CN117417070A - Device for removing antibiotic resistance genes in aquaculture water area and rapid detection method - Google Patents
Device for removing antibiotic resistance genes in aquaculture water area and rapid detection method Download PDFInfo
- Publication number
- CN117417070A CN117417070A CN202311365924.7A CN202311365924A CN117417070A CN 117417070 A CN117417070 A CN 117417070A CN 202311365924 A CN202311365924 A CN 202311365924A CN 117417070 A CN117417070 A CN 117417070A
- Authority
- CN
- China
- Prior art keywords
- antibiotic resistance
- primer
- water
- ultraviolet
- tube
- 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 62
- 230000003115 biocidal effect Effects 0.000 title claims abstract description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 51
- 238000009360 aquaculture Methods 0.000 title claims abstract description 16
- 244000144974 aquaculture Species 0.000 title claims abstract description 16
- 238000001514 detection method Methods 0.000 title abstract description 21
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 13
- 239000000523 sample Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 18
- 239000002028 Biomass Substances 0.000 claims description 17
- 239000012528 membrane Substances 0.000 claims description 12
- 101100223892 Escherichia coli sulI gene Proteins 0.000 claims description 10
- 101000767160 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Intracellular protein transport protein USO1 Proteins 0.000 claims description 10
- 239000003610 charcoal Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 9
- 230000007246 mechanism Effects 0.000 claims description 9
- 238000011330 nucleic acid test Methods 0.000 claims description 9
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 239000002131 composite material Substances 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 150000004714 phosphonium salts Chemical group 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 7
- 108010090804 Streptavidin Proteins 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000011901 isothermal amplification Methods 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 239000002351 wastewater Substances 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- 238000000975 co-precipitation Methods 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 239000013024 dilution buffer Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 3
- 238000005342 ion exchange Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 239000011535 reaction buffer Substances 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 3
- 239000010902 straw Substances 0.000 claims description 3
- 108091036078 conserved sequence Proteins 0.000 claims description 2
- 230000005389 magnetism Effects 0.000 claims description 2
- 239000013589 supplement Substances 0.000 claims description 2
- 241001529936 Murinae Species 0.000 claims 1
- 240000008042 Zea mays Species 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000209149 Zea Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 101100367123 Caenorhabditis elegans sul-1 gene Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091036333 Rapid DNA Proteins 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F9/00—Multistage treatment of water, waste water or sewage
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/28—Treatment of water, waste water, or sewage by sorption
- C02F1/283—Treatment of water, waste water, or sewage by sorption using coal, charred products, or inorganic mixtures containing them
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/30—Treatment of water, waste water, or sewage by irradiation
- C02F1/32—Treatment of water, waste water, or sewage by irradiation with ultraviolet light
- C02F1/325—Irradiation devices or lamp constructions
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/48—Treatment of water, waste water, or sewage with magnetic or electric fields
- C02F1/484—Treatment of water, waste water, or sewage with magnetic or electric fields using electromagnets
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Environmental & Geological Engineering (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Water Supply & Treatment (AREA)
- Hydrology & Water Resources (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (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 discloses an antibiotic resistance gene removing device and a rapid detection method in aquaculture water, and relates to the technical field of environmental microorganism pollution treatment and detection.
Description
Technical Field
The invention relates to the technical field of environmental microorganism pollution treatment and detection, in particular to an antibiotic resistance gene removal device and a rapid detection method in a cultivation water area.
Background
Since the 20 th century, a large number of antibiotics including penicillin, streptomycin, etc. have been discovered successively, whereby humans have opened the antibiotic era, gradually taking up absolute advantage in combating bacteria. However, with an excessive dependence on the use of antibiotics, antibiotic resistance genes (Antibiotics resistance genes, ARGs) are constantly present and accumulate in large amounts in environmental microorganisms, resulting in an increasing problem of microbial resistance. The horizontal and vertical transfer of ARGs between microorganisms makes the power of the primary antibiotic drugs diminished, even ineffective. The antibiotic resistance gene is used as a new environmental pollutant, affects biosafety and seriously threatens human health.
