CN111235146A - 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 20 wt% 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 20 wt% 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 20 wt% 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 20 wt% SDS solution is 1 g: 0.3 mL;
in the second SDS treatment, the proportion of the precipitate to 20 wt% SDS-DNA extraction buffer solution is 1g (wet weight) to 0.1 mL;
in the second SDS treatment, the ratio of the precipitate to 20 wt% SDS-DNA extraction buffer was 1g (wet weight) to 0.1 mL.
The 20 wt% SDS-DNA extraction buffer solution is obtained by mixing a 20 wt% 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 70 vol%, 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 70 vol%, 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) high purity of intracellular and extracellular DNA, and suitability for subsequent molecular biologyAnd (5) studying. 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
① weighing CER, placing in centrifuge tube A, adding phosphate buffer solution, shaking, standing, and carefully removing part of supernatant after activation of CER is finished;
② placing a proper amount of sludge mixed liquor in a centrifuge tube B, removing all supernatant carefully after centrifugation, supplementing phosphate buffer solution, mixing evenly, transferring the sludge mixed liquor to a centrifuge tube A in the step ①;
③ transferring the sludge mixed solution and CER in the centrifuge tube A in the step ② to a wide-mouth flat-bottom glass bottle, adding a rotor, and placing the mixture in a magnetic stirring water bath for reaction;
④ after the reaction, transferring the mixed liquid sludge and CER into a centrifuge tube C, centrifuging, carefully transferring the supernatant, and performing membrane treatment to obtain the crude extract of extracellular DNA of sludge.
(3) Purification of sludge extracellular DNA
① taking crude extract of extracellular DNA of sludge, placing in centrifuge tube D, adding CTAB, reversing, mixing, standing in water bath, centrifuging, and carefully removing supernatant;
② adding high-salt TE buffer solution into the centrifuge tube D in the step ①, reversing and mixing, adding precooled isopropanol, reversing and mixing, standing on ice, centrifuging, and carefully removing supernate;
③ adding TE buffer solution into the centrifuge tube D in the step ②, reversing and mixing evenly, adding a mixed solution of phenol, chloroform and isoamylol, reversing and mixing evenly again, centrifuging, and carefully transferring the upper-layer water phase into the centrifuge tube E;
④ adding chloroform and isoamylol into the centrifuge tube E in the step ③, reversing, mixing evenly, centrifuging, and carefully transferring the upper aqueous phase into a centrifuge tube F;
⑤ adding sodium acetate and precooled absolute ethyl alcohol into the centrifuge tube F in the step ④, reversing, uniformly mixing, standing on ice, centrifuging, and carefully removing supernatant;
⑥ Add ethanol to centrifuge tube F in step ⑤ after centrifugation, the supernatant was carefully removed and the DNA pellet was retained.
⑦ repeat step ⑥ once;
③ sterile enzyme-free water is added into the centrifuge tube F filled with DNA sediment in the step ⑦, and the purified sludge extracellular DNA solution is obtained.
(4) Extraction of sludge intracellular DNA
① after carefully removing CER from centrifuge tube C in steps (2) - ④, the sludge pellet was transferred to centrifuge tube G;
② adding DNA extraction buffer solution and proteinase K into the centrifuge tube G, and oscillating;
③ adding SDS into the centrifuge tube G, standing in a water bath, and reversing for several times;
④, centrifuging after the water bath is finished, keeping the sludge to be precipitated in a centrifuge tube G, and transferring the supernatant into a centrifuge tube H;
⑤ adding DNA extraction buffer solution and SDS, slightly whirling and mixing, and standing in water bath;
⑥, centrifuging after the water bath is finished, keeping the sludge to be precipitated in a centrifuge tube G, and transferring the supernatant to a centrifuge tube H in the step ④;
⑦ repeating the steps ⑤ - ⑥ once, and finally obtaining the supernatant in the centrifuge tube H, namely the crude extract of the intracellular DNA of the sludge.
