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
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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
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<213> Artificial Sequence (Artificial Sequence)
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cgcaccggaa acatcgctgc ac 22
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<213> Artificial Sequence (Artificial Sequence)
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tgaagttccg ccgcaaggct cg 22
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<213> Artificial Sequence (Artificial Sequence)
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tccggtggag gccggtatct gg 22
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cgggaatgcc atctgccttg ag 22
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<210>6
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<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
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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.
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