrichment times and formula y=569.635+4.671x of tetA genes, wherein x is tetA enrichment times, and y is that ARGs is always enriched with
Multiple, and then obtain soil antibiotic resistance level of pollution.
The purpose resistant gene length is respectively：The length of tetA genes is 71bp.
Pcr amplification reaction system in the step (1) is 25 μ L, including the template DNA (1-10ng) of 2 μ L, respectively
The forward and reverse primer (10 μM) of 0.5 μ L, 2 × Premix Ex Taq of 12.5 μ LTMThe nothing of (Dalian treasured biotech firm) and 9.5 μ L
Bacterium distilled water；PCR amplification condition is：95 DEG C of pre-degeneration 10min；95 DEG C of 40 cycles keep 20s (denaturation), 57 DEG C of holdings
20s (annealing) and 72 DEG C of holding 30s (extension)；72 DEG C keep 5min (fully extended).
Transformed competence colibacillus Bacillus coli cells in the step (2) the specific steps are：Competence is taken out from -80 DEG C of refrigerators
Cell, thaw 5min on ice, and 40 μ L competent cells are shifted to reaction tube is connected after touching mixing, tap centrifuge tube mixing and in
20min is placed on ice, later in 42 DEG C of water-bath 45-50s, is then transferred quickly to place 2min on ice, and the SOC of 950 μ L is added
Culture solution in 37 DEG C of shaken cultivations (150 revs/min) 1.5 hours, then will each convert 100 μ L of culture medium to connecting reaction tube
It is coated onto on two LB/ ammonia benzyl/IPTG/X-Gal tablets, tablet is incubated overnight (16-24 hours) in 37 DEG C, put after taking out tablet
4 DEG C of colour developing 10h, select hickie bacterium colony.
In the step (3), the formula that plasmid concentration is converted into the copy number of target gene in every μ L plasmid solutions is：Matter
Grain concentration (ng/ μ L) × 6.02 × 1023/ [(Insert Fragment length+3015) × 660 × 109]。
It is a kind of detection soil antibiotic resistance level of pollution kit, comprising forward primer sequence such as SEQIDNo.1 with
Soil DNA and resistant gene standard items are carried out at the same time quantitative pcr amplification tetA genes by reverse primer sequences such as SEQIDNo.2,
According to the enrichment times of tetA genes and formula y=569.635+4.671x, and then obtain soil antibiotic resistance level of pollution.
Beneficial effects of the present invention：
According between specific resistance gene tetA enrichment times and the total enrichment times of ARGs have extremely significant positive correlation
(P<0.001), the level that soil antibiotic resistance pollutes is detected and is commented according to the concentration level of this gene in soil
Valence has greatly saved human and material resources, and can preferably react soil antibiotic resistance level of pollution.
Description of the drawings
Relationships of the Fig. 1 between tetA genetic enrichments multiple and the total enrichment times of ARGs.
Specific implementation mode
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Take park native from 14 places, wherein 6 parks are irrigated with sewage disposal plant effluent, 6 parks
It is to be irrigated with fresh water, 2 national park soil for being not affected by Human impact are as a contrast.Each park acquires 3 parts of soil.It adopts
The pedotheque of collection is immediately placed in ice chest, takes back and every part of soil is removed the impurity such as tree root, stone and insect simultaneously behind laboratory
It is sufficiently mixed uniformly, is then store in -20 DEG C of refrigerators, and utilize MoBio PowerSoil DNA extracts reagents in one week
Box directly extracts DNA from soil.
Select primer pair forward primer sequence such as SEQIDNo.1 and reverse primer sequences such as SEQIDNo.2, tetA-F 5 '-
CTCACCAGCCTGACCTCGAT-3 '/tetA-R 5 '-CACGTTGTTATAGAAGCCGCATAG-3 ' are to tetracycline
(Tetracycline) class resistant gene tetA carries out PCR amplification.