(5) Purification of intracellular DNA from sludge
① placing crude extract of intracellular DNA in centrifuge tube I, adding mixed solution of phenol, chloroform and isoamylol, mixing, centrifuging, and transferring the upper water phase to centrifuge tube J;
② adding chloroform and isoamylol into the centrifuge tube J, reversing and mixing evenly, centrifuging, and carefully transferring the upper aqueous phase into a centrifuge tube K;
③ adding isopropanol into centrifuge tube K, standing at room temperature for precipitation, centrifuging, and carefully removing supernatant;
④ adding ethanol into the centrifuge tube K, centrifuging, carefully removing the supernatant, and keeping the DNA precipitate;
⑤ repeat step ④ once.
⑥ sterile enzyme-free water is added into the centrifuge tube K filled with the DNA sediment in the step ⑤, and the purified sludge intracellular DNA solution is obtained.
(6) Detection of intracellular and extracellular drug resistance genes of sludge
①, 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 the actual sewage plant;
② taking the plasmid standard of the drug-resistant gene and 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 and the threshold cycle number of the fluorescence of the drug-resistant gene or the 16S rDNA amplification;
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.
③ comparing with the quantitative standard curve of the drug-resistant gene and 16S rDNA, obtaining the copy number concentration of the drug-resistant gene and 16S rDNA in the sludge intracellular or extracellular DNA of the template according to the amplification fluorescence threshold of the drug-resistant gene and 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 the step (2), the centrifugal tube A, B and the volume of the C are 10mL, the sub-step ① of the centrifugal tube is a CER brand and model of Dowex Marathon C sodium type with the particle size of 20-50 meshes, the mass of the CER is 2g, and the phosphate buffer solution is prepared from 137mM NaCl, 2.7mM KCl and 10mM Na2HPO4And 1.8mM KH2PO4The components of the composition are as follows,adjusting the pH value of a water bath to 7.2 by adopting NaOH and HCl, adjusting the volume of the phosphate buffer solution to 5mL, adjusting the activation time of the CER to 1h, adjusting the volume of the removed supernatant to 4mL, adjusting the substep ② to 29mg of MLVSS (calculated as 70g of CER/g of MLVSS), adjusting the volume of the supplemented phosphate buffer solution to 4mL, adjusting the substep ③ to 20mL of wide-mouth flat-bottom glass bottle, adjusting the diameter of the rotor to 1.5mm, adjusting the water bath temperature to 4 ℃, adjusting the rotating speed of a magnetic stirrer to 600rpm, adjusting the reaction time to 6h, adjusting the centrifugation conditions in the substep ④ to 10000g, 4 ℃ and 15min, and adjusting the pore diameter of the filter membrane to 0.22 mu m.
In the step (3), the volumes of the centrifuge tube D, E and the centrifuge tube F are 2mL, the times of inversion and mixing are 5-10 times, the volume of the extracellular DNA crude extraction liquid is 0.5mL, the mass concentration of CTAB is 1%, the volume of CTAB is 0.5mL, the temperature of the water bath is 65 ℃, the time of the water bath is 30min, the centrifugation conditions are 10000g, 20 ℃ and 10min, the volume of the high-salt TE buffer solution is 10mMTris-HCl, 0.1mM EDTA and 1M NaCl, the pH is adjusted to 8.0 by NaOH and HCl, the volume of the high-salt TE buffer solution is 0.5mL, the volume of the anhydrous isopropanol is 0.3mL, the precooling temperature of the anhydrous isopropanol is 4 ℃, the time is 1h, the conditions are 10000g, 4 ℃ and 10min, the temperature of the sub-step is adjusted by 10mM EDTA and HCl, the volume of the aqueous phase is adjusted by 0.50 mL, the concentration of the ethanol layer is 0.5mL, the volume of the ethanol-based on the ethanol layer is 50 mL, the volume of the ethanol-based on the concentration of the ethanol-water-ethanol-water-ethanol-water-ethanol-water-ethanol-water-ethanol-water-ethanol.