Due to the wide use of antibiotics in aquaculture industry, microorganisms in aquaculture water widely generate antibiotic resistance, and the antibiotic resistance genes enter fishes through body surface contact, fish gill respiration, food chains and other modes, so that the food safety and biosafety of human beings are threatened. Furthermore, the antibiotic resistance gene may be transmitted and amplified by means of vertical and horizontal gene transfer. More seriously, public health risks are greatly exacerbated if these resistance genes are transferred into harmful bacteria of humans, animals and plants. Therefore, the removal of antibiotic resistance genes in aquaculture waters is an important measure for protecting human health and ecological safety.
At present, many methods for removing antibiotic resistance genes in water are available, but all are experimental stages of scientific research. In addition, for antibiotic resistance gene detection, the current mainstream method is a real-time fluorescence quantitative PCR method, and has higher requirements on equipment, personnel and sites, and professional personnel are required to carry out the detection under laboratory conditions, so that the method is simple to operate, has obvious effect on the evaluation of the removal effect of the antibiotic resistance gene in a culture water area, and the method of combining isothermal amplification with a lateral flow chromatography test strip is a rapid, simple and easy-to-operate gene detection method.
Disclosure of Invention
The invention aims to provide a device for removing antibiotic resistance genes in aquaculture water and a rapid detection method, so as to solve the problems in the prior art in the background art.
In order to achieve the above purpose, the invention provides a device for removing antibiotic resistance genes in aquaculture water and a rapid detection method.
The antibiotic resistance gene removing device comprises a water inlet pipe, a filter pipe mechanism, an electromagnetic device, a connecting pipe, an ultraviolet irradiation pipe and a water outlet pipe which are sequentially connected from top to bottom;
the filter pipe mechanism, the electromagnetic device and the ultraviolet irradiation pipe are all used for removing antibiotic resistance genes in the cultivation wastewater.
Preferably, the filter pipe mechanism comprises a filter pipe and a magnetic biomass charcoal filter screen which is arranged in the filter pipe and is used for filtering and is magnetic in an electrified state;
the electromagnetic device comprises an electromagnetic generator, an electromagnetic layer arranged in the electromagnetic generator, and a direct current power line arranged at two ends of the electromagnetic generator and electrically connected with the electromagnetic generator;
the ultraviolet irradiation tube comprises a transparent glass window arranged in the ultraviolet lamp device and an irradiation tube arranged at the bottom of the transparent glass window;
the ultraviolet lamp device comprises an ultraviolet generator and an ultraviolet lamp arranged at the front end of the ultraviolet generator.
Preferably, the adsorption material used for the magnetic biomass charcoal filter screen is a magnetic biomass charcoal-quaternary phosphonium salt composite material, the input amount of the adsorption material is 10-200mg/L, the magnetic biomass charcoal-quaternary phosphonium salt composite material is prepared by taking corn straw biomass charcoal as a porous carrier and then adopting a coprecipitation method-ion exchange method, and the magnetic biomass charcoal-quaternary phosphonium salt composite material can effectively degrade antibiotic resistance genes in the environment.
Preferably, the ultraviolet irradiation tube is a pipeline type ultraviolet disinfection module or an open channel ultraviolet disinfection module, and the ultraviolet irradiation dose is 5-160mJ/cm 2 The antibiotic resistance gene in the environment can be effectively degraded under ultraviolet irradiation.
The invention also discloses an application of the device for removing the antibiotic resistance genes in the aquaculture water, which can be used for removing the antibiotic resistance genes sul1 and int1 genes in the aquaculture water.