Above-mentioned pcr amplification reaction system is 25 μ L, and including the template DNA (1-10ng) of 2 μ L, each 0.5 μ L's is positive and negative
To primer (10 μM), 2 × Premix Ex Taq of 12.5 μ LTMThe aseptic double-distilled water of (Dalian treasured biotech firm) and 9.5 μ L.
Above-mentioned PCR amplification condition is：95 DEG C of pre-degeneration 10min；95 DEG C of 40 cycles keep 20s (denaturation), 57 DEG C of guarantors
It holds 20s (annealing) and 72 DEG C keeps 30s (extension)；72 DEG C keep 5min (fully extended).
PCR product after amplification is detected by 1% agarose gel electrophoresis, and according to DNA marker to gene
Fragment length is compared, and the length of tetA genes is 71bp.Corresponding electrophoretic band in Ago-Gel is carried out to cut glue time
It receives, the glue purification QIAquick Gel Extraction Kit of Promega companies is used in combination to carry out purifying recycling.
PCR product after purification be used for build clone library, be first by after purification PCR product and Promega companies
PGEM-T easy carriers be attached, coupled reaction system be 10 μ L, including：The connection buffer solution of 5 μ L, the T of 1 μ L
Carrier, the T4DNA ligases of 1 μ L, about 1 μ L PCR product after purification (specific volume can be according to PCR product concentration tune
It is whole), remaining volume is supplied with aseptic double-distilled water.Followed by transformation experiment, step is：Impression is taken out from -80 DEG C of refrigerators
State cell, thaw 5min on ice, shifts 40 μ L competent cells to reaction tube is connected after touching mixing, tap centrifuge tube mixing is simultaneously
It in placing 20min on ice, later in 42 DEG C of water-bath 45-50s, is then transferred quickly to place 2min on ice, is added 950 μ l's
SOC culture solutions in 37 DEG C of shaken cultivations (150 revs/min) 1.5 hours, then will each convert culture medium 100 to connecting reaction tube
μ L are coated onto on two LB/ ammonia benzyl/IPTG/X-Gal tablets, and tablet is incubated overnight (16-24 hours) in 37 DEG C, after taking out tablet
4 DEG C of colour developing 10h are put, hickie bacterium colony (positive colony) is selected, is enlarged culture to it using the LB culture solutions containing ammonia benzyl, carries
It takes plasmid and send biotech firm that Insert Fragment is sequenced, using Nanodrop 2000 to the correct plasmid of sequencing result
Concentration and purity are measured, and calculate the copy number per target gene in μ L plasmid solutions according to following formula：
It is per the target gene copy number (copy/μ L) entrained by μ L plasmid solutions：Plasmid concentration (ng/ μ L) × 6.02 ×
1023/ [(Insert Fragment length+3015) × 660 × 109]。
Plasmid containing target gene is serially diluted by 10 times of gradients, as ARGs fluorescent quantitative PCR experiments
Standard items.
Standard items are prepared, followed by fluorescent quantitative PCR experiment.Select primer pair forward primer sequence such as
SEQIDNo.1 and reverse primer sequences such as SEQIDNo.2, tetA-F 5 '-CTCACCAGCCTGACCTCGAT-3 '/tetA-R
5 '-CACGTTGTTATAGAAGCCGCATAG-3 ' expand tetA genes.
Fluorescent quantitative PCR reaction system is 25 μ L, including the template DNA (1-10ng) of 2 μ L, each 0.5 μ L's
Forward and reverse primer (10 μM), 2 × SYBR Premix Ex Taq of 12.5 μ LTM(Dalian treasured biotech firm) and 9.5 μ L's is sterile
Distilled water.
Fluorescent quantitative PCR condition is：95 DEG C of pre-degeneration 10min, 95 DEG C of holding 20s (denaturation) of 40 cycles, 57
It DEG C keeps 20s (annealing) and 72 DEG C keeps 30s (extension, step reading SYBR fluorescence signals).
