CN111560446A - Quantitative detection method for free-state antibiotic resistance genes in sewage - Google Patents
Quantitative detection method for free-state antibiotic resistance genes in sewage Download PDFInfo
- Publication number
- CN111560446A CN111560446A CN202010214165.4A CN202010214165A CN111560446A CN 111560446 A CN111560446 A CN 111560446A CN 202010214165 A CN202010214165 A CN 202010214165A CN 111560446 A CN111560446 A CN 111560446A
- Authority
- CN
- China
- Prior art keywords
- antibiotic resistance
- free
- concentration
- sewage
- ethanol
- 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.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 107
- 230000003115 biocidal effect Effects 0.000 title claims abstract description 95
- 239000010865 sewage Substances 0.000 title claims abstract description 67
- 238000001514 detection method Methods 0.000 title abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 111
- 239000000706 filtrate Substances 0.000 claims abstract description 29
- 239000006228 supernatant Substances 0.000 claims abstract description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000002244 precipitate Substances 0.000 claims abstract description 22
- 238000002156 mixing Methods 0.000 claims abstract description 20
- 239000011259 mixed solution Substances 0.000 claims abstract description 15
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 14
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 14
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 11
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000013612 plasmid Substances 0.000 claims description 45
- 101100223892 Escherichia coli sulI gene Proteins 0.000 claims description 30
- 235000019441 ethanol Nutrition 0.000 claims description 30
- 238000003753 real-time PCR Methods 0.000 claims description 28
- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 17
- 239000010802 sludge Substances 0.000 claims description 17
- 238000000137 annealing Methods 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 239000000126 substance Substances 0.000 claims description 16
- 238000005119 centrifugation Methods 0.000 claims description 12
- 238000011144 upstream manufacturing Methods 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 10
- 238000004445 quantitative analysis Methods 0.000 claims description 10
- 238000004925 denaturation Methods 0.000 claims description 8
- 230000036425 denaturation Effects 0.000 claims description 8
- 238000010812 external standard method Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 claims description 4
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 238000012360 testing method Methods 0.000 abstract description 11
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000009826 distribution Methods 0.000 abstract description 3
- 239000003344 environmental pollutant Substances 0.000 abstract description 3
- 231100000719 pollutant Toxicity 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- ANAKGLHGKIVHLU-UHFFFAOYSA-N azanium;ethanol;acetate Chemical compound [NH4+].CCO.CC([O-])=O ANAKGLHGKIVHLU-UHFFFAOYSA-N 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000012869 ethanol precipitation Methods 0.000 abstract description 2
- 238000011529 RT qPCR Methods 0.000 abstract 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 33
- 239000000499 gel Substances 0.000 description 9
- 238000004364 calculation method Methods 0.000 description 7
- 239000013600 plasmid vector Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
Images
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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of sewage treatment, in particular to a quantitative detection method of free-state antibiotic resistance genes in sewage. The method comprises the following steps: mixing the sewage filtrate with ammonium acetate, adding ethanol, mixing, and standing; centrifuging, removing supernatant, adding ethanol water solution, and mixing to obtain ethanol/precipitate mixed solution; centrifuging, removing supernatant, and adding sterile enzyme-free water to obtain free DNA; and detecting the concentration of the free-state antibiotic resistance gene. The invention defines a quantitative detection method of the free-state antibiotic resistance genes with low concentration in the sewage plant, combines the technologies of ammonium acetate-ethanol precipitation, qPCR test and the like, overcomes the bottleneck that the concentration of the free-state antibiotic resistance genes in the sewage plant is low and the interference factors are complex, and provides a sensitive and reliable analysis means for the research of the space-time distribution and removal characteristics of the free-state antibiotic resistance genes of the novel trace pollutants in the sewage plant.
Description
Technical Field
The invention relates to the technical field of sewage treatment, in particular to a quantitative detection method of free-state antibiotic resistance genes in sewage.
Background
The world health organization and the organizations such as the U.S. disease and control center have listed bacterial "antibiotic resistance" as one of the greatest threats to human health in the twenty-first century. Antibiotic resistance genes are the underlying cause of bacteria acquiring antibiotic resistance. Among the numerous environmental media, sewage treatment systems have proven to be one of the important repositories for antibiotic resistance genes and also to be the major source of the spread of antibiotic resistance gene contamination to the environment.
In sewage treatment systems, antibiotic resistance gene-related studies are mostly spread around activated sludge, and few studies are focused on the antibiotic resistance gene in a sewage free state (i.e., the antibiotic resistance gene which is free from activated sludge cells in sewage). The free antibiotic resistance gene can be permanently existed in sewage, can not be thoroughly eliminated by advanced treatment processes such as ultraviolet disinfection, membrane filtration, chlorine disinfection and the like, and can be horizontally transferred into environmental bacteria and pathogenic bacteria. Finally, free antibiotic resistance genes can diffuse into the environment with effluent from sewage plants, creating potential ecological and human health risks.
The biggest difficulty in realizing the comprehensive analysis of the environmental behavior characteristics of the free antibiotic resistance genes in the sewage plant is to construct a separation and quantitative detection method of the free antibiotic resistance genes. Because the concentration of free DNA in a sewage plant is extremely low, and external interference factors (such as salts, organic matters and other impurities in sewage) for separating the free DNA are complex, great challenges are brought to the subsequent quantitative analysis of free antibiotic resistance genes.
Disclosure of Invention
In view of the above, the present invention provides a method for quantitatively detecting free antibiotic resistance genes in sewage. The method overcomes the bottlenecks of low concentration of free-state antibiotic resistance genes and complex interference factors in the sewage plant, and provides a sensitive and reliable analysis means for the research on the space-time distribution and the removal characteristics of the free-state antibiotic resistance genes of the novel trace pollutants in the sewage plant.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a quantitative detection method of free-state antibiotic resistance genes in sewage, which comprises the following steps:
step 1, pretreating a sample to obtain a filtrate; the sample is sewage and/or sludge mixed liquor;
step 2, separation of free DNA:
step 2-1, reversing and uniformly mixing the obtained filtrate and an ammonium acetate aqueous solution, then adding absolute ethyl alcohol, reversing and uniformly mixing, and standing;
step 2-2, removing supernatant after centrifugation, adding an ethanol aqueous solution into the precipitate, and uniformly mixing to obtain an ethanol/precipitate mixed solution;
step 2-3, centrifuging the ethanol/precipitate mixed solution, removing supernatant, and adding sterile enzyme-free water to obtain free DNA;
and 3, amplifying the antibiotic resistance gene and the 16S rDNA by using the free DNA as a template and adopting a real-time fluorescent quantitative polymerase chain reaction (qPCR), and detecting the concentration of the free antibiotic resistance gene by using an external standard method.
Preferably, the pretreatment in step 1 is: centrifuging the sludge mixed liquor, and keeping a supernatant; filtering the supernatant and/or the sewage by a filter membrane to obtain a filtrate.
Preferably, the pore diameter of the filter membrane is 0.2 to 0.3 μm.
Preferably, the pore size of the filter is 0.22. mu.m.
Preferably, in the step 2-1, the volume ratio of the filtrate, the ammonium acetate aqueous solution and the absolute ethyl alcohol is (7-9): 3-5): 22-26.
Preferably, in step 2-1, the volume ratio of the filtrate, the ammonium acetate aqueous solution and the absolute ethyl alcohol is 2:1: 6.
Preferably, the concentration of the ammonium acetate aqueous solution is 7-8M.
Preferably, the concentration of the aqueous ammonium acetate solution is 7.5M.
Preferably, the absolute ethyl alcohol is precooled absolute ethyl alcohol at the temperature of 0-4 ℃.
Preferably, the absolute ethanol is pre-cooled absolute ethanol at 4 ℃.
Preferably, the standing temperature is 0-4 ℃ and the standing time is 50-70 min.
Preferably, the temperature of the standing is 0 ℃ and the time is 1 h.
Preferably, the centrifugation is carried out at 0-4 deg.C and 13000-15000 g for 20-40 min.
Preferably, the centrifugation is carried out at 14000g for 30min at 4 ℃.
Preferably, the volume ratio of the filtrate in the step 2-1 to the ethanol aqueous solution in the step 2-2 is (7-9): (1-3).
Preferably, the volume ratio of the filtrate in the step 2-1 to the ethanol aqueous solution in the step 2-2 is 4: 1.
Preferably, the ethanol aqueous solution has a volume percentage concentration of 70-80%.
Preferably, the ethanol aqueous solution has a concentration of 70% by volume.
Preferably, step 2-2 is specifically: centrifuging, removing supernatant, adding 1/2 ethanol water solution into precipitate, mixing, and transferring the ethanol/precipitate mixture from container 1 to container 2; centrifuging the ethanol/precipitate mixture in container 2 and removing the supernatant; adding the rest ethanol water solution into the container 1 again, mixing uniformly, and transferring the obtained ethanol/precipitate mixed solution from the container 1 to a container 2; the adding amount of the ethanol water solution is 0.6-0.8 mL/time.
Preferably, the volume of the container 1 is 40 to 60 mL.
Preferably, the volume of the container 1 is 50 mL.
Preferably, the volume of the container 2 is 1.5 to 5 mL.
Preferably, the volume of the container 2 is 2 mL.
Preferably, containers 1 and 2 are centrifuge tubes.
Preferably, the volume ratio of the filtrate in step 2-1 to the sterile, enzyme-free water in step 2-3 is 8: (50-100).
Preferably, the volume ratio of the filtrate in step 2-1 to the sterile, enzyme-free water in step 2-3 is 1: 10.
preferably, the antibiotic resistance genes are sul1 and sul 2.
Preferably, the primer sequences used for amplification in step 3 are:
the nucleotide sequence of the upstream primer of sul1 is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the downstream primer of sul1 is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the upstream primer of sul2 is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the downstream primer of sul2 is shown as SEQ ID NO: 4 is shown in the specification;
the nucleotide sequence of the 16S rDNA upstream primer is shown in SEQ ID NO: 5 is shown in the specification;
the nucleotide sequence of the 16S rDNA downstream primer is shown in SEQ ID NO: and 6.
Preferably, the amplification conditions of qPCR are: treating with uracil-DNA glycosylase at 50 deg.C for 2 min; hot starting at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing for 15s, and extension at 72 ℃ for 1min for 40 cycles; the annealing temperatures of sul1 and sul2 were 60 ℃ and the annealing temperature of 16S rDNA was 55 ℃.
Preferably, the qPCR reaction system is as follows:
| reagent | Volume (μ L) |
| PCR premix (2X) | 10 |
| Forward primer (10. mu.M) | 0.6 |
| Reverse primer (10. mu.M) | 0.6 |
| DNA template (1 ng/. mu.L) | 2 |
| Sterile enzyme-free water | 6.8 |
| Total up to | 20。 |
Preferably, in the preparation of the plasmid standard used in the external standard method, the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 7min at 72 ℃;
the PCR reaction system is as follows:
| reagent | Volume (μ L) |
| Taq polymerase (5U/. mu.L) | 0.1 |
| Buffer (containing 20mM Mg)2+) | 2 |
| dNTPs (2.5 mM each) | 1.6 |
| Forward primer (10. mu.M) | 0.4 |
| Reverse primer (10. mu.M) | 0.4 |
| DNA template (1 ng/. mu.L) | 1 |
| Sterile enzyme-free water | 14.5 |
| Total up to | 20。 |
In the invention, the external standard method specifically comprises the following steps:
carrying out qPCR (quantitative polymerase chain reaction) experiments by taking antibiotic resistance genes and 16S rDNA plasmid standard products as templates; drawing a quantitative standard curve of the antibiotic resistance gene and the 16S rDNA according to the corresponding relation between the copy number concentration of the plasmid standard substance and the cycle number of the antibiotic resistance gene or the 16S rDNA amplified fluorescence threshold; the preparation method of the plasmid standard product comprises the following steps: using free DNA in sewage as a template, and adopting common PCR to amplify a target gene; performing agarose gel electrophoresis on a common PCR product, cutting a target gene band under a gel imager, and purifying by adopting a gel purification kit; after adjusting the gel-purified target gene to a suitable concentration, ligating the gene to a plasmid vector, and transforming the plasmid vector into competent cells; after a positive clone transformant is selected, a plasmid standard substance is extracted by a plasmid extraction kit and stored in a refrigerator;
and (3) comparing the quantitative standard curve of the antibiotic resistance gene and the 16S rDNA, obtaining the copy number concentration of the antibiotic resistance gene and the 16S rDNA in the free DNA of the template according to the amplification fluorescence threshold of the antibiotic resistance gene and the 16S rDNA in the free DNA, and further calculating the absolute concentration and the relative concentration of the free antibiotic resistance gene in the sewage sample.
In the present invention, the copy number concentration range of the antibiotic resistance gene or the 16S rDNA plasmid standard is 102～108copies, number of gradients 6, gradient value 10 times.
The invention provides a quantitative detection method of free-state antibiotic resistance genes in sewage. The method comprises the following steps: pretreating a sample to obtain a filtrate; the sample is sewage and/or sludge mixed liquor; the obtained filtrate and the ammonium acetate aqueous solution are reversed and mixed evenly, then absolute ethyl alcohol is added, reversed and mixed evenly, and the mixture is kept stand; centrifuging, removing supernatant, adding ethanol water solution into the precipitate, and mixing to obtain ethanol/precipitate mixed solution; centrifuging the ethanol/precipitate mixed solution, removing supernatant, and adding sterile enzyme-free water to obtain free DNA; and (3) taking the free DNA as a template, amplifying the antibiotic resistance gene and the 16S rDNA by adopting qPCR, and detecting the concentration of the free antibiotic resistance gene by using an external standard method. The invention has the technical effects that:
the invention defines a quantitative detection method of the free antibiotic resistance genes with low concentration in the sewage plant, combines the technologies of ammonium acetate-ethanol precipitation, qPCR test and the like, overcomes the bottleneck of low concentration of the free antibiotic resistance genes and complex interference factors in the sewage plant, and provides a sensitive and reliable analysis means for the research of the space-time distribution and removal characteristics of the free antibiotic resistance genes of the novel trace pollutants in the sewage plant.
Drawings
Figure 116 qPCR test representative quantitative standard curves for S rDNA, sul1 and sul 2;
FIG. 2 is the absolute concentration of free antibiotic resistance genes in the influent and effluent of seven actual sewage plants;
FIG. 3 is the relative concentrations of free antibiotic resistance genes in the influent and effluent of seven actual sewage plants;
FIG. 4 is the absolute concentration of free antibiotic resistance genes in the water of biochemical ponds in seven actual sewage plants;
FIG. 5 is the relative concentrations of free antibiotic resistance genes in the water of biochemical ponds in seven actual sewage plants.
Detailed Description
The invention discloses a quantitative detection method of free-state antibiotic resistance genes in sewage, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein may be made and equivalents employed without departing from the spirit and scope of the invention.
The invention provides a quantitative detection method of free-state antibiotic resistance genes in a sewage plant, which comprises the following steps:
(1) sample pretreatment
Taking sludge mixed liquid and a sewage sample from an actual sewage plant. And after the sludge mixed liquor is centrifugally separated, the supernatant is reserved. Filtering the supernatant and sewage with a filter membrane, and storing the filtrate in a refrigerator.
(2) Isolation of free DNA
Putting the filtrate obtained in the step (1) into a centrifuge tube A. Ammonium acetate was added and mixed by inversion. Adding precooled absolute ethyl alcohol, reversing and uniformly mixing the mixture again, and standing the mixture on ice;
② after centrifugation, carefully remove the supernatant. Add pre-cooled ethanol to centrifuge tube a. After slight mixing, the ethanol/precipitate mixture was transferred to a new centrifuge tube B;
③ after centrifuging the centrifuge tube B, carefully removing the supernatant. Add the pre-cooled ethanol to centrifuge tube a again. Slightly mixing, and transferring the ethanol/precipitate mixed solution into a centrifuge tube B;
fourthly, centrifuging the centrifuge tube B, and carefully removing the supernatant. Adding a proper volume of sterile enzyme-free water to obtain a concentrated free DNA solution.
(3) Quantitative detection of free antibiotic resistance gene
Firstly, taking free DNA as a template, and adopting real-time fluorescent quantitative polymerase chain reaction (qPCR) to amplify antibiotic resistance genes and 16S rDNA in an actual sewage plant;
and secondly, performing qPCR (quantitative polymerase chain reaction) experiments by taking antibiotic resistance genes and 16S rDNA plasmid standard products as templates. Drawing a quantitative standard curve of the antibiotic resistance gene and the 16S rDNA according to the corresponding relation between the copy number concentration of the plasmid standard substance and the cycle number of the antibiotic resistance gene or the 16S rDNA amplified fluorescence threshold;
remarks explanation: the preparation method of the plasmid standard substance comprises the steps of taking free DNA in sewage as a template and adopting common PCR to amplify target genes; performing agarose gel electrophoresis on a common PCR product, cutting a target gene band under a gel imager, and further purifying by using a gel purification kit; after adjusting the gel-purified gene of interest to an appropriate concentration, it is ligated to a plasmid vector and transformed into competent cells. After selecting the positive clone transformant, extracting a plasmid standard substance by using a plasmid extraction kit, and storing the plasmid standard substance in a refrigerator.
And thirdly, comparing a quantitative standard curve of the antibiotic resistance gene and the 16S rDNA, acquiring the copy number concentration of the antibiotic resistance gene and the 16S rDNA in the free DNA of the template according to the amplification fluorescence threshold of the antibiotic resistance gene and the 16S rDNA in the free DNA, and further calculating the absolute concentration and the relative concentration of the free antibiotic resistance gene in the sewage sample.
Preferably, the sludge mixed liquor in the step (1) is taken from a membrane biological reaction tank or an aerobic tank of an actual sewage plant; sewage is taken from inlet water and outlet water of an actual sewage plant;
preferably, the sewage/sludge mixed liquor in the step (1) is placed in a sterilized brown glass bottle and is transported to a laboratory within 2 hours by using an ice bag;
preferably, the volume of the sludge mixed liquor, the inlet water and the outlet water in the step (1) is 10 mL;
preferably, the pore size of the filter membrane in the step (1) is 0.22 μm;
preferably, the refrigerator temperature in the step (1) is set to-20 ℃;
preferably, the centrifugation conditions in step (1) are as follows: 5000g, 4 ℃ and 5 min.
Preferably, the volume of the filtrate in the steps (2) to (r) is 8 mL;
preferably, the volume of the centrifuge tube A in the steps (2) -phi is 50 mL;
preferably, the concentration of ammonium acetate in the steps (2) - (r) is 7.5M;
preferably, the volume of ammonium acetate in the steps (2) -r is 4 mL;
preferably, the volume of the absolute ethyl alcohol in the steps (2) to (r) is 24 mL;
preferably, the precooling temperature of the absolute ethyl alcohol in the steps (2) to (r) is 4 ℃;
preferably, the ice-bath time in the steps (2) -phi is 1 h;
preferably, the centrifugation conditions in the steps (2) - ② are as follows: 14000g for 30min at 4 ℃;
preferably, the precooling temperature of the ethanol in the steps (2) - ② and (2) - ③ is 4 ℃, and the concentration of the ethanol is 70 percent;
preferably, the volume of the centrifugal tube B in the steps (2) - ② and (2) - ③ is 2 mL;
preferably, the centrifugation conditions in the steps (2) - ③ and (2) -fourthly are as follows: 14000g for 30min at 4 ℃;
preferably, the volume of the sterile and enzyme-free water in the steps (2) to (4) is 80. mu.L.
Preferably, the types of the target antibiotic resistance genes in the steps (3) -r are sul1 and sul 2;
preferably, the primer sequences in the qPCR test in steps (3) -r are:
nucleotide sequence of sul1 upstream primer: CGCACCGGAAACATCGCTGCAC, respectively;
nucleotide sequence of sul1 downstream primer: TGAAGTTCCGCCGCAAGGCTCG, respectively;
nucleotide sequence of sul2 upstream primer: TCCGGTGGAGGCCGGTATCTGG, respectively;
nucleotide sequence of sul2 downstream primer: CGGGAATGCCATCTGCCTTGAG, respectively;
nucleotide sequence of 16s rDNA upstream primer: ACTCCTACGGGAGGCAGCAG, respectively;
nucleotide primer sequence of 16s rDNA downstream primer: ATTACCGCGGCTGCTGG, respectively;
preferably, the qPCR test conditions in steps (3) - ① are uracil-DNA glycosylase treatment at 50 ℃ for 2min and Dual-Lock at 95 ℃TMPerforming hot start for 2min by using Taq DNA polymerase; denaturation at 95 ℃ for 15s, annealing for 15s, and extension at 72 ℃ for 1min for 40 cycles. The annealing temperatures of sul1 and sul2 were 60 ℃ and the annealing temperature of 16S rDNA was 55 ℃;
preferably, the volume of the qPCR test reaction system in the steps (3) -phi is 20 mu L, and the specific composition is as follows:
preferably, the copy number concentration range of the antibiotic resistance gene or the 16S rDNA plasmid standard in the steps (3) - ② is 102～108copies, number of gradients 6, gradient value 10 times.
Preferably, the target genes amplified by the common PCR in the remark specification comprise sul1, sul2 and 16 SrDNA; the Gel purification Kit is TaKaRaMiniBESTAgare Gel DNA Extraction Kit; the plasmid vector is pMD 18-T; the competent cell is Escherichia coli JM 109; the plasmid extraction kit is MiniBESTPlasmodidpurification kit; the refrigerator storage temperature is-20 ℃.
Preferably, the calculation formula of the copy number concentration of the plasmid standard substance in the steps (3) - ② is Cc＝Cm×NA/[(LpMD18-T+Lx)×M0]In the formula CcCopy number concentration of plasmid standards, copies/. mu.L; cmIs the plasmid standard quality concentration, ng/μ L; l ispMD18-TIs the length of pMD 18-T plasmid vector, 2692 bp; l isxFor the length of the inserted target gene, bp; m0The average molecular weight of each base pair is 660g/M, NA is the Avogastron constant, 6.02 × 1023。
Preferably, the primer sequences used for ordinary PCR amplification of the target genes (sul1, sul2 and 16rDNA) in the remark notes refer to step (3).
Preferably, the general PCR reaction conditions in the remark description are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 7 min.
Preferably, the volume of the common PCR reaction system in the remark description is 20 μ L, and the specific composition is as follows:
preferably, the calculation formula of the absolute concentration of the free-state antibiotic resistance gene in the sewage sample in the step (3) -III is as follows: absolute concentration of free antibiotic resistance gene ═ copy number concentration of free antibiotic resistance gene × sample concentration multiple/free DNA recovery rate.
Preferably, the calculation formula of the concentration times of the sample is as follows: sample concentration fold (filtrate volume/sterile enzyme-free water volume), see claim step (2) for specific parameters.
The free DNA recovery rate is calculated by the following formula: free DNA recovery rate-actual mass concentration of plasmid standard/theoretical mass concentration of plasmid standard. Wherein the theoretical mass concentration of the plasmid standard substance is 100 or 1 ng/mu L; the actual mass concentration of the plasmid standard can be determined by substituting the plasmid standard for the filtrate in step (2) of claim 1.
Preferably, the calculation formula of the relative concentration of the free-state antibiotic resistance genes in the sewage sample in the step (3) -III is as follows: the relative concentration of the free-form antibiotic resistance gene is equal to the copy number concentration of the free-form antibiotic resistance gene/16 SrDNA copy number concentration.
The reagent or instrument used in the method for quantitatively detecting the free-state antibiotic resistance gene in the sewage can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 quantitative determination of free antibiotic resistance Gene in Sewage plant in actual Sewage plant
The method comprises the following steps: sample pretreatment
Samples of sewage were taken from the water inlet and outlet points of seven actual sewage plants in Wuxi city, Jiangsu province, and placed in sterilized brown glass bottles and transported to the laboratory within 2h using ice boxes. Filtering with 0.22 μm filter membrane, collecting filtrate, and storing in a refrigerator at-20 deg.C.
Step two: isolation and detection of free DNA
(1) And 8mL of the filtrate obtained in the step one is taken and placed in a 50mL centrifuge tube. 4mL of 7.5M ammonium acetate was added and mixed by inversion. Adding 24mL of pre-cooled anhydrous ethanol at 4 ℃, reversing the mixture again, uniformly mixing the mixture, and standing the mixture on ice for 1 hour;
(2) after centrifugation at 14,000g for 30min at 4 ℃, the supernatant was carefully removed. 1mL of pre-cooled 70% ethanol at 4 ℃ was added to the centrifuge tube. After slight mixing, the ethanol/precipitate mixture was transferred to a new 2mL centrifuge tube;
(3) after centrifugation at 14,000g for 30min at 4 ℃, the supernatant was carefully removed. 1mL of 4 ℃ pre-cooled 70% ethanol was again added to the 50mL centrifuge tube of step (1). After slight mixing, completely transferring the ethanol/precipitate mixed solution into the 2mL centrifuge tube in the step (2);
(4) after centrifugation at 14,000g for 30min at 4 ℃, the supernatant was carefully removed. Adding 80 mu L of sterile enzyme-free water to obtain a concentrated free DNA solution;
step three: quantitative detection of free antibiotic resistance gene
(1) Taking the free DNA in the step two as a template, and amplifying antibiotic resistance genes (sul1 and sul2) and 16S rDNA in an actual sewage plant by adopting a qPCR test;
the primer sequences in the qPCR assay were:
the nucleotide sequence of the upstream primer of sul1 is CGCACCGGAAACATCGCTGCAC;
the nucleotide sequence of the downstream primer of sul1 is TGAAGTTCCGCCGCAAGGCTCG;
the nucleotide sequence of the upstream primer of sul2 is TCCGGTGGAGGCCGGTATCTGG;
the nucleotide sequence of the downstream primer of sul2 is CGGGAATGCCATCTGCCTTGAG;
the nucleotide sequence of the 16S rDNA upstream primer is ACTCCTACGGGAGGCAGCAG;
the nucleotide primer sequence of the 16S rDNA downstream primer is ATTACCGCGGCTGCTGG.
The qPCR test conditions were: treating with uracil-DNA glycosylase at 50 deg.C for 2 min; 95 ℃ Dual-LockTMPerforming hot start for 2min by using Taq DNA polymerase; denaturation at 95 ℃ for 15s, annealing at 15s, and extension at 72 ℃ for 1min for 40 cycles. The annealing temperatures of sul1 and sul2 were 60 ℃ and the annealing temperature of 16S rDNA was 55 ℃.
The volume of the qPCR test reaction system is 20 mu L, and the specific composition is as follows:
TABLE 1 qPCR test reaction System
(2) Antibiotic resistance genes (sul1 and sul2) and 16S rDNA plasmid standards were prepared, and the copy number concentration of the plasmid standards was calculated. Sequentially diluting plasmid standard substance with known copy number by 10 times gradient, and selecting concentration range at 10%8～102Plasmid standards of copies six gradients were subjected to qPCR testing. Drawing a quantitative standard curve of the antibiotic resistance gene and the 16SrDNA according to the corresponding relation between the copy number concentration of the plasmid standard substance and the cycle number of the antibiotic resistance gene or the 16S rDNA amplification fluorescence threshold, wherein the representative quantitative standard curve is shown in figure 1;
remarks explanation:
① the calculation formula of the copy number concentration of the plasmid standard substance is Cc＝Cm×NA/[(LpMD18-T+Lx)×M0]In the formula CcCopy number concentration of plasmid standards, copies/. mu.L; cmIs the plasmid standard quality concentration, ng/μ L; l ispMD18-TIs the length of pMD 18-T plasmid vector, 2692 bp; l isxFor the length of the inserted target gene, bp; m0The average molecular weight of each base pair is 660g/M, NA is the Avogastron constant, 6.02 × 1023。
② the preparation steps of the plasmid standard substance comprise: taking the free DNA in the sewage plant in the step two as a template, and adopting common PCR to amplify target genes (sul1, sul2 and 16 SrDNA); performing agarose Gel electrophoresis on a common PCR product, cutting a target gene band under a Gel imager, and purifying by using a Gel purification Kit (TaKaRaMiniBestagarase Gel DNAextraction Kit); after adjusting the gel-purified gene of interest to an appropriate concentration, it was ligated to a plasmid vector (pMD 18-T) and transformed into competent cells (Escherichia coli JM 109). After positive clone transformants were picked, the Plasmid-upgraded standards were extracted with a Plasmid extraction Kit (MiniBEST Plasmid Purification Kit) and stored in a-20 ℃ freezer.
③ in the preparation of plasmid standard substance, the common PCR reaction conditions are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 7 min.
In the preparation of the plasmid standard substance, 20 mu L of common PCR reaction system is adopted, and the specific composition is as follows:
TABLE 2 general PCR reaction System volume
| Reagent | Volume (μ L) |
| Ex Taq(5U/μL) | 0.1 |
| 10 × Ex Taq Buffer (containing 20mM Mg2+) | 2 |
| dNTP mix (2.5 mM each) | 1.6 |
| Forward primer (10. mu.M) | 0.4 |
| Reverse primer (10. mu.M) | 0.4 |
| DNA template (1 ng/. mu.L) | 1 |
| Sterile enzyme-free water | 14.5 |
| Total up to | 20 |
(3) And (3) comparing the quantitative standard curve of the antibiotic resistance gene and the 16S rDNA, and acquiring the copy number concentration of the antibiotic resistance gene and the 16S rDNA in the free DNA of the template according to the amplification fluorescence threshold of the antibiotic resistance gene and the 16S rDNA in the free DNA. Finally, the absolute concentration of the free antibiotic resistance gene in the sample is calculated according to the formula "the absolute concentration of the free antibiotic resistance gene is the copy number concentration of the free antibiotic resistance gene x the concentration multiple of the sample/the recovery rate of the free DNA" (wherein, the calculation formula of the concentration multiple of the sample is that the concentration multiple of the sample is the volume of filtrate/the volume of sterile enzyme-free water, i.e. 8mL/80 uL is 100 times; and the calculation formula of the recovery rate of the free DNA is that the recovery rate of the free DNA is the actual mass concentration of the plasmid standard/the theoretical mass concentration of the plasmid standard, wherein, the theoretical mass concentration of the plasmid standard is 10 ng/mL; the actual mass concentration of the plasmid standard is obtained according to the step after the filtrate in the second step is replaced by the sul1 plasmid standard. the relative concentration of the free antibiotic resistance gene is the copy number concentration of the free antibiotic resistance gene/16S rDNA according to the formula Copy number concentration "the relative concentration of the free antibiotic resistance gene in the sample was calculated.
The detection results are shown in FIG. 2 and FIG. 3, the absolute concentration mean value ranges of the free sul1 and sul2 in the inlet water of the actual sewage plant are respectively 2.8 × 102～2.3×105copies/mL, undetectable-3.3 × 104The relative concentration mean ranges of free sul1 and sul2 are respectively 0.003-0.519 copies/copies16S rDNA and 0.003-0.100 copies/copies16S rDNA, and the absolute concentration mean ranges of free sul1 and sul2 in the effluent water of an actual sewage plant are respectively 1.3 × 103～9.5×104copies/mL, undetectable-2.8 × 104copies/mL; the relative concentration mean ranges of free sul1 and sul2 are respectively: 0.109-0.619 copies/copies16S rDNA, and no-1.402 copies/copies16S rDNA.
Example 2 quantitative determination of free antibiotic resistance genes in sludge mixtures in actual Sewage plants
The difference between this example and example 1 is that the sample is a sludge mixed solution from a biochemical tank (membrane biological reaction tank or aerobic tank), so the sludge mixed solution is centrifuged (5,000g, 4 ℃, 5min), the supernatant is retained, the supernatant is filtered with a 0.22 μm filter membrane, and the filtrate sample required for quantitative detection of free antibiotic resistance genes is finally obtained, the other operations are the same as example 1, and the absolute concentration mean ranges of free sul1 and sul2 in the sludge mixed solution in the actual sewage plant are 1.2 × 103～1.4×107copies/mL、2.1×102～4.2×107copies/mL; the relative concentration mean ranges of sul1 and sul2 in free state are respectively: 0.043-3.598 copies/copies16S rDNA, 0.039-0.782 copies/copies16S rDNA.
Comparative example 1
The difference between the comparative example and the example 1 is that 16mL of 4 ℃ precooled absolute ethanol is directly added into 8mL of filtrate in the separation step (2) of the free DNA, in addition, sul1 plasmid standard substance is representatively selected to replace the filtrate in the second step of the example 1, other operations are the same as the example 1, and finally, the absolute concentration mean value range of the free sul1 in the sludge mixed liquid in the actual sewage plant is obtained, wherein the absolute concentration mean value range is not detected to be 5.4 × 105copies/mL。
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of southern China
<120> quantitative detection method of free-state antibiotic resistance gene in sewage
<130>MP2002408
<160>6
<170>SIPOSequenceListing 1.0
<210>1
<211>22
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
cgcaccggaa acatcgctgc ac 22
<210>2
<211>22
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tgaagttccg ccgcaaggct cg 22
<210>3
<211>22
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tccggtggag gccggtatct gg 22
<210>4
<211>22
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
cgggaatgcc atctgccttg ag 22
<210>5
<211>20
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
<210>6
<211>17
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
attaccgcgg ctgctgg 17
Claims (10)
1. A method for quantitatively detecting free-state antibiotic resistance genes in sewage is characterized by comprising the following steps:
step 1, pretreating a sample to obtain a filtrate; the sample is sewage and/or sludge mixed liquor;
step 2, separation of free DNA:
step 2-1, reversing and uniformly mixing the obtained filtrate and an ammonium acetate aqueous solution, then adding absolute ethyl alcohol, reversing and uniformly mixing, and standing;
step 2-2, removing supernatant after centrifugation, adding an ethanol aqueous solution into the precipitate, and uniformly mixing to obtain an ethanol/precipitate mixed solution;
step 2-3, centrifuging the ethanol/precipitate mixed solution, removing supernatant, and adding sterile enzyme-free water to obtain free DNA;
and 3, amplifying the antibiotic resistance gene and the 16SrDNA by using qPCR (quantitative polymerase chain reaction) by using the free DNA as a template, and detecting the concentration of the free antibiotic resistance gene by using an external standard method.
2. The quantitative determination method according to claim 1, wherein the pretreatment in step 1 is: centrifuging the sludge mixed liquor, and keeping a supernatant; filtering the supernatant and/or the sewage by a filter membrane to obtain a filtrate; the aperture of the filter membrane is 0.2-0.3 μm.
3. The quantitative determination method of claim 1, wherein in step 2-1, the volume ratio of the filtrate, the ammonium acetate aqueous solution and the absolute ethyl alcohol is (7-9): (3-5): (22-26); the concentration of the ammonium acetate aqueous solution is 7-8M; the absolute ethyl alcohol is precooled absolute ethyl alcohol at the temperature of 0-4 ℃; the standing temperature is 0-4 ℃, and the standing time is 50-70 min.
4. The quantitative determination method according to claim 1, wherein the centrifugation is performed at 0 to 4 ℃ and 13000 to 15000g for 20 to 40 min.
5. The quantitative determination method of claim 1, wherein the concentration of the ethanol aqueous solution is 70 to 80% by volume.
6. The quantitative determination method according to claim 1, wherein the step 2-2 is specifically: centrifuging, removing supernatant, adding ethanol water solution into precipitate, mixing, and transferring the obtained ethanol/precipitate mixed solution from container 1 to container 2; centrifuging the ethanol/precipitate mixture in container 2 and removing the supernatant; adding the ethanol aqueous solution into the container 1 again, uniformly mixing, and transferring the obtained ethanol/precipitate mixed solution from the container 1 to a container 2; the adding amount of the ethanol water solution is 0.6-0.8 mL/time.
7. The quantitative determination method according to claim 1, wherein the volume ratio of the filtrate in step 2-1 to the sterile, enzyme-free water in step 2-3 is 8: (50-100).
8. The quantitative determination method of claim 7, wherein the antibiotic resistance genes are sul1 and sul2, and the primer sequences used for amplification in step 3 are:
the nucleotide sequence of the sul1 upstream primer is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the sul1 downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the sul2 upstream primer is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the sul2 downstream primer is shown as SEQ ID NO: 4 is shown in the specification;
the nucleotide sequence of the 16SrDNA upstream primer is shown as SEQ ID NO: 5 is shown in the specification;
the nucleotide sequence of the 16SrDNA downstream primer is shown as SEQ ID NO: and 6.
9. The quantitative determination method of claim 1, wherein the amplification conditions of qPCR are: treating with uracil-DNA glycosylase at 50 deg.C for 2 min; hot starting at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing for 15s, and extension at 72 ℃ for 1min for 40 cycles; the annealing temperatures of sul1 and sul2 were 60 ℃ and the annealing temperature of 16SrDNA was 55 ℃;
the qPCR reaction system was as follows:
In the preparation of the plasmid standard used in the external standard method, the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 7min at 72 ℃;
the PCR reaction system is as follows:
10. the quantitative determination method according to any one of claims 1 to 9, wherein the external standard method is specifically:
carrying out qPCR (quantitative polymerase chain reaction) experiments by taking antibiotic resistance genes and plasmid standard products of 16SrDNA as templates; according to the corresponding relation between the copy number concentration of the plasmid standard substance and the fluorescence threshold cycle number of the antibiotic resistance gene or 16SrDNA amplification, drawing a quantitative standard curve of the antibiotic resistance gene and the 16 SrDNA;
and (3) comparing the quantitative standard curve of the antibiotic resistance gene and the 16SrDNA, acquiring the copy number concentration of the antibiotic resistance gene and the 16SrDNA in the template free DNA according to the amplification fluorescence threshold values of the antibiotic resistance gene and the 16SrDNA in the free DNA, and further calculating the absolute concentration and the relative concentration of the free antibiotic resistance gene in the sewage sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010214165.4A CN111560446A (en) | 2020-03-24 | 2020-03-24 | Quantitative detection method for free-state antibiotic resistance genes in sewage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010214165.4A CN111560446A (en) | 2020-03-24 | 2020-03-24 | Quantitative detection method for free-state antibiotic resistance genes in sewage |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111560446A true CN111560446A (en) | 2020-08-21 |
Family
ID=72074128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010214165.4A Pending CN111560446A (en) | 2020-03-24 | 2020-03-24 | Quantitative detection method for free-state antibiotic resistance genes in sewage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111560446A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114262715A (en) * | 2021-12-28 | 2022-04-01 | 中国环境科学研究院 | Method for evaluating resistance gene environmental health risk in compost product |
| CN117699958A (en) * | 2024-02-02 | 2024-03-15 | 青岛海湾中水有限公司 | Sewage treatment system and sewage treatment method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705298A (en) * | 2009-11-05 | 2010-05-12 | 中国农业大学 | Quick bacterium examination kit and detection method thereof |
| CN102534002A (en) * | 2012-01-09 | 2012-07-04 | 中山大学 | Method for identifying antibiotic resistance bacterial strains in aquaculture organisms and culture environment |
| CN102876772A (en) * | 2012-06-20 | 2013-01-16 | 山东省海洋水产研究所 | Method for detecting tetracycline resistance gene in seawater |
| CN104694653A (en) * | 2014-11-25 | 2015-06-10 | 南京大学 | Method for removing antibiotics resistance genes (ARGs) in sewage based on disinfecting technology |
| CN107034127A (en) * | 2017-06-15 | 2017-08-11 | 黄捷 | A kind of test tube and method for being used to extract microbial gene and Inorganic chemical substance in soil |
| CN109652512A (en) * | 2019-01-28 | 2019-04-19 | 南京师范大学 | The quantitative detecting method of drug resistant gene in Taihu Lake water body and deposit |
-
2020
- 2020-03-24 CN CN202010214165.4A patent/CN111560446A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705298A (en) * | 2009-11-05 | 2010-05-12 | 中国农业大学 | Quick bacterium examination kit and detection method thereof |
| CN102534002A (en) * | 2012-01-09 | 2012-07-04 | 中山大学 | Method for identifying antibiotic resistance bacterial strains in aquaculture organisms and culture environment |
| CN102876772A (en) * | 2012-06-20 | 2013-01-16 | 山东省海洋水产研究所 | Method for detecting tetracycline resistance gene in seawater |
| CN104694653A (en) * | 2014-11-25 | 2015-06-10 | 南京大学 | Method for removing antibiotics resistance genes (ARGs) in sewage based on disinfecting technology |
| CN107034127A (en) * | 2017-06-15 | 2017-08-11 | 黄捷 | A kind of test tube and method for being used to extract microbial gene and Inorganic chemical substance in soil |
| CN109652512A (en) * | 2019-01-28 | 2019-04-19 | 南京师范大学 | The quantitative detecting method of drug resistant gene in Taihu Lake water body and deposit |
Non-Patent Citations (1)
| Title |
|---|
| 李蕊: "水环境游离DNA表征及其应用探索", 《中国优秀硕士学位论文选文数据库 基础科学辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114262715A (en) * | 2021-12-28 | 2022-04-01 | 中国环境科学研究院 | Method for evaluating resistance gene environmental health risk in compost product |
| 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 |
| CN117699958A (en) * | 2024-02-02 | 2024-03-15 | 青岛海湾中水有限公司 | Sewage treatment system and sewage treatment method |
| CN117699958B (en) * | 2024-02-02 | 2024-04-19 | 青岛海湾中水有限公司 | Sewage treatment system and sewage treatment method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106544443B (en) | Pseudomonas aeruginosa dry efflorescence LAMP (loop-mediated isothermal amplification) rapid detection kit and use method thereof | |
| CN111560446A (en) | Quantitative detection method for free-state antibiotic resistance genes in sewage | |
| CN110684866A (en) | Primer pair, kit and method for detecting chicken infectious anemia virus | |
| CN111549182A (en) | Probe and kit for detecting SARS-CoV-2 | |
| CN101333568B (en) | Quantitative determination process for enterovirus in environment water body | |
| CN106929578A (en) | The evaluation method of planktonic bacteria group in a kind of Taihu Lake water body | |
| CN108004334B (en) | Quadruple fluorescent PCR primer group, probe group, kit and method for detecting four pathogenic bacteria in drinking water | |
| CN106399497B (en) | Pistacia chinensis bunge microsatellite loci, primers and application | |
| CN109355404B (en) | Primer, kit and detection method for isothermal detection of listeria monocytogenes based on polymerase helix reaction | |
| CN116083316A (en) | Alcaligenes faecalis and application thereof in algae removal | |
| CN111073986A (en) | Rapid constant-temperature detection method for salmonella, primer group and application | |
| CN115873930A (en) | Double-target-point drug-resistant salmonella detection method based on RPA-Cas12a | |
| CN114958835A (en) | Combination product and kit for detecting bacterial rice blight bacteria | |
| CN111235146B (en) | Method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge and method for detecting drug-resistant genes carried by sludge | |
| CN110295163B (en) | Reagent and method for extracting rice rhizosphere iron membrane microorganism DNA | |
| CN108823207B (en) | Bn-miR43 of ramie and application thereof | |
| CN112831603A (en) | Kit and method for detecting food-borne pathogenic bacteria based on multiple PCR (polymerase chain reaction) technology | |
| CN101724703B (en) | Oxya specific molecular marker DNA sequence and application thereof | |
| CN110923340A (en) | Method for detecting anammox bacteria | |
| CN112063759A (en) | RT-LAMP primer, kit and detection method for simultaneously detecting multiple viruses of banana | |
| CN113337637A (en) | Primer group and kit for molecular detection of SARS-CoV-2 coronavirus | |
| CN111378724A (en) | RNA amplification detection method and detection kit | |
| CN113355330B (en) | ssDNA aptamer for specifically recognizing Weissella viridescens and screening method and application thereof | |
| Eschweiler et al. | DNA-analysis to study the microbial diversity in recharged groundwater | |
| CN106755423B (en) | Specific sequence-based escherichia coli and shigella detection primer, kit and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200821 |