The invention also discloses a method for detecting the gene removal effect of the antibiotic resistance gene removal device in the cultivation water area, which comprises the following steps:
the first step: according to the antibiotic resistance genes sul1 and int1 gene sequences in the CARD database, isothermal amplification (RPA) primer design is carried out on a target gene conserved sequence by using MEGA5.0 software, and the primer is modified, so that the primer can be suitable for double-target nucleic acid test paper;
and a second step of: taking 500mL of water sample from the water inlet pipe and the water outlet pipe respectively, and then passing through a 0.22 mu M filter membrane;
and a third step of: the method comprises the steps of rapidly extracting nucleic acid, shearing a filter membrane with a water sample filtered, placing the filter membrane into a 2mL centrifuge tube, adding 300mL of buffer A, fully and uniformly mixing the filter membrane with the buffer A by using a handheld electric grinder for 30 seconds, adding 300mL of buffer B, and repeatedly blowing for 20 times by using a liquid transfer device; the mixed solution can be used for the subsequent RPA reaction;
fourth step: amplifying RPA at constant temperature, adding 25 mu L of reaction buffer solution into a recombinant enzyme freeze-dried ball reaction tube, adding 2 mu L of upstream primer and 2 mu L of downstream primer, adding 4 mu L of template, and adding water to supplement to 50 mu L;
fifth step: placing the reaction system in the fifth step into a thermostat or a water bath, and incubating for 20 minutes at 39 ℃;
sixth step: taking 4 mu L of reaction product, taking 36 mu L of dilution buffer matched with RPA, uniformly mixing, adding all the mixture into a sample adding hole of the double-target nucleic acid test paper, waiting for 3-8 minutes, and observing the result.
Preferably, the upstream primer of RPA of sul1 is F1, the downstream primer R1, and the probe is probe1, the sequence is as follows;
F1:5’-TCTGAATCTCACCGAGGACTCCTTCTTCGAT-3’；
R1:5’-GTCTAAGAGCGGCGCAATACGTCTGATCTCA-3’；
Probe1:
5’-GATCGAAATGCTGCGAGTCGGATCAGACGTCGTGGATGTCGGACCGGCCGCCA-3’；
the RPA upstream primer of the int1 is F2, the downstream primer is R2, the probe is probe2, and the sequence is as follows:
F2:5’-CTGTCGCGTGCACGGGCATGGTGGCTGAAG-3’；
R2:5’-TCTACGGCACGTTTGAAGGCGCGCTGAAAGGTCT-3’；
Probe2:
5’-GCATTCCTGGCCGTGGTTCTGGGTTTTTGCGCAGCACACGCATTCGACCGATCC-3’；
preferably, the 5' end of the primer R1 is modified and connected with streptavidin bio, the 5' end of the primer R2 is modified and connected with DIG antibody, FAM fluorescent labels are added to the 5' ends of the probes Probe1 and Probe2, C3spacer is added to the 3' end of the Probe, and 1 base is replaced by dSpacer at a position 30-35bp away from the 5' end of the Probe, specifically as follows:
R1:5’-Bio-GTCTAAGAGCGGCGCAATACGTCTGATCTCA-3’；
R2:5’-DIG-TCTACGGCACGTTTGAAGGCGCGCTGAAAGGTCT-3’
Probe1:5’-FAM-GATCGAAATGCTGCGAGTCGGATCAGACGTCG(dSpacer)
GGATGTCGGACCGGCCGCCA-C3spacer-3’
Probe2:5’-FAM-GCATTCCTGGCCGTGGTTCTGGGTTTTTGC(dSpacer)
GCAGCACACGCATTCGACCGATCC-C3spacer-3’
preferably, the detection line T1 of the double-target nucleic acid test paper is coated with an anti-DIG antibody, the sample binding pad is provided with anti-FITC mouse monoclonal antibody coupled colloidal gold, and the detection line T2 is coated with streptavidin.
The invention has the following beneficial effects:
1. the low-cost recyclable adsorption material adopted by the invention can effectively remove the antibiotic resistance genes in the cultivation wastewater, so that the industrialized removal of the antibiotic resistance genes is possible, and the low-cost method has advantages when being popularized, and can have important significance for the pollution control of the antibiotic resistance genes in cultivation water areas.
2. According to the invention, the antibiotic resistance gene indicator genes sul1 and int1 are arranged, so that the abundance of the two target genes in the culture water area is high, and the two target genes can be used as indicator genes to more effectively indicate the removal condition of the antibiotic resistance genes.