Use TaqMan probe method with probe TM1389F (5 '-FAM-CTTGTACACACCGCCCGTC-TAMRA-3 ') and
Primer BACT1369F (5 '-CGGTGAATACGTTCYCGG-3 ')/PROK1492R (5 '-GGWTACCTTGTTACGACTT-3 ')
To bacterial 16 S rRNA genes carry out real-time fluorescence quantitative PCR analysis [bibliography Suzuki M T, Taylor L T,
DeLong E F.Quantitative analysis of small-subunit rRNA genes in mixed
microbial populations via 5'-nuclease assays.Applied and Environmental
Microbiology,2000,66(11):4605-4614]。
ARGs concentration levels are calculated according to following formula：
Δ Ct=Ct(ARGs)-Ct(16S) (a)
Δ Δ Ct=Δs Ct(to be measured)-ΔCt(control) (b)
FC=2[-(ΔΔCt)] (c)
Δ Ct be sample to be tested target gene Ct values (recurring number that is undergone when fluorescence signal reaches given threshold) with
The difference of 16SrRNA gene C t values, Δ Δ Ct refer to the target gene Δ Ct values of sample to be tested and the Δ Ct values of control sample
Difference, FC values refer to enrichment times of the target gene abundance compared to control sample of sample to be tested.
This research is expanded with 87 kinds of ARGs of identical soil pair, by detect the concentration levels of tetA genes with it is total
The correlation of ARGs concentration levels (adductions of 87 kinds of resistant gene enrichment times), as a result such as Fig. 1.
It can be seen that there is extremely significant positive between soil tetA genetic enrichments multiple and the total enrichment times of ARGs by upper figure
Pass relationship (P<0.001), thus can according to the concentration levels of tetA genes in soil to soil antibiotic resistance level of pollution into
Row detection and evaluation.
Verify example：
Certain park soil is chosen, and acquires three parts of soil, using same procedure to above-mentioned 87 kinds of ARGs and bacterial 16 S rRNA
Gene abundance is detected, and chooses the national park soil of the studies above as a contrast, according to formula (a), (b) and (c) it is right
The enrichment times of the tetA genes of this three parts of soil are calculated, while according to formula y=569.635+4.671x (wherein x
For tetA enrichment times, y is the total enrichment times of ARGs) soil antibiotic resistance level of pollution is predicted.Soil is surveyed
The total enrichment times of ARGs be respectively 2321.612,651.486 and 533.531, and predict the total enrichment times of ARGs be respectively
1042.197,602.695 and 574.655.By one-way analysis of variance, find between two groups of data without significant difference, card
Bright two groups of data are comparable；Found by correlation analysis, have between two groups of data extremely significant positive correlation (R=1.00,
P<0.01)。
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not
Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (4)
1. a kind of method of tetA genes in detecting soil antibiotic resistance level of pollution, it is characterized in that：It specifically includes following
The step of：
(1) forward primer sequence such as SEQ ID No.1 and reverse primer sequences such as SEQ ID No.2 are selected, to purpose resistance base
Because tetA carries out PCR amplification；The length of tetA genes is 71bp；
(2) the purpose resistant gene after gel extraction purifying amplification, resistant gene is connected in carrier T, then conversion impression
State Bacillus coli cells select positive clone molecule, with the LB culture solutions containing ammonia benzyl after applying LB/ ammonia benzyl/IPTG/X-Gal tablets
It is cultivated；
(3) concentration for the plasmid that detection is extracted from the LB culture bacterium solutions containing ammonia benzyl, converses entrained by unit volume plasmid
Plasmid containing target gene is serially diluted, as resistant gene standard by the copy number of resistant gene by 10 times of gradients
Product；
(4) soil DNA is extracted, soil DNA and resistant gene standard items are carried out at the same time quantitative pcr amplification tetA genes, according to
The enrichment times and formula y=569.635+4.671x of tetA genes, wherein x are tetA enrichment times, and y is ARGs always enrichments times
Number, and then obtain soil antibiotic resistance level of pollution.