In step (4), the centrifuge tubes G and H have a volume of 2mL, and the DNA extraction buffers of substeps ② and ⑤ are prepared from 100mM Tris-HCl, 100mM EDTA, and 100mM Na2HPO4The centrifugal conditions of the substeps ④ and ⑥ are 10000g, 20 ℃ and 10min, the substep ② is that the volume of the DNA extraction buffer solution is 810 mu L (calculated by 2.7mL of DNA extraction buffer solution/g of wet sludge), the concentration of the protease K is 10mg/mL, the volume of the protease K is 3 mu L, the oscillation temperature is 37 ℃, the oscillation speed is 225rpm, the oscillation time is 30min, the substep ③ is that the mass concentration of the SDS is 20%, the volume of the SDS is 90 mu L, the temperature of the water bath is 65 ℃, the time of the water bath is 2h, the time interval of inversion and uniform mixing is 15min, the substep ⑤ is that the volume of the DNA extraction buffer solution is 270 mu L (calculated by 2.7mL of DNA extraction buffer solution/g of wet sludge), the mass concentration of the SDS is 20%, the volume of the SDS is 30 mu L, the temperature of the pot is 65 ℃, and the time of the water bath is 10 min.
In the step (5), the volumes of the centrifuge tube I, J and the centrifuge tube K are 2mL, the volume of the substep ① is 0.8mL of crude extraction liquid of intracellular DNA of the sludge, the volume ratio of the mixed liquid of phenol, chloroform and isoamyl alcohol is 25: 24: 1, the volume ratio of the mixed liquid of phenol, chloroform and isoamyl alcohol is 0.8mL, the times of inversion and mixing are 5-10 times, the centrifugation conditions are 14000g, 4 ℃ and 10min, the volume of the transferred upper layer aqueous phase is 0.7mL, the substep ② is 24: 1, the volume ratio of the mixed liquid of chloroform and isoamyl alcohol is 0.7mL, the times of inversion and mixing are 5-10 times, the centrifugation conditions are 14000g, 4 ℃ and 10min, the volume of the transferred upper layer aqueous phase is 0.6mL, the substep ③ is 0.36mL, the volume of isopropanol is 1h, the centrifugation conditions are 14000g, 20 ℃ and 10min, the temperature of the substep ④ is 1404 ℃ and the concentration of the ethanol is 14070 ℃ and the volume of the precooled ethanol is 100. mu.82 mL.
In step (6), the copy number concentration range of the drug-resistant gene or 16S rDNA plasmid standard in substep ② is 102～108copies, number of gradients 6, gradient value 10 times.
In step (6), substep ② indicates that the target gene for general PCR amplification includes 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 JM109, the Plasmid Extraction Kit is MiniBEST Plasmid Purification Kit, and the refrigerator storage temperature is-20 ℃.
In step (6), its substep ② remarks that the formula for calculating the copy number concentration of the plasmid standard 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; m0Is the average molecular weight of each base pair, 660 g/M; NA is Avgalois constant, 6.02X 1023。
In step (6), its substep ② remarks that the general PCR reaction conditions were pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing for 30s, and extension at 72 ℃ for 30s for 35 cycles, and extension at 72 ℃ for 7 min.
In step (6), the calculation formula of the relative concentration of the intracellular and extracellular drug-resistant genes in the sludge in substep ③ is that the relative concentration of the drug-resistant genes is the copy number concentration of the drug-resistant genes/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 is characterized in that a sludge extracellular DNA extraction reagent is added according to the equal proportion of the precipitation mass of wet sludge, and 300mg of wet sludge is taken 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.) 70 vol% 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 TaqDNA 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. Amplifying fluorescence according to plasmid standard copy number concentration and drug-resistant gene or 16S rDNAThe quantitative standard curve of the drug resistance gene and 16SrDNA was plotted against the threshold cycle number, and a representative quantitative standard curve is shown in FIG. 4.