3. The isothermal amplification RPA combined double-target nucleic acid lateral flow chromatography test strip detection technology does not need professional gene amplification equipment, so that the on-site detection and the self-detection of a terminal farmer are possible.
4. The rapid DNA extraction buffers A and B tested were matched to complete DNA extraction within 1 minute. And can well carry out subsequent LAMP reaction.
Drawings
FIG. 1 is a front view of an antibiotic resistance gene removal apparatus.
FIG. 2 is a cross-sectional view of an antibiotic resistance gene removal apparatus.
FIG. 3 is a schematic cross-sectional view of an ultraviolet irradiation tube of the antibiotic resistance gene removal apparatus.
FIG. 4 is a top view of an ultraviolet irradiation tube of the antibiotic resistance gene removal apparatus.
FIG. 5 is a schematic diagram of the detection of a dual-target antibiotic resistance gene.
Fig. 6 is a graph of the detection result.
In the figure: 1. the device comprises a water inlet pipe, a filter pipe mechanism, a 201 filter pipe, a 202 magnetic biomass charcoal filter screen, 3, an electromagnetic layer 302 of an electromagnetic device 301, an electromagnetic generator 303, a direct current power line 4, an ultraviolet irradiation pipe 401, a transparent glass window, a 402 irradiation pipe 5, an ultraviolet lamp device, 501, an ultraviolet generator, 502, an ultraviolet lamp, 6, a water outlet pipe, 7 and a connecting pipe.
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.
Referring to fig. 1-5, the present invention provides a technical solution: an antibiotic resistance gene removing device comprises a water inlet pipe 1, a filter pipe mechanism 2, an electromagnetic device 3, a connecting pipe 7, an ultraviolet irradiation pipe 5 and a water outlet pipe 6 which are sequentially connected from top to bottom;
the filter pipe mechanism 2, the electromagnetic device 3 and the ultraviolet irradiation pipe 5 are all used for removing antibiotic resistance genes in the cultivation wastewater.
As an embodiment of the present invention, the filter tube mechanism 2 includes a filter tube 201 and a magnetic biomass charcoal filter screen 202 disposed in the filter tube 201 for filtering and having magnetism in an energized state;
the electromagnetic device 3 comprises an electromagnetic generator 302, an electromagnetic layer 301 arranged in the electromagnetic generator 302, and a direct current power line 303 arranged at two ends of the electromagnetic generator 302 and electrically connected with the electromagnetic generator 302;
the ultraviolet irradiation tube 4 comprises a transparent glass window 401 arranged in the ultraviolet lamp device 5 and an irradiation tube 402 arranged at the bottom of the transparent glass window 401;
the ultraviolet lamp device 5 includes an ultraviolet generator 501 and an ultraviolet lamp 502 disposed at the front end of the ultraviolet generator 501.
As an embodiment of the invention, the adsorption material used by the magnetic biomass charcoal filter screen 202 is a magnetic biomass charcoal-quaternary phosphonium salt composite material, the input amount of the adsorption material is 10-200mg/L, and the magnetic biomass charcoal-quaternary phosphonium salt composite material is prepared by taking corn straw biomass charcoal as a porous carrier and then adopting a coprecipitation method-ion exchange method.
As an embodiment of the invention, the ultraviolet irradiation tube (4) is a pipeline type ultraviolet disinfection module or an open channel ultraviolet disinfection module, and the ultraviolet irradiation dose is 5-160mJ/cm 2 。
The invention also discloses an application of the device for removing the antibiotic resistance genes in the aquaculture water, which can be used for removing the antibiotic resistance genes sul1 and int1 genes in the aquaculture water.