2. the method as described in claim 1, it is characterized in that：Pcr amplification reaction system in the step (1) is 25 μ L,
Include 2 μ L template DNA be 1-10ng, the forward and reverse primer of each 0.5 μ L, a concentration of 10 μM, 2 × Premix of 12.5 μ L
The aseptic double-distilled water of Ex TaqTM and 9.5 μ L；
PCR amplification condition is：95 DEG C of pre-degeneration 10min；95 DEG C of holding 20s of 40 cycles, 60 DEG C keep 20s and 72 DEG C of holding
30s, 72 DEG C of holding 5min.
3. the method as described in claim 1, it is characterized in that：Transformed competence colibacillus Bacillus coli cells tool in the step (2)
Body step is：Competent cell is taken out from -80 DEG C of refrigerators, thaw 5min on ice, and 40 μ L competent cells are shifted after touching mixing
To connection reaction tube, taps centrifuge tube mixing and in placing 20min on ice, later in 42 DEG C of water-bath 45-50s, then turn rapidly
It moves to and places 2min on ice, the SOC culture solutions of 950 μ L are added to connecting reaction tube, at 37 DEG C, 150 revs/min of shaken cultivations 1.5
Hour, then each 100 μ L of conversion culture medium are coated onto on two LB/ ammonia benzyl/IPTG/X-Gal tablets, by tablet in 37 DEG C of mistakes
Night cultivates, and puts 4 DEG C of colour developing 10h after taking out tablet, selects hickie bacterium colony.
4. the method as described in claim 1, it is characterized in that：In the step (3), it is molten that plasmid concentration is converted into every μ L plasmids
The formula of the copy number of target gene is in liquid：Plasmid concentration (ng/ μ L) × 6.02 × 1023/ [(Insert Fragment length+3015)
×660×109]。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510526038.7A CN105018637B (en) | 2015-08-24 | 2015-08-24 | Application and method of the tetA genes in detecting soil antibiotic resistance level of pollution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510526038.7A CN105018637B (en) | 2015-08-24 | 2015-08-24 | Application and method of the tetA genes in detecting soil antibiotic resistance level of pollution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105018637A CN105018637A (en) | 2015-11-04 |
| CN105018637B true CN105018637B (en) | 2018-08-31 |
Family
ID=54408935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510526038.7A Expired - Fee Related CN105018637B (en) | 2015-08-24 | 2015-08-24 | Application and method of the tetA genes in detecting soil antibiotic resistance level of pollution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105018637B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501325A (en) * | 2020-12-09 | 2021-03-16 | 国家海洋环境监测中心 | Quantitative detection method for antibiotic resistance gene of high-flux aquaculture |
| CN113388688A (en) * | 2021-06-16 | 2021-09-14 | 济南大学 | Method and system for detecting spread of manure source drug-resistant bacteria in soil |
| CN114262715B (en) * | 2021-12-28 | 2023-07-28 | 中国环境科学研究院 | Method for evaluating environmental health risk of resistance genes in compost products |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561342A (en) * | 2015-01-23 | 2015-04-29 | 杭州迪安医学检验中心有限公司 | Primers and kit for detecting tetracycline drug-resistance genes of bacteria |
-
2015
- 2015-08-24 CN CN201510526038.7A patent/CN105018637B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561342A (en) * | 2015-01-23 | 2015-04-29 | 杭州迪安医学检验中心有限公司 | Primers and kit for detecting tetracycline drug-resistance genes of bacteria |
Non-Patent Citations (3)
| Title |
|---|
| 北京地区农田土壤抗生素抗性基因分布状况的初步研究;王佳佳;《2015年中国环境科学学会学术年会论文集（第一卷）》;20150806;135-140 * |
| 环境因素对土壤中几种典型四环素抗性基因形成的影响;张俊;《环境科学》;20141130;第35卷(第11期);4267-4274 * |