Remarks explanation:
① the calculation formula of the copy number concentration of the plasmid standard substance is 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; m0Is the average molecular weight of each base pair, 660 g/M; NA is Avgalois constant, 6.02X 1023。
② Plasmid standard preparation comprises using purified intracellular and extracellular DNA as template, amplifying target gene (sul1, sul2, tetC, tetM, tetO, tetX and 16S rDNA) by common PCR, subjecting the common PCR product to Agarose gel electrophoresis, cutting target gene band under gel imager, purifying by gel Purification Kit (TaKaRa MiniBEST Agarose gel DNA Extraction Kit), adjusting the gel purified target gene to appropriate concentration, connecting to Plasmid vector (pMD 18-T), transforming into competent cell (Escherichia coli JM109), selecting positive clone transformant, extracting (MiniBEST Plasmid Purification Kit) quality-improving Plasmid standard with Plasmid Extraction Kit, and storing in refrigerator at-20 deg.C.
③ the ordinary PCR reaction conditions for preparing plasmid standard are pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing for 30s, extension at 72 deg.C for 30s, 35 cycles, and extension at 72 deg.C for 7 min.
④ plasmid standard preparation, the volume of the common PCR reaction system adopts 20uL, and the specific composition is:
(3) comparing a quantitative standard curve of the drug-resistant gene and the 16S rDNA, and obtaining the template sludge cell according to the amplification fluorescence threshold of the drug-resistant gene and the 16S rDNA in the sludge intracellular or extracellular DNAThe copy number concentrations of the drug-resistant gene and 16S rDNA in the intracellular or extracellular DNA, and the relative concentrations of the drug-resistant gene in the sludge cells or extracellular cells were calculated (see fig. 5 and 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 (9)
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 with the activated cation exchange resin, stirring and reacting at 0-4 ℃ and 500-800 rpm for 4-8 h, and centrifuging to separate a supernatant a and a 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 20 wt% of sodium dodecyl benzene sulfonate and treatment by using 20 wt% of sodium dodecyl benzene sulfonate-DNA extraction buffer solution, and separating a supernatant b to obtain a crude sludge intracellular DNA extraction solution.
2. The method according to claim 1, wherein the cation exchange resin is of the Dowex Marathon sodium C type, and 60 to 80g of the cation exchange resin is mixed per g of the volatile sludge mixed liquor suspended solids.
3. The method according to claim 1 or 2, characterized in that the precipitate a is mixed with DNA extraction buffer and proteinase K, and is treated with sodium dodecyl benzene sulfonate three times after enzymolysis;
the treatment of the sodium tert-dodecyl benzene sulfonate comprises the following steps:
first treatment: mixing the enzymolysis product with 20 wt% 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 20 wt% 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 20 wt% sodium dodecyl benzene sulfonate solution-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.
4. The method according to any one of claims 1 to 3, wherein the crude extract of the extracellular DNA of the sludge is sequentially treated with cetyl trimethyl ammonium bromide, a high-salt TE buffer solution, isopropanol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol, then precipitated with sodium acetate-absolute ethanol, and washed with 70 vol% ethanol to obtain the extracellular DNA.
5. The method according to any one of claims 1 to 4, wherein the crude extract of intracellular DNA of the sludge is treated by phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol in sequence, precipitated by isopropanol and washed by 70 vol% ethanol to obtain intracellular DNA.
6. The method according to any one of claims 1 to 5, wherein the extraction process is performed by a live/dead cell staining method to assess whether the extracellular DNA is contaminated with intracellular DNA.
7. 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 to 6 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.
8. The detection method according to claim 7, wherein the drug resistance gene comprises: sul1, sul2, tetC, tetM, tetO and tetX.
9. The detection method according to claim 7, wherein 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.
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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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