As an embodiment of the present invention, a method for detecting the gene removal effect of an antibiotic resistance gene removal device in a cultivation water area, comprising the steps of:
the first step: selecting an Antibiotic Resistance Gene (ARG) indicator gene and a reference gene in the aquaculture water;
and a second step of: according to the existing indicator gene sequence and reference gene in the CARD database, performing conservative sequence comparison on the indicator gene sequence by using MEGA5.0 software, finding out the conservative sequence, then performing recombinase-mediated isothermal amplification (RPA) primer design, and modifying the primer to enable the primer to be suitable for double-target nucleic acid test paper;
and a third step of: taking 500mL of water sample from the water inlet pipe and the water outlet pipe respectively, and then passing through a 0.22 mu M filter membrane;
fourth step: the method comprises the steps of rapidly extracting nucleic acid, shearing a filter membrane with a water sample filtered, placing the filter membrane into a 2mL centrifuge tube, adding 300mL of buffer A, fully and uniformly mixing the filter membrane with the buffer A by using a handheld electric grinder for 30 seconds, adding 300mL of buffer B, and repeatedly blowing for 20 times by using a liquid transfer device; the mixed solution can be used for the subsequent RPA reaction;
fifth step: according to the RPA kit instruction, adding 25 mu L of reaction buffer solution, 2 mu L of upstream primer, 2 mu L of downstream primer, 4 mu L of template and the balance of water into a recombinant enzyme freeze-dried ball reaction tube, and supplementing to 50 mu L;
sixth step: placing the reaction system of the fifth step in a thermostat or a water bath for 30 minutes at 35 ℃; seventh step: taking 4 mu L of reaction product, taking 36 mu L of dilution buffer matched with RPA, uniformly mixing, adding all the mixture into a sample adding hole of the double-target nucleic acid test paper, waiting for 3-8 minutes, and observing the result.
An Antibiotic Resistance Gene (ARG) indicator gene is sul1, and the reference gene is int1; the upstream primer and the downstream primer of RPA of sul1 are F1 and R1, and the probe is probe1, and the sequence is as follows;
F1:5’-TCTGAATCTCACCGAGGACTCCTTCTTCGAT-3’；
R1:5’-GTCTAAGAGCGGCGCAATACGTCTGATCTCA-3’；
Probe1:
5’-GATCGAAATGCTGCGAGTCGGATCAGACGTCGTGGATGTCGGACCGGCCGCCA-3’；
the RPA upstream primer of the int1 is F2, the downstream primer is R2, the probe is probe2, and the sequence is as follows:
F2:5’-CTGTCGCGTGCACGGGCATGGTGGCTGAAG-3’；
R2:5’-TCTACGGCACGTTTGAAGGCGCGCTGAAAGGTCT-3’；
Probe2:
5’-GCATTCCTGGCCGTGGTTCTGGGTTTTTGCGCAGCACACGCATTCGACCGATCC-3’；
the primer R1 is modified and connected with streptavidin bio at the 5' end, the primer R2 is modified and connected with a TAMRA antibody at the 5' end, FAM fluorescent markers are added at the 5' ends of the probes Probe1 and Probe2, C3spacer is added at the 3' end of the Probe, and dSpacer is used for replacing 1 base at a position 30-35bp away from the 5' end of the Probe, specifically as follows:
R1:5’-Bio-GTCTAAGAGCGGCGCAATACGTCTGATCTCA-3’；
R2:5’-TAMRA-TCTACGGCACGTTTGAAGGCGCGCTGAAAGGTCT-3’
Probe1:5’-FAM-GATCGAAATGCTGCGAGTCGGATCAGACGTCG(dSpacer)
GGATGTCGGACCGGCCGCCA-C3spacer-3’
Probe2:5’-FAM-GCATTCCTGGCCGTGGTTCTGGGTTTTTGC(dSpacer)
GCAGCACACGCATTCGACCGATCC-C3spacer-3’
the detection line T1 of the double-target nucleic acid test paper of the rapid detection method of the antibiotic resistance gene is coated with an anti-DIG antibody, the sample binding pad is provided with anti-FITC mouse monoclonal antibody coupling colloidal gold, and the detection line T2 is coated with streptavidin. The processed data and results are shown in fig. 6.