| 禽畜养殖场土壤抗生素抗性基因污染的初步研究;邹世春;《中山大学学报（自然科学版）》;20121130;第51卷(第6期);87-91 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105018637A (en) | 2015-11-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gupta et al. | Phylogenetic framework and molecular signatures for the class Chloroflexi and its different clades; proposal for division of the class Chloroflexi class. nov. into the suborder Chloroflexineae subord. nov., consisting of the emended family Oscillochloridaceae and the family Chloroflexaceae fam. nov., and the suborder Roseiflexineae subord. nov., containing the family Roseiflexaceae fam. nov. | |
| Shang et al. | Effects of organic fertilizer on soil nutrient status, enzyme activity, and bacterial community diversity in Leymus chinensis steppe in Inner Mongolia, China | |
| Karr et al. | Remarkable diversity of phototrophic purple bacteria in a permanently frozen Antarctic lake | |
| CN105112519A (en) | CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method | |
| CN103725796B (en) | Mark parting fluorescence PCR detection kit and preparation thereof in a kind of bovine viral diarrhea virus | |
| Jia et al. | Environmental fate of tetracycline resistance genes originating from swine feedlots in river water | |
| CN105018637B (en) | Application and method of the tetA genes in detecting soil antibiotic resistance level of pollution | |
| Nwachukwu et al. | Metagenomics: a tool for exploring key microbiome with the potentials for improving sustainable agriculture | |
| Kim et al. | Detection of Brucella canis and Leptospira interrogans in canine semen by multiplex nested PCR | |
| Naganandhini et al. | Persistence of pathogenic and non-pathogenic Escherichia coli strains in various tropical agricultural soils of India | |
| Zerzghi et al. | Influence of long‐term land application of Class B biosolids on soil bacterial diversity | |
| Hou et al. | Characterization of prokaryotic and eukaryotic microbial community in pacific white shrimp ponds | |
| Achyar et al. | In silico study of developing a method for detecting pathogenic bacteria in refillable drinking water samples | |
| Guron et al. | Manure-based amendments influence surface-associated bacteria and markers of antibiotic resistance on radishes grown in soils with different textures | |
| CN102337344A (en) | Quantitative detection method of escherichia coli in soil and assay kit thereof | |
| Nguyen et al. | Impacts of initial fertilizers and irrigation systems on paddy methanogens and methane emission | |
| Wang et al. | The divergence of cpcBA-IGS sequences between Dolichospermum and Aphanizomenon (Cyanobacteria) and the molecular detection of Dolichospermum flos-aquae in Taihu Lake, China | |
| Zhao et al. | Optimization of photosynthetic bacteria wastewater treatment and study of microbial species diversity | |
| Shen et al. | Using an intervening sequence of Faecalibacterium 16S rDNA to identify poultry feces | |
| Zhou et al. | Bacterial features in tilapia (Oreochromis niloticus) and environments in a goose-tilapia polyculture model | |
| CN105018636B (en) | Application and method of the ermC genes in soil antibiotic resistance level of pollution is detected | |
| Kang et al. | Tetracycline resistance genes are more prevalent in wet soils than in dry soils | |
| Kamusoko et al. | Purification and amplification of DNA from cellulolytic bacteria: Application for biogas production from crop residues | |
| CN102676678B (en) | PCR (Polymerase Chain Reaction) primer group for distinguishing five types of shellfish bait microalgae | |
| Qi et al. | Bacterial communities under long-term conventional and transgenic cotton farming systems using V3-V5 and V5-V9 of 16S rDNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Han Xuemei Inventor after: Liang Yu Inventor after: Li Helian Inventor after: Chen Ying Inventor after: Zhang Mingliang Inventor before: Han Xuemei Inventor before: Liang Yu Inventor before: Li Helian Inventor before: Chen Ying |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180831 Termination date: 20200824 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |