CN111235146B - Method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge and method for detecting drug-resistant genes carried by sludge - Google Patents
Method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge and method for detecting drug-resistant genes carried by sludge Download PDFInfo
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Abstract
The invention relates to the technical field of sewage treatment, in particular to a method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge and a method for detecting intracellular and extracellular drug resistance genes in sludge. The method provided by the invention combines an ion exchange resin method and a sodium dodecyl benzene sulfonate-high salt method to extract extracellular and intracellular DNA of sludge, and the intracellular and extracellular DNA extracted by the method has no obvious cross contamination. The specific primer pair is adopted for extracting and amplifying products, so that the intracellular and extracellular drug resistance genes in the sludge can be successfully distinguished, the space-time distribution characteristics of the drug resistance genes with different forms in the sludge are expected to be explored, and a basis is provided for developing corresponding drug resistance gene risk assessment schemes and elimination means in the future.
Description
Technical Field
The invention relates to the technical field of sewage treatment, in particular to a method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge and a method for detecting drug-resistant genes carried by the same.
Background
The world health organization and the organizations such as the U.S. disease and control center have consistently listed bacterial "drug resistance" as one of the greatest threats to human health in the twenty-first century. The united nations environmental agency lists "drug resistance" as one of six emerging environmental problems worldwide. The drug resistance gene is the root cause of the acquired drug resistance of the bacteria, so the accurate quantification of the drug resistance gene is important for developing a drug resistance risk assessment scheme and a drug resistance elimination means. Among the numerous environmental media, sewage treatment systems have proven to be one of the important repositories for drug resistance genes.
In a sewage treatment system, the drug resistance genes in the sludge can be divided into intracellular drug resistance genes and extracellular drug resistance genes according to physical forms. The drug resistance genes have different forms and different environmental behavior characteristics. However, the current researches on the distribution rule and the growth and decline characteristics of drug-resistant genes in sewage treatment systems almost consider the sludge as a whole, and do not distinguish the forms. The research of the drug-resistant genes with different forms mainly relates to the extraction of DNA with different forms in the early stage and the quantitative detection of the drug-resistant genes with different forms in the later stage. The quantitative detection means of the drug-resistant gene is relatively mature and uniform, but the extraction of complete and non-cross-contamination DNA with different forms is still a very challenging task so far. Considering different propagation modes, propagation modes and elimination difficulties of intracellular and extracellular drug resistance genes in sludge, a quantitative detection method of drug resistance genes with different forms needs to be developed.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for separating intracellular and extracellular DNAs in sludge and a method for detecting drug-resistant genes carried by the same, wherein the method can realize good separation of intracellular and extracellular DNAs, and does not have significant cross contamination, so that accurate detection of intracellular and extracellular antibiotic genes in sludge can be realized.
The invention provides a method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge, which is characterized by comprising the following steps of:
mixing the sludge precipitate with activated Cation Exchange Resin (CER), stirring and reacting at 0-4 ℃ and 500-800 rpm for 4-8 h, and centrifuging and separating supernatant a and precipitate a;
filtering the supernatant a by an acetate fiber membrane to obtain a crude extract of sludge extracellular DNA;
and mixing the precipitate a with an intracellular DNA extraction buffer solution, sequentially carrying out enzymolysis by using protease K, treatment by using sodium dodecyl benzene sulfonate (SDS) and treatment by using an SDS-DNA extraction buffer solution, and separating a supernatant b to obtain a crude extract of the intracellular DNA of the sludge.
In the present invention, the sludge precipitate is resuspended in phosphate buffer and then mixed with activated CER. The sludge sediment refers to sediment obtained after a sludge sample is centrifuged, and the volume of the sediment is equal to that of the sludge sample. The phosphate buffer comprises: water and 137mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na2HPO4And 1.8mmol/L KH2PO4And (4) forming. The pH of the buffer salt was 7.2.
In the invention, the CER is of Dowex Marathon C sodium type, and each g of volatile sludge mixed liquid suspended solids (MLVSS) is mixed with 60-80 g of the CER. The activating reagent of the resin adopts phosphate buffer solution, and the activation is carried out at room temperature, specifically 18-30 ℃. The activation time was 1 h. After activation, 10,000g, 4 ℃ centrifugation for 5min, remove the supernatant, obtain the activated resin.
In the specific embodiment, after the mixed solution of the sludge and the phosphate is stirred and reacted for 6 hours at the temperature of 4 ℃ and the rpm of 600, 10,000g of the mixed solution is centrifuged for 15min at the temperature of 4 ℃ to separate a supernatant a and a precipitate a;
when the supernatant a is filtered by an acetate fiber membrane, the aperture of the filter membrane is 0.22 mu m.
Mixing the precipitate a with an intracellular DNA extraction buffer solution and proteinase K, and carrying out three times of SDS treatment after enzymolysis;
the proportion of the sediment a, the DNA extraction buffer solution and the protease K is 1g (wet weight), 2.7mL and 10 mu L;
the intracellular DNA extraction buffer solution comprises water and 100mM Tris-HCl, 100mM EDTA and 100mM Na2HPO41.5M NaCl and 1% CTAB, pH 8.0.
The concentration of the protease K is 10mg/mL, and the enzymolysis condition is 37 ℃, and the stirring is carried out at 225rpm for 30 min. After enzymolysis, centrifuging and discarding the supernatant to obtain the enzymolysis product.
The three SDS treatments included:
first treatment: mixing the enzymolysis product with 20wt% SDS solution, incubating for 2h at 65 ℃, centrifuging for 10min at 10000rpm, and separating precipitate and supernatant c;
and (3) second treatment: mixing the precipitate after the first treatment with 20wt% SDS-DNA extraction buffer solution, incubating at 65 ℃ for 10min, centrifuging at 10000rpm for 10min, and separating the precipitate from supernatant d;
and (3) third treatment: mixing the precipitate after the second treatment with 20wt% SDS-DNA extraction buffer solution, incubating at 65 ℃ for 10min, centrifuging at 10000rpm for 10min, and taking supernatant e;
and combining the supernatants c, d and e to obtain a supernatant b.
In the first SDS treatment, the ratio of the enzymolysis product to 20wt% SDS solution is 1 g: 0.3 mL;
in the second SDS treatment, the proportion of the precipitate to 20wt% SDS-DNA extraction buffer solution is 1g (wet weight) to 0.1 mL;
in the second SDS treatment, the ratio of the precipitate to 20wt% SDS-DNA extraction buffer was 1g (wet weight) to 0.1 mL.
The 20wt% SDS-DNA extraction buffer solution is obtained by mixing a 20wt% SDS solution and a DNA extraction buffer solution in a volume ratio of 1: 9.
In the invention, the crude DNA extract inside and outside the sludge cell is also subjected to a purification step.
In the invention, the crude extract of the sludge extracellular DNA is sequentially treated by Cetyl Trimethyl Ammonium Bromide (CTAB), high-salt TE buffer solution, isopropanol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol, then precipitated by sodium acetate-absolute ethyl alcohol and washed by an ethanol solution, and finally the purified extracellular DNA is obtained.
In some embodiments, the CTAB processing comprises: mixing the sludge extracellular crude extract with 1 wt% CTAB solution of the same volume, standing at 65 deg.C for 30min, centrifuging at 10,000g and 4 deg.C for 10min, and collecting precipitate b.
In some embodiments, the high salt TE buffer treatment comprises: mixing the precipitate b with high-salt TE buffer solution, adding anhydrous isopropanol, standing at 0-4 deg.C for 1h, centrifuging at 4 deg.C for 10min at 10,000g, and collecting precipitate c. Wherein the high-salt TE buffer solution comprises water and 10mM Tris-HCl, 0.1mM EDTA and 1M NaCl, and the pH value is 8.0; the volume of the high-salt TE buffer solution is equal to that of the CTAB solution, and the volume ratio of the isopropyl alcohol to the high-salt TE buffer solution is 0.6: 1.
In some embodiments, the phenol-chloroform-isoamyl alcohol treatment comprises: and mixing the precipitate c with TE buffer solution, and adding a mixed solution of phenol, chloroform and isoamylol. After mixing, the mixture was centrifuged at 10,000rpm for 10min at 4 ℃ and then the upper aqueous phase a was taken. The TE buffer solution comprises water, 10mM Tris-HCl and 0.1mM EDTA, and the pH value is 8.0; the volume ratio of the mixed solution of phenol, chloroform and isoamylol is 25: 24: 1; the volume ratio of the TE buffer solution to the mixed solution of phenol, chloroform and isoamylol is 1: 1.
In some embodiments, the chloroform-isoamyl alcohol treatment comprises: mixing the upper water phase a with chloroform-isoamyl alcohol, centrifuging at 10,000rpm for 5min at 4 deg.C, and collecting the upper water phase b; the volume ratio of the chloroform to the isoamylol mixed solution is 24: 1; the volume ratio of the chloroform-isoamyl alcohol mixed solution to the upper aqueous phase a is 1: 1.
In some embodiments, the precipitation treatment with sodium acetate-absolute ethanol comprises: mixing the upper water phase b with 3mol/L sodium acetate solution and precooled absolute ethyl alcohol, standing for 1h at 0-4 ℃, centrifuging for 10min at 4 ℃ by 14,000g, and taking the precipitate d. The volume ratio of the upper-layer water phase b to the sodium ethoxide solution to the absolute ethyl alcohol is 10: 1: 20.
In some embodiments, the ethanol wash treatment comprises: the precipitate d was washed with ethanol solution. The concentration of the ethanol solution is 70vol%, and the washing times of the ethanol solution are 2 times.
In the invention, the crude extract of the sludge intracellular DNA is sequentially treated by phenol-chloroform-isoamylol and chloroform-isoamylol, precipitated by isopropanol and washed by ethanol solution to obtain purified intracellular DNA.
In some embodiments, the phenol-chloroform-isoamyl alcohol treatment comprises: mixing the crude extract of intracellular DNA with the mixture of phenol, chloroform and isoamylol, centrifuging at 10,000rpm for 10min at 4 deg.C, and collecting the upper water phase c. The mixing step is reversed and uniformly mixed, and the reversing times are 5-10 times. The volume ratio of the mixed solution of phenol, chloroform and isoamylol is 25: 24: 1; the volume ratio of the phenol, chloroform and isoamylol mixed solution to the sludge intracellular DNA crude extraction solution is 1: 1.
In some embodiments, the chloroform-isoamyl alcohol treatment comprises: and (3) mixing the upper aqueous phase c with the chloroform-isoamylol mixed solution in the same volume, centrifuging for 10min at the room temperature of 10,000g, and taking the upper aqueous phase d. The volume ratio of the chloroform to the isoamyl alcohol mixed solution is 24: 1.
In some embodiments, precipitating with isopropanol comprises: the upper aqueous phase d was mixed with isopropanol and after 1h of precipitation at room temperature, the supernatant was removed by centrifugation at 14,000rpm for 10min at room temperature. The volume ratio of the upper aqueous phase d to the isopropanol is 1: 0.6.
In some embodiments, the ethanol wash treatment comprises: the precipitate d was washed with ethanol solution. The concentration of the ethanol solution is 70vol%, and the washing times of the ethanol solution are 2 times.
In the present invention, the extraction method further comprises the step of evaluating whether the extracellular DNA is contaminated by the intracellular DNA by a live/dead cell staining method.
The invention provides a method for detecting intracellular and extracellular drug resistance genes in sludge, which takes sludge intracellular and extracellular DNAs separated by the method as templates; respectively carrying out fluorescent quantitative polymerase chain reaction (qPCR) amplification by using primers of target drug-resistant genes, and analyzing the distribution condition of intracellular and extracellular drug-resistant genes in the sludge according to the amplification result.
In the present invention, the drug resistance gene comprises: sul1, sul2, tetC, tetM, tetO and tetX.
In the present invention, the amplification system comprises:
the amplification procedure comprises:
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 primer sequence in the qPCR test is as follows:
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 tetC upstream primer nucleotide sequence is CTTGAGAGCCTTCAACCCAG;
the nucleotide sequence of the tetC downstream primer is ATGGTCGTCATCTACCTGCC;
the tetM upstream primer nucleotide sequence is ACAGAAAGCTTATTATATAAC;
the nucleotide sequence of the tetM downstream primer is TGGCGTGTCTATGATGTTCAC;
the tetO upstream primer nucleotide sequence is ACGGARAGTTTATTGTATACC;
the nucleotide sequence of the tetO downstream primer is TGGCGTATCTATAATGTTGAC;
the tetX upstream primer nucleotide sequence is AGCCTTACCAATGGGTGTAAA;
the nucleotide sequence of the tetX downstream primer is TTCTTACCTTGGACATCCCG;
the nucleotide sequence of the 16S rDNA upstream primer is ACTCCTACGGGAGGCAGCAG;
the nucleotide primer sequence of the 16S rDNA downstream primer is ATTACCGCGGCTGCTGG.
The invention combines an ion exchange resin method and an SDS-high salt method to extract extracellular and intracellular DNA of sludge. The ion exchange resin method can remove Ca in the sludge extracellular matrix through chemical action2+And Mg2+Plasma is adopted to efficiently destroy the interaction between extracellular DNA and other polymers, and the influence on the integrity of cells is small; and the recovery rate of the intracellular DNA extracted by the SDS-high salt method is high. The sludge extracellular and intracellular DNA purified by the CTAB and/or phenol-chloroform-isoamylol method has high purity, and is suitable for qPCR detection of drug-resistant genes. Therefore, the invention can meet the basic requirements of extracting intracellular and extracellular DNAs in the sludge: (1) the recovery rate of the extracellular DNA is high, the extracellular DNA can represent an extracellular DNA library, and no intracellular DNA pollution exists; (2) recovering intracellular and extracellular DNAs simultaneously; (3) the purity of the intracellular DNA and the extracellular DNA is high, and the method is suitable for subsequent molecular biology research. The method can successfully distinguish the intracellular and extracellular drug resistance genes in the sludge, is expected to be used for exploring the space-time distribution characteristics of the drug resistance genes with different forms in the sludge, and provides a basis for developing corresponding drug resistance gene risk assessment schemes and eliminating means in the future.
Drawings
FIG. 1 shows the intracellular DNA extraction and cell integrity of sludge in homogeneous extraction (extraction matrix 0.1M phosphate, pH8.0, error bars indicate standard deviation of 3 replicates);
FIG. 2 shows the intracellular DNA extraction and cellular integrity of sludge in the enzymatic degradation extraction process (error bars indicate standard deviations of 3 replicates);
FIG. 3 is the intracellular DNA extraction and cellular integrity of sludge in CER extraction (error bars indicate standard deviation of 3 replicates);
FIG. 4 is a qPCR test representative quantitative standard curve for 16S rDNA, sul1, sul2, tetC, tetM, tetO and tetX;
FIG. 5 is the relative concentrations of extracellular drug resistance genes of sludge in six actual sewage plants;
FIG. 6 is the relative concentrations of intracellular drug resistance genes of sludge in six actual sewage plants.
Detailed Description
The invention provides a separation method of intracellular and extracellular DNAs in sludge and a detection method of drug-resistant genes carried by the same, and a person skilled in the art can realize the separation method by properly improving 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 methods and applications of this 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 methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention aims to provide a method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge and a method for quantitatively detecting drug-resistant genes carried by the same. The invention adopts the following technical scheme:
(1) sludge sample pretreatment
And (4) taking a sludge mixed liquid sample in an actual sewage plant, and carrying out centrifugal separation. The supernatant was carefully removed, leaving the sludge pellet to be stored in a refrigerator.
(2) Extraction of sludge extracellular DNA
Weighing CER, placing the CER in a centrifuge tube A, adding phosphate buffer solution, shaking up and standing. After activation of the CER was complete, a portion of the supernatant was carefully removed;
② taking a proper amount of sludge mixed liquid, and placing the sludge mixed liquid in a centrifuge tube B. After centrifugation, the whole supernatant was carefully removed and supplemented with phosphate buffer. After uniformly mixing, transferring the sludge mixed solution into a centrifuge tube A in the step I;
transferring the sludge mixed liquor and CER in the centrifuge tube A to a wide-mouth flat-bottom glass bottle, adding a rotor, and placing the centrifuge tube A in a magnetic stirring water bath for reaction;
and fourthly, after the reaction is finished, transferring the mixed liquid sludge and the CER into a centrifugal tube C. After centrifugation, supernatant is carefully removed and membrane treatment is carried out, thus obtaining crude extract of sludge extracellular DNA.
(3) Purification of sludge extracellular DNA
Firstly, taking crude extraction liquid of sludge extracellular DNA, and placing the crude extraction liquid in a centrifuge tube D. Adding CTAB, reversing, mixing, standing in a water bath. After centrifugation, the supernatant was carefully removed;
and secondly, adding a high-salt TE buffer solution into the centrifuge tube D in the step I. After mixing by inversion, pre-cooled isopropanol was added. The mixture was inverted again and then allowed to stand on ice. After centrifugation, the supernatant was carefully removed;
and thirdly, adding TE buffer solution into the centrifuge tube D in the second step, and reversing and mixing uniformly. Adding the mixture of phenol, chloroform and isoamyl alcohol, and reversing and mixing evenly. After centrifugation, the upper aqueous phase was carefully transferred to centrifuge tube E;
fourthly, adding chloroform and isoamylol into the centrifugal tube E in the third step. And (4) reversing and mixing. After centrifugation, the upper aqueous phase was carefully transferred to centrifuge tube F;
fifthly, adding sodium acetate and precooled absolute ethyl alcohol into the centrifuge tube F in the step IV, reversing and uniformly mixing, and standing on ice. After centrifugation, the supernatant was carefully removed;
sixthly, adding ethanol into the centrifuge tube F in the fifth step. After centrifugation, the supernatant was carefully removed, and the DNA pellet was retained.
Seventhly, repeating the steps once;
and thirdly, adding sterile enzyme-free water into the centrifugal tube F filled with the DNA precipitate in the step (seventhly), and obtaining the purified sludge extracellular DNA solution.
(4) Extraction of sludge intracellular DNA
Carefully removing CER in the centrifuge tube C in the step (2) to the step (iv), and transferring the sludge precipitate to a centrifuge tube G;
adding DNA extraction buffer solution and protease K into the centrifuge tube G, and oscillating;
continuing adding SDS into the centrifuge tube G, standing in a water bath for several times at intervals;
fourthly, after the water bath is finished, carrying out centrifugal treatment, keeping sludge to be precipitated in a centrifugal tube G, and transferring supernatant into a centrifugal tube H;
fifthly, continuously adding DNA extraction buffer solution and SDS into the centrifuge tube D. Slightly swirling and mixing uniformly, and standing in a water bath kettle;
sixthly, after the water bath is finished, performing centrifugal treatment, keeping sludge to be precipitated in a centrifugal tube G, and transferring supernatant to a centrifugal tube H in the step IV;
seventhly, repeating the steps for one time. And finally, obtaining the supernatant in the centrifugal tube H as the crude extraction liquid of the intracellular DNA of the sludge.
(5) Purification of intracellular DNA from sludge
Firstly, crude extraction liquid of intracellular DNA of sludge is taken and placed in a centrifugal tube I. Adding the mixture of phenol, chloroform and isoamyl alcohol, and reversing and mixing evenly. After centrifugation, the upper aqueous phase was carefully transferred to centrifuge tube J;
② adding chloroform and isoamylol into the centrifuge tube J. And (4) reversing and mixing. After centrifugation, the upper aqueous phase is carefully transferred to a centrifuge tube K;
and thirdly, continuously adding isopropanol into the centrifuge tube K, and standing and precipitating at room temperature. After centrifugation, the supernatant was carefully removed;
fourthly, adding ethanol into the centrifuge tube K. After centrifugation, the supernatant was carefully removed, leaving the DNA pellet;
fifthly, repeating the step (iv) once.
Sixthly, adding sterile enzyme-free water into the centrifuge tube K filled with the DNA precipitate in the fifth step to obtain a purified sludge intracellular DNA solution.
(6) Detection of intracellular and extracellular drug resistance genes of sludge
Using the pure sludge extracellular DNA in the step (3) or the pure sludge intracellular DNA in the step (5) as a template, and adopting qPCR to amplify the drug-resistant gene and 16S rDNA in an actual sewage plant;
secondly, taking the drug-resistant gene and the plasmid standard product of 16S rDNA as a template to carry out qPCR experiment. Drawing a quantitative standard curve of the drug-resistant gene and the 16S rDNA according to the corresponding relation between the copy number concentration of the plasmid standard substance and the drug-resistant gene or the 16S rDNA amplification fluorescence threshold cycle number;
remarks explanation: the preparation method of the plasmid standard substance comprises the steps of taking sludge DNA 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 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 positive cloning transformants are picked, plasmid standard substances are extracted by a plasmid extraction kit and stored in a refrigerator.
Thirdly, comparing the quantitative standard curve of the drug-resistant gene and the 16S rDNA, obtaining the copy number concentration of the drug-resistant gene and the 16S rDNA in the sludge intracellular or extracellular DNA of the template according to the amplification fluorescence threshold of the drug-resistant gene and the 16S rDNA in the sludge intracellular or extracellular DNA, and calculating the relative concentration of the drug-resistant gene in the sludge intracellular or extracellular.
In the step (1), the sludge mixed liquor is obtained from an actual sewage plant; placing the sludge mixed liquor in a sterilized brown glass bottle, and transporting the sludge mixed liquor to a laboratory by adopting an ice bag; the centrifugation conditions were: 5000g, 4 ℃ and 5 min; the refrigerator temperature was set at 4 ℃.
In step (2), the centrifuge tube A, B and C have a volume of 10 mL. The CER is of a Dowex Marathon C sodium type with a size of 20-50 meshes; the mass of the CER is 2 g; the phosphate buffer solution is composed of 137mM NaCl, 2.7mM KCl and 10mM Na2HPO4And 1.8mM KH2PO4Adjusting the pH value to 7.2 by adopting NaOH and HCl in a water bath; the volume of the phosphate buffer solution is 5 mL; the activation time of the CER is 1 h; the volume of the removed supernatant was 4 mL. Substep (c) of about 29mg of said MLVSS (in terms of 70g CER/g MLVSS); the volume of the supplemental phosphate buffer was 4 mL. The volume of the wide-mouth flat-bottom glass bottle is 20 mL; the diameter of the rotor is 1.5 mm; the temperature of the water bath is 4 ℃; the rotating speed of the magnetic stirrer is 600 rpm; the reaction time is 6 h; the centrifugation conditions in the substep IV are as follows: 10000g, 4 ℃, 15 min; the aperture of the filter membrane is 0.22 μm.