TABLE 1 Sul1 genes and intI Gene target sequences
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. The device for removing the antibiotic resistance gene in the cultivation water area is characterized by comprising a water inlet pipe (1), a filter pipe mechanism (2), an electromagnetic device (3), a connecting pipe (7), an ultraviolet irradiation pipe (5) and a water outlet pipe (6) which are sequentially connected from top to bottom;
the filter tube mechanism (2), the electromagnetic device (3) and the ultraviolet irradiation tube (5) are all used for removing antibiotic resistance genes in the cultivation wastewater.
2. An apparatus for removing antibiotic resistance gene in a cultivation water area according to claim 1, wherein said filter tube means (2) comprises a filter tube (201) and a magnetic biomass charcoal filter screen (202) provided in the filter tube (201) for filtration and having magnetism in an energized state;
the electromagnetic device (3) comprises an electromagnetic generator (302), an electromagnetic layer (301) arranged in the electromagnetic generator (302) and a direct current power line (303) arranged at two ends of the electromagnetic generator (302) and electrically connected with the electromagnetic generator (302);
the ultraviolet irradiation tube (4) comprises a transparent glass window (401) arranged in the ultraviolet lamp device (5) and an irradiation tube (402) arranged at the bottom of the transparent glass window (401);
the ultraviolet lamp device (5) comprises an ultraviolet generator (501) and an ultraviolet lamp (502) arranged at the front end of the ultraviolet generator (501).
3. The device for removing antibiotic resistance genes in a cultivation water area according to claim 2, wherein the adsorption material used by the magnetic biomass charcoal filter screen (202) is a magnetic biomass charcoal-quaternary phosphonium salt composite material, the input amount of the adsorption material is 10-200mg/L, and the magnetic biomass charcoal-quaternary phosphonium salt composite material is prepared by taking corn straw biomass charcoal as a porous carrier and then adopting a coprecipitation method-ion exchange method.
4. An apparatus for removing antibiotic resistance gene in a cultivation water area according to claim 3, wherein the ultraviolet irradiation tube (4) is a pipe type ultraviolet disinfection module or an open channel ultraviolet disinfection module, and the ultraviolet irradiation dose is 5-160mJ/cm 2 。
5. Use of an antibiotic resistance gene removal device in a aquaculture water according to claim 1, characterized in that it can be used for the removal of the antibiotic resistance genes sul1 and int1 genes in aquaculture water.
6. A method for detecting the gene-removal effect of the antibiotic resistance gene-removal apparatus in a aquaculture water according to claim 1, comprising the steps of:
the first step: according to the antibiotic resistance genes sul1 and int1 gene sequences in the CARD database, isothermal amplification primer design is carried out on a target gene conserved sequence by using MEGA5.0 software, and the primer is modified, so that the primer can be suitable for double-target nucleic acid test paper;
and a second step of: taking 500mL of water sample from the water inlet pipe and the water outlet pipe respectively, and then passing through a 0.22 mu M filter membrane;
and a third step of: the method comprises the steps of rapidly extracting nucleic acid, shearing a filter membrane with a water sample filtered, placing the filter membrane into a 2mL centrifuge tube, adding 300mL of buffer A, fully and uniformly mixing the filter membrane with the buffer A by using a handheld electric grinder for 30 seconds, adding 300mL of buffer B, and repeatedly blowing for 20 times by using a liquid transfer device; the mixed solution can be used for the subsequent RPA reaction;
fourth step: amplifying RPA at constant temperature, adding 25 mu L of reaction buffer solution into a recombinant enzyme freeze-dried ball reaction tube, adding 2 mu L of upstream primer and 2 mu L of downstream primer, adding 4 mu L of template, and adding water to supplement to 50 mu L;
fifth step: placing the reaction system in the fifth step into a thermostat or a water bath, and incubating for 20 minutes at 39 ℃;
sixth step: taking 4 mu L of reaction product, taking 36 mu L of dilution buffer matched with RPA, uniformly mixing, adding all the mixture into a sample adding hole of the double-target nucleic acid test paper, waiting for 3-8 minutes, and observing the result.
7. The method according to claim 6, wherein the upstream primer of RPA of the antibiotic resistance gene sul1 is F1, the downstream primer is R1, and the sequences of the probe1d are as follows;
F1:5’-TCTGAATCTCACCGAGGACTCCTTCTTCGAT-3’；
R1:5’-GTCTAAGAGCGGCGCAATACGTCTGATCTCA-3’；
Probe1:
5’-GATCGAAATGCTGCGAGTCGGATCAGACGTCGTGGATGTCGGACCGGC CGCCA-3’；
the upstream primer of RPA of the antibiotic resistance gene int1 is F2, the downstream primer is R2, and the sequences of the probe2 are respectively as follows:
F2:5’-CTGTCGCGTGCACGGGCATGGTGGCTGAAG-3’；
R2:5’-TCTACGGCACGTTTGAAGGCGCGCTGAAAGGTCT-3’；
Probe2:
5’-GCATTCCTGGCCGTGGTTCTGGGTTTTTGCGCAGCACACGCATTCGACC GATCC-3’。
8. the method according to claim 7, wherein the 5' -end modification of the primer R1 is linked with streptavidin bio, the 5' -end modification of the primer R2 is linked with DIG antibody, FAM fluorescent labels are added to the 5' -ends of the probes Probe1 and Probe2, C3spacer is added to the 3' -end of the Probe, and 1 base is replaced by dSpacer at a position 30-35bp away from the 5' -end of the Probe, specifically as follows:
R1:5’-Bio-GTCTAAGAGCGGCGCAATACGTCTGATCTCA-3’；
R2:5’-DIG-TCTACGGCACGTTTGAAGGCGCGCTGAAAGGTCT-3’
Probe1:5’-FAM-GATCGAAATGCTGCGAGTCGGATCAGACGTCG(dSpacer)GGATGTCGGACCGGCCGCCA-C3spacer-3’
Probe2:5’-FAM-GCATTCCTGGCCGTGGTTCTGGGTTTTTGC(dSpacer)
GCAGCACACGCATTCGACCGATCC-C3spacer-3’。
9. the method of claim 6, wherein the test line T1 of the dual-target nucleic acid test paper is coated with an anti-DIG antibody, the sample binding pad is provided with anti-FITC murine monoclonal antibody coupled colloidal gold, and the test line T2 is coated with streptavidin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311365924.7A CN117417070A (en) | 2023-10-20 | 2023-10-20 | Device for removing antibiotic resistance genes in aquaculture water area and rapid detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311365924.7A CN117417070A (en) | 2023-10-20 | 2023-10-20 | Device for removing antibiotic resistance genes in aquaculture water area and rapid detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117417070A true CN117417070A (en) | 2024-01-19 |
Family
ID=89531940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311365924.7A Pending CN117417070A (en) | 2023-10-20 | 2023-10-20 | Device for removing antibiotic resistance genes in aquaculture water area and rapid detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117417070A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009291668A (en) * | 2008-06-02 | 2009-12-17 | Hiroshima Univ | Water area environment improving material and its use |
| CN107064040A (en) * | 2017-06-23 | 2017-08-18 | 江苏省环境科学研究院 | The efficiently concentrating and separation method of traces of antibiotic in water environment |
| CN110226604A (en) * | 2019-07-05 | 2019-09-13 | 中国科学院南京土壤研究所 | A kind of magnetic green substance charcoal-quaternary alkylphosphonium salt sterilization material, method of preparation and use |
| CN110526529A (en) * | 2019-10-11 | 2019-12-03 | 南京大学 | The advanced purification process of ARGs in a kind of removal sewage |
| CN214734668U (en) * | 2021-04-14 | 2021-11-16 | 江苏省农业科学院 | But reuse sewage antibiotic resistance gene's remove device |
| CN116479094A (en) * | 2023-03-10 | 2023-07-25 | 天津科技大学 | Method for simultaneously detecting drug-resistant bacteria by double targets based on Pfago combined LAMP amplification one-tube method and application |
-
2023
- 2023-10-20 CN CN202311365924.7A patent/CN117417070A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009291668A (en) * | 2008-06-02 | 2009-12-17 | Hiroshima Univ | Water area environment improving material and its use |
| CN107064040A (en) * | 2017-06-23 | 2017-08-18 | 江苏省环境科学研究院 | The efficiently concentrating and separation method of traces of antibiotic in water environment |