In step (3), the centrifuge tube D, E and F have a volume of 2 mL; the inversion and mixing times are 5-10 times; the substeps of the centrifugation are 14000g, 4 ℃ and 5 min. The volume of the crude extract of the extracellular DNA is 0.5 mL; the mass concentration of CTAB is 1%; the volume of CTAB is 0.5 mL; the water bath temperature is 65 ℃; the water bath time is 30 min; centrifugation conditions were 10000g, 20 ℃ and 10 min. The sub-step is that the high-salt TE buffer solution consists of 10mM Tris-HCl, 0.1mM EDTA and 1M NaCl, and the pH value is adjusted to 8.0 by adopting NaOH and HCl; the volume of the high-salt TE buffer solution is 0.5 mL; the volume of the anhydrous isopropanol is 0.3 mL; the precooling temperature of the anhydrous isopropanol is 4 ℃; the ice bath time is 1 h; the centrifugation conditions were 10000g, 4 ℃ and 10 min. The TE buffer solution in the step III consists of 10mM Tris-HCl and 0.1mM EDTA, and the pH value is adjusted to 8.0 by adopting NaOH and HCl; the volume of the TE buffer solution is 0.6 mL; the volume ratio of the mixed solution of phenol, chloroform and isoamylol is 25: 24: 1; the volume of the mixed solution of phenol, chloroform and isoamylol is 0.6 mL; the volume of the transferred upper aqueous phase was 0.5 mL. In the substep, the volume ratio of the chloroform to isoamylol mixed solution is 24: 1; the volume of the chloroform-isoamylol mixed solution is 0.5 mL; the volume of the transferred upper aqueous phase was 0.4 mL. The sodium acetate concentration is 3M; the volume of the sodium acetate is 40 mu L; the ethanol precooling temperature is 4 ℃; the volume of the ethanol is 0.88 mL; the ice-bath time was 1 h. The pre-cooling temperature of the ethanol is 4 ℃; the ethanol concentration is 70 vol%; the volume of ethanol is 1 mL. The volume of the sterile and enzyme-free water is 50-100 mu L.
In step (4), the centrifuge tubes G and H were 2mL in volume. The substeps of two and five are that the DNA extraction buffer solution is prepared from 100mM Tris-HCl, 100mM EDTA and 100mM Na2HPO41.5M NaCl and 1% CTAB, adjusted to pH8.0 with NaOH and HCl. The substeps of the method comprise: 10000g, 20 ℃ and 10 min. The volume of the DNA extraction buffer solution is 810 mu L (calculated by 2.7mL DNA extraction buffer solution/g wet sludge); the concentration of the protease K is 10 mg/mL; the volume of the proteinase K is 3 mu L; the oscillation temperature is 37 ℃; the oscillation rotating speed is 225 rpm; the oscillation time is 30 min. The substep of the method is that the mass concentration of the SDS is 20 percent; the SDS bodyThe product is 90 mu L; the water bath temperature is 65 ℃; the water bath time is 2 h; the time interval for the inversion mixing was 15 min. The volume of the DNA extraction buffer solution is 270 mu L (calculated according to 2.7mL DNA extraction buffer solution/g wet sludge); the mass concentration of SDS is 20%; the SDS volume was 30 μ L; the temperature of the water bath kettle is 65 ℃; the water bath time is 10 min.
In step (5), the centrifuge tube I, J and K were 2mL in volume. The volume of a crude extraction liquid of the sludge intracellular DNA is 0.8 mL; the volume ratio of the mixed solution of phenol, chloroform and isoamylol is 25: 24: 1; the volume of the mixed solution of phenol, chloroform and isoamylol is 0.8 mL; the inversion and mixing times are 5-10 times; the centrifugation conditions were: 14000g, 4 ℃ and 10 min; the volume of the transferred upper aqueous phase was 0.7 mL. The volume ratio of the chloroform to isoamylol mixed solution is 24: 1; the volume of the chloroform-isoamylol mixed solution is 0.7 mL; the inversion and mixing times are 5-10 times; the centrifugation conditions were: 14000g, 4 ℃ and 10 min; the volume of the transferred upper aqueous phase was 0.6 mL. The volume of the isopropanol is 0.36 mL; the precipitation time is 1 h; the centrifugation conditions were: 14000g, 20 ℃ and 10 min. The pre-cooling temperature of the ethanol is 4 ℃; the ethanol concentration is 70 vol%; the volume of the ethanol is 1 mL; the centrifugation conditions were: 14000g, 4 ℃ and 5 min. The volume of the sterile enzyme-free water is 50-100 mu L.
In the step (6), the substep is that the copy number concentration range of the drug-resistant gene or 16S rDNA plasmid standard substance is 102～108copies, number of gradients 6, gradient value 10 times.
In the step (6), the substep of remarking indicates that the target genes amplified by the ordinary PCR comprise sul1, sul2 and 16S rDNA; the Gel purification Kit is TaKaRa MiniBEST Agarose Gel DNA Extraction Kit; the plasmid vector is pMD 18-T; the competent cell is Escherichia coli JM 109; the Plasmid extraction Kit is a MiniBEST Plasmid Purification Kit; the refrigerator storage temperature is-20 ℃.
In step (6), its substep 2 notes the plasmidThe formula for calculating the copy number concentration of the standard substance is as follows: 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; m0Is the average molecular weight of each base pair, 660 g/M; NA is Avgalois constant, 6.02X 1023。
In the step (6), the substep of remarking indicates that the general 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; extension at 72 ℃ for 7 min.
In the step (6), the calculation formula of the relative concentration of intracellular and extracellular drug resistance genes in the sludge is as follows: the relative concentration of the drug-resistant gene is the copy number concentration of the drug-resistant gene/16S rDNA copy number concentration.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
EXAMPLE 1 extraction and purification of intracellular and extracellular DNA from sludge
1. Extraction of sludge extracellular DNA by ion exchange resin
(1) 30 parts of 2g CER are weighed and placed in 30 10mL centrifuge tubes, 5mL phosphate buffer is added, the mixture is shaken up and then is kept stand for activating the resin for 1 h.
(2) After centrifugation at 10,000g at 4 ℃ for 5min, 4mL of the supernatant was carefully removed.
(3) An appropriate volume of sludge mixture (containing 29mg of MLVSS) was centrifuged at 4,000g at 4 ℃ for 5min, and the supernatant carefully removed. Phosphate buffer was supplemented to 5 mL. After mixing, the mixture was transferred to the above CER-containing centrifuge tube.
(4) The CER-sludge mixture was transferred to a 20mL flat bottom flask, added to a 1.5mm rotor and placed in a 4 ℃ magnetic water bath.
(5) Transferring the resin-sludge mixed solution into a 10mL centrifugal tube for 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 hours respectively, centrifuging for 15min at 10,000g and 4 ℃, carefully taking out supernatant and passing through a 0.22 mu m acetate fiber membrane to obtain crude extracellular DNA extracting solution of sludge.
2. Sludge extracellular DNA purification
(1) 0.5mL of crude extract of extracellular DNA of sludge is taken and placed in a 1.5mL centrifuge tube, 0.5mL of 1% CTAB is added, shaking is carried out uniformly, and then standing is carried out for 30min at 65 ℃.
(2) After centrifugation at 10,000g for 10min at 4 ℃, the supernatant was carefully removed.
(3) Add 0.5mL of high salt TE buffer to the pellet from step (2). After mixing by inversion, 0.3mL of pre-chilled (4 ℃ C.) isopropanol was added. The mixture was inverted again and left on ice for 1 h.
(4) After centrifugation at 10,000g for 10min at 4 ℃, the supernatant was carefully removed.
(5) After adding 0.6mL of TE buffer to the pellet of step (4), the mixture was inverted and mixed. After addition of 0.6mL phenol-chloroform-isoamyl alcohol (25: 24: 1, v/v), the mixture was again mixed by inversion.
(6) After centrifugation at 10,000rpm for 10min at 4 ℃, the upper aqueous phase (about 0.5mL) was carefully removed to a new 2mL centrifuge tube.
(7) Add equal volume of chloroform to isoamyl alcohol (24: 1, v/v) to the 2mL centrifuge tube from step (6). After mixing by inversion, the mixture was centrifuged at 10,000rpm at 4 ℃ for 5 min. The upper aqueous phase (about 0.4mL) was again transferred to a new 2mL centrifuge tube.
(8) Add 40. mu.L of 3M sodium acetate and 0.88mL of pre-cooled (4 ℃ C.) absolute ethanol to the 2mL centrifuge tube from step (7), mix by inversion and stand on ice for 1 h.
(9) After centrifugation at 14,000g for 10min at 4 ℃, the supernatant was carefully removed.
(10) To the precipitate from step (9) was added 1mL of pre-cooled (4 ℃ C.) 70% ethanol. After centrifugation at 14,000rpm for 5min at 4 ℃, the supernatant was carefully removed.
(11) Repeating the step (10) once.
(12) And adding 100 mu L of sterile enzyme-free water into the sediment obtained by centrifugation to obtain pure sludge intracellular DNA solution.
3. Sludge intracellular DNA extraction and purification
And (4) continuously using the sludge sediment after centrifugation when the extraction of the extracellular DNA of the sludge is finished for extracting the intracellular DNA of the sludge. The sludge intracellular DNA extraction method comprises the step of adding the DNA in equal proportion according to the wet sludge precipitation qualityAdding sludge extracellular DNA extraction reagent, here taking 300mg wet mud as an example. The sludge intracellular DNA extraction buffer comprises water and 100mM Tris-HCl, 100mM EDTA and 100mM Na2HPO41.5M NaCl and 1% CTAB, pH 8.0.
(1) mu.L of sludge intracellular DNA extraction buffer and 3. mu.L of proteinase K (10mg/mL) were added and shaken horizontally at 225rpm at 37 ℃ for 30 min.
(2) Adding 90 mu L of 20% SDS, and carrying out water bath in a water bath kettle at 65 ℃ for 2h, and reversing 5-10 times at intervals of 15 min.
(3) Centrifuge at 10,000rpm for 10min at room temperature and transfer the supernatant to a new 2mL centrifuge tube.
(4) To the pellet from step (3), 270. mu.L of extraction buffer and 30. mu.L of 20% SDS were added. Mixing by gentle vortex, and water-bathing in 65 deg.C water bath for 10 min.
(5) Centrifuge at 10,000rpm for 10min at room temperature and transfer the supernatant to a 2mL centrifuge tube in step (3).
(6) Repeating the steps (4) and (5) once. And combining the supernatant obtained by the 3 times of extraction to obtain the crude extract of the intracellular DNA of the sludge.
(7) 0.8mL of crude extract of intracellular DNA of sludge is taken and mixed with phenol, chloroform and isoamylol (25: 24: 1, v/v) in equal volume by inversion. After centrifugation at 10,000rpm for 5min at 4 ℃, the upper aqueous phase (0.7mL) was carefully transferred to a new 2mL centrifuge tube.
(8) Add 0.7mL of chloroform/isoamyl alcohol (24: 1, v/v) to the 2mL centrifuge tube from step (7). After mixing by inversion, the mixture was centrifuged at 10,000g for 5min at room temperature. The upper aqueous phase (0.6mL) was again transferred to a new 2mL centrifuge tube.
(9) Add 0.6mL of isopropanol to the 2mL centrifuge tube from step (8) and precipitate at room temperature for 1 h. After centrifugation at 14,000rpm for 10min at room temperature, the supernatant was carefully removed.
(10) 1mL of pre-chilled (4 ℃ C.) 70vol% ethanol was added. After centrifugation at 14,000rpm for 5min at 4 ℃, the supernatant was carefully removed.
(11) Repeating the step (10) once.
(12) And adding 100 mu L of sterile enzyme-free water into the sediment obtained by centrifugation to obtain pure sludge intracellular DNA solution.
Comparative example 1
This comparative example differs from example 1 in that: and extracting sludge extracellular DNA by adopting a homogenization method. The other steps are the same as in example 1. The specific steps of extracting sludge extracellular DNA by a homogenization method are as follows:
an appropriate volume of sludge mixed liquor (containing 29mg of MLVSS) was taken and centrifuged at 4000rpm for 5min at 4 ℃ in a 10mL centrifuge tube. Discarding the supernatant;
with 0.12M NaH2PO4(pH 8.0) at 250rpm at 25 ℃ for 10 min; centrifuging at 10,000rpm at 4 deg.C for 5min, and collecting supernatant; repeating the operation for 3 times, combining the supernatants for 3 times, and passing through a 0.22 μm cellulose acetate membrane to obtain crude extract of extracellular DNA of sludge.
Comparative example 2
This comparative example differs from example 1 in that: and (3) extracting sludge extracellular DNA by adopting enzymatic degradation. The other steps are the same as in example 1. The specific steps for extracting sludge extracellular DNA through enzymatic degradation are as follows:
an appropriate volume of sludge mixed liquor (containing 29mg of MLVSS) was taken and centrifuged at 4000rpm for 5min at 4 ℃ in a 10mL centrifuge tube. After discarding the supernatant, 0.85% NaCl solutions containing 5, 50, 100, 200 and 500. mu.g/mL proteinase K were added, respectively, and the mixture was treated at 37 ℃ for 1 hour. Then, centrifuging for 15min at 10,000rpm at 4 ℃, taking the supernatant to pass through a 0.22 mu m acetate fiber membrane, thus obtaining the crude extract of the extracellular DNA of the sludge.
Assessment of intracellular and extracellular DNA cross contamination of sludge
And evaluating sludge intracellular DNA cross contamination in the sludge extracellular DNA extraction process by adopting a live/dead cell staining method. LIVE/DEAD cell staining experiments were performed using the kit LIVE/DEAD Baclight BacterialViabilitykit.
The results show that: the extraction amount of extracellular DNA and the integrity of cells of sludge in the extraction methods of example 1 and comparative examples 1 to 2 are shown in FIGS. 1 to 3. The CER extraction method and the homogeneous extraction method (the extraction medium is 0.1M phosphate, pH8.0) have high extraction efficiency on the sludge extracellular DNA. The extracellular DNA extraction amount of the sludge in the homogeneous extraction method can reach 3.8mg/g MLVSS, and no obvious cell lysis phenomenon is observed. Enzymatic degradation extracts sludge extracellular DNA poorly (fig. 2), probably because proteinase K failed to completely eliminate the interaction between proteins and sludge intracellular DNA.For CER extraction, the maximum extraction was close to 5.6mg/g MLVSS after 6h of extraction and no significant cell lysis occurred within 6h (FIG. 3). This indicates that the CER extraction method can remove Ca in the sludge extracellular matrix by chemical action2+And Mg2+The plasma disrupts the interaction between sludge extracellular DNA and other polymers with little effect on cellular integrity.
As a result, the effect of CER on extracellular DNA extraction is better than that of the homogeneous method and the enzymatic method. The CER extraction method requires control of CER dosage, stirring intensity and extraction time. When the extraction conditions of 70g CER/MLVSS, 600rpm and 6h are adopted, the extraction amount of the sludge extracellular DNA is higher (reaching 5.6mg/g MLVSS), and no obvious sludge intracellular DNA cross contamination is caused.
Example 2 detection of intracellular and extracellular drug resistance genes in sludge
(1) Using the pure sludge extracellular DNA or the pure sludge intracellular DNA in the example 1 as a template, and adopting qPCR to amplify the sludge intracellular and extracellular drug resistance genes (sul1, sul2, tetC, tetM, tetO and tetX) and 16S rDNA in an actual sewage plant;
the primer sequence in the qPCR test is as follows:
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 tetC upstream primer nucleotide sequence is CTTGAGAGCCTTCAACCCAG;
the nucleotide sequence of the tetC downstream primer is ATGGTCGTCATCTACCTGCC;
the tetM upstream primer nucleotide sequence is ACAGAAAGCTTATTATATAAC;
the nucleotide sequence of the tetM downstream primer is TGGCGTGTCTATGATGTTCAC;
the tetO upstream primer nucleotide sequence is ACGGARAGTTTATTGTATACC;
the nucleotide sequence of the tetO downstream primer is TGGCGTATCTATAATGTTGAC;
the tetX upstream primer nucleotide sequence is AGCCTTACCAATGGGTGTAAA;
the nucleotide sequence of the tetX downstream primer is TTCTTACCTTGGACATCCCG;
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 for sul1, sul2, tetC and tetX were 60 ℃ and for tetM, tetO and 16S rDNA were 55 ℃.
The volume of the qPCR test reaction system is 20 mu L, and the qPCR test reaction system specifically comprises the following components:
(2) drug resistance genes (sul1, sul2, tetC, tetM, tetO, and tetX) and 16S rDNA plasmid standards were prepared, and the copy number concentration of the plasmid standards was calculated. Sequentially carrying out 10-fold gradient dilution on plasmid standard substances with known copy numbers, and selecting a concentration range of 108～102Plasmid standards of copies six gradients were subjected to qPCR testing. According to the corresponding relation between the copy number concentration of the plasmid standard substance and the fluorescence threshold cycle number of the drug-resistant gene or 16S rDNA amplification, a quantitative standard curve of the drug-resistant gene and the 16S rDNA is drawn, and a representative quantitative standard curve is shown in figure 4.
Remarks explanation:
the calculation formula of the copy number concentration of the plasmid standard substance is as follows: ce＝Cm×NA/[(LpMD18-T+Lx)×M0]In the formula CcCopy number concentration of plasmid standards, copies/. mu.L; gmIs 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; m0An average molecular weight of 660 g-M; NA is Avgalois constant, 6.02X 1023。
② the preparation steps of the plasmid standard substance comprise: using purified intracellular and extracellular DNA as a template, and adopting common PCR to amplify target genes (sul1, sul2, tetC, tetM, tetO, tetX and 16S rDNA); 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 (TaKaRa miniBEST Agarose Gel DNA Extraction 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 cloning transformants were picked, the Plasmid-quality standards were extracted with a Plasmid extraction Kit (MiniBEST Plasmid Purification Kit) and stored in a freezer at-20 ℃.
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, 20uL of common PCR reaction system volume is adopted, and the plasmid standard substance specifically comprises the following components:
(3) and (3) comparing the quantitative standard curves of the drug-resistant gene and the 16S rDNA, obtaining the copy number concentration of the drug-resistant gene and the 16S rDNA in the sludge intracellular or extracellular DNA of the template according to the amplification fluorescence threshold of the drug-resistant gene and the 16S rDNA in the sludge intracellular or extracellular DNA, and calculating the relative concentration of the drug-resistant gene in the sludge intracellular or extracellular (see figure 5 and figure 6). The results show that the relative concentration mean values of the sludge extracellular sul1, sul2, tetC, tetM, tetO and tetX in the actual sewage plant are respectively as follows: 0.087 to 3.156, 0.351 to 2.955, 0.016 to 0.130, 6.9 × 10-4～0.011、3.3×10-40.011, 0.005-0.092 copies/copies 16S rDNA; the relative concentration mean values of sul1, sul2, tetC, tetM, tetO and tetX in the sludge cells are respectively as follows: 0.133 to 7.152, 0.461 to 13.946, 0.005 to 1.403, 4.2 × 10-4～1.405、0.001～0.448、0.007～4.771copies/copies 16S rDNA。
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (5)
1. The method for separating intracellular and extracellular DNAs (deoxyribonucleic acids) in sludge is characterized by comprising the following steps of:
mixing the sludge precipitate and phosphate buffer solution after heavy suspension with activated cation exchange resin, stirring and reacting at the temperature of 0-4 ℃ and 600rpm for 6 hours, and centrifuging and separating supernatant a and precipitate a; the cation exchange resin is of a Dowex Marathon C sodium type, and each g of volatile sludge mixed liquid suspended solid is mixed with 70g of the cation exchange resin; the sludge sediment refers to sediment obtained after a sludge sample is centrifuged;
filtering the supernatant a by an acetate fiber membrane to obtain a crude extract of sludge extracellular DNA;
treating the crude extract of the sludge extracellular DNA with cetyl trimethyl ammonium bromide, a high-salt TE buffer solution, isopropanol, phenol-chloroform-isoamylol and chloroform-isoamylol in sequence, precipitating with sodium acetate-absolute ethyl alcohol, and washing with 70vol% ethanol to obtain extracellular DNA;
mixing the precipitate a with intracellular DNA extraction buffer solution and protease K, carrying out enzymolysis, carrying out treatment by sodium dodecyl benzene sulfonate for three times, and separating supernatant b to obtain sludge intracellular DNA crude extraction solution;
the treatment of the sodium tert-dodecyl benzene sulfonate comprises the following steps:
first treatment: mixing the enzymolysis product with 20wt% sodium dodecyl benzene sulfonate solution, incubating at 65 ℃ for 2h, centrifuging at 10000rpm for 10min, and separating precipitate and supernatant c;
and (3) second treatment: mixing the precipitate after the first treatment with 20wt% sodium dodecyl benzene sulfonate solution-DNA extraction buffer solution, incubating at 65 ℃ for 10min, centrifuging at 10000rpm for 10min, and separating the precipitate and supernatant d;
and (3) third treatment: mixing the precipitate after the second treatment with 20wt% sodium dodecyl benzene sulfonate solution-DNA extraction buffer solution, incubating at 65 ℃ for 10min, centrifuging at 10000rpm for 10min, and taking supernatant e;
mixing the supernatant c, d and e to obtain a supernatant b;
and treating the crude extract of the sludge intracellular DNA sequentially by phenol-chloroform-isoamylol and chloroform-isoamylol, precipitating by isopropanol, and washing by 70vol% ethanol to obtain the intracellular DNA.
2. The method of claim 1, wherein the extraction is performed by staining the extracellular DNA with live/dead cells to determine whether the extracellular DNA is contaminated with intracellular DNA.
3. A method for detecting intracellular and extracellular drug resistance genes in sludge, which is characterized in that the method of any one of claims 1-2 is used for separating intracellular and extracellular DNAs in the sludge; and respectively carrying out amplification by using primers of target drug-resistant genes, and analyzing the existence condition of intracellular and extracellular drug-resistant genes in the sludge according to the amplification result.
4. The detection method according to claim 3, wherein the drug resistance genes include sul1, sul2, tetC, tetM, tetO and tetX.
5. The detection method according to claim 3, wherein the amplification system comprises:
The amplification procedure comprises:
treating with uracil-DNA glycosylase at 50 deg.C for 2 min;
double-Lock Taq DNA polymerase hot start at 95 ℃ for 2 min;
denaturation at 95 ℃ for 15s, annealing at 15s, and extension at 72 ℃ for 1min for 40 cycles.
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| CN104911178A (en) * | 2015-06-19 | 2015-09-16 | 厦门大学 | Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample |
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| CN108569831A (en) * | 2018-04-25 | 2018-09-25 | 中原工学院 | A kind of extraction separation method of effective activated sludge exoprotein and application |
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| CN104911178A (en) * | 2015-06-19 | 2015-09-16 | 厦门大学 | Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample |
| CN106480015A (en) * | 2016-09-23 | 2017-03-08 | 天津大学 | A kind of method of extracellular dna in high efficiency extraction deposit |
| CN108569831A (en) * | 2018-04-25 | 2018-09-25 | 中原工学院 | A kind of extraction separation method of effective activated sludge exoprotein and application |
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