| CN110226604A (en) * | 2019-07-05 | 2019-09-13 | 中国科学院南京土壤研究所 | A kind of magnetic green substance charcoal-quaternary alkylphosphonium salt sterilization material, method of preparation and use |
| CN110526529A (en) * | 2019-10-11 | 2019-12-03 | 南京大学 | The advanced purification process of ARGs in a kind of removal sewage |
| CN214734668U (en) * | 2021-04-14 | 2021-11-16 | 江苏省农业科学院 | But reuse sewage antibiotic resistance gene's remove device |
| CN116479094A (en) * | 2023-03-10 | 2023-07-25 | 天津科技大学 | Method for simultaneously detecting drug-resistant bacteria by double targets based on Pfago combined LAMP amplification one-tube method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ica et al. | Characterization of mono-and mixed-culture Campylobacter jejuni biofilms | |
| Zhang et al. | Rapid cultivation of free-living planktonic anammox cells | |
| CN103993007B (en) | The simple and easy method of a kind of high efficiency extraction DNA from pedotheque | |
| CN1957089B (en) | Measuring contamination | |
| CN102181572B (en) | Mycobacterium tuberculosis nucleic acid detection kit by utilizing RNA isothermal amplification | |
| Donde et al. | Understanding wastewater treatment mechanisms: a review on detection, removal, and purification efficiencies of faecal bacteria indicators across constructed wetlands | |
| CN106434921B (en) | A kind of microbial sources tracking molecular marked compound and its high-flux detection method detected for a variety of fecal pollution sources | |
| CN103468806B (en) | The method for quick of scallop pathogenic Vibrio splindidus Vibrio splendidus | |
| CN111088380A (en) | Brucella LF-RPA detection primer, probe and detection kit | |
| CN108546779A (en) | RPA- Sidestream chromatographies detection primer, probe and the kit of bovine rota | |
| Wu et al. | Antibiotic resistance genes in Chishui River, a tributary of the Yangtze River, China: Occurrence, seasonal variation and its relationships with antibiotics, heavy metals and microbial communities | |
| CN107502678A (en) | A kind of method and device for detecting blue algae producing microcystic toxins | |
| CN117417070A (en) | Device for removing antibiotic resistance genes in aquaculture water area and rapid detection method | |
| CN105018343B (en) | Ortho states microorganism oil gas and the automation sample processing device and Automation workstation of hydrate exploration technology | |
| Li et al. | Comparing diversity patterns and processes of microbial community assembly in water column and sediment in Lake Wuchang, China | |
| CN116203094A (en) | Electrochemical staged detection method for single base mutant of multiple helicobacter pylori | |
| CN107488718A (en) | Detect C60Nanoparticles generate the method and device influenceed to Microcystin | |
| CN108588183A (en) | A kind of detection reaction system of calcein visualization LAMP detection Klebsiella Pneumoniaes | |
| Chou et al. | Anaerobic ammonium oxidation improvement via a novel capsule bioreactor | |
| Wen et al. | Comparative Study of Two Methods of Enteric Virus Detection and Enteric Virus Relationship with Bacterial Indicator in Poyang Lake, Jiangxi, China | |
| CN203117162U (en) | Integrated membrane biosensor for quickly detecting escherichia coli | |
| Hamner et al. | Unexpected prevalence of eae-positive Escherichia coli in the Animas River, Durango, Colorado | |
| CN106591462B (en) | Primer, kit and identification method for identifying genetic relationship between Yangtze river finless porpoise individuals | |
| CN104862413A (en) | Fluorescence detection primer of A type and B type wolbachia as well as detection method and detection kit thereof | |
| CN110616279A (en) | Kit for synchronously and quantitatively detecting 3 important shrimp pathogens |
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 |