CN111471750A - Detection method of bacterial strain for rapidly degrading nitrogen in sewage and application thereof - Google Patents

Abstract

The invention belongs to the technical field of environmental engineering, and discloses a method for detecting a bacterial strain that rapidly degrades nitrogen in sewage and an application thereof. Prepare the standard sample of the strain by using the samples of the bacterial strains with no virulence; establish a standard curve corresponding to the concentration of the standard sample of the strain and the number of critical cycles; perform PCR amplification and electrophoresis on the samples of the strain to establish the concentration of the samples of the strain corresponding to the number of critical cycles. Standard curve: According to the standard curve corresponding to the concentration of the strain sample and the critical cycle number, the number of copies of the strain gene in the strain sample is obtained, that is, the number of strains in the sample of the strain that rapidly degrades nitrogen in sewage is obtained. The detection standard product of the bacterial strain provided by the invention has high sensitivity, simple preparation method, good purity and wide detection range, can rapidly and quantitatively detect the nitrogen-degrading bacterial strain in sewage, and realizes reduction of the cost of denitrification of sewage.

Description

A kind of detection method and application of strains that rapidly degrade nitrogen in sewage

technical field

The invention belongs to the technical field of environmental engineering, and in particular relates to a detection method and application of a bacterial strain that rapidly degrades nitrogen in sewage.

Background technique

At present, the closest existing technology: In recent decades, people have gradually paid attention to environmental protection, and the state has increased its efforts to prevent and control polluted water bodies. The problem of eutrophication is still serious, and the safety of drinking water is still threatened. With the expansion of sewage treatment plants, some industrial sewage has also been included in sewage treatment plants. The pollutants are complex and diverse, and the concentration of nutrients such as nitrogen and phosphorus is too high. The existing process basically does not set nitrogen and phosphorus removal. Deep processing. In addition, with the increase of sewage volume, the sewage treatment plant has exceeded its original processing capacity, affecting the normal operation of sewage treatment facilities, resulting in excessive discharge of nitrogen and phosphorus in the effluent. Wastes from agricultural production activities, livestock and poultry breeding, aquaculture and rural residents' life enter the water body through surface runoff, mid-stream flow, farmland drainage and underground leakage, causing serious pollution to the water body. In particular, excessive application of chemical fertilizers in agricultural production, improper disposal of livestock and poultry manure, high-density aquaculture and excessive feeding of feed, kitchen waste in residential areas and excrement and urine in toilets will all cause nitrogen pollution in water bodies. Non-point source pollution has the characteristics of scattered sources and many points, wide pollution range, large amount of pollutants, and more complex pollutant components and processes, so nitrogen pollution is more difficult to control. The process of denitrification in sewage by traditional denitrification technology is complicated; while the new biological denitrification technology has high cost and is difficult to popularize and use.

To sum up, the problems existing in the prior art are: the process of denitrification from sewage by the traditional denitrification technology is complicated; while the newly emerging biological denitrification technology has high cost and is difficult to popularize and use.

SUMMARY OF THE INVENTION

Aiming at the problems existing in the prior art, the present invention provides a method for detecting a strain that rapidly degrades nitrogen in sewage and its application.

The present invention is achieved in this way, a method for detecting a bacterial strain that rapidly degrades nitrogen in sewage, and the method for detecting a bacterial strain that rapidly degrades nitrogen in sewage comprises the following steps:

In step 1, a sample of a strain that rapidly degrades nitrogen in sewage is obtained, and a standard product of a strain that rapidly degrades nitrogen in sewage is obtained;

Step 2, establishing a standard curve corresponding to the concentration of the strain standard product that rapidly degrades nitrogen in the sewage and the critical cycle number;

In step 3, PCR amplification and electrophoresis confirmation are performed on the samples of the strains that rapidly degrade nitrogen in sewage in step 1, and a standard curve corresponding to the concentration of the samples of strains that rapidly degrade nitrogen in sewage and the critical cycle number is established;

Step 4: According to the standard curve corresponding to the concentration of the sample of the bacterial strain that rapidly degrades nitrogen in the sewage and the critical cycle number, the number of copies of the gene of the strain in the sample of the bacterial strain that rapidly degrades nitrogen in the sewage is obtained, that is, the obtained sample is obtained. The number of strains in a sample of strains described as rapidly degrading nitrogen in sewage.

Further, the method for obtaining the samples of the strains that rapidly degrade nitrogen in sewage is:

(1) Collect 5 mL of sewage, drop it into a conical flask containing 30 mL of enriched culture solution, and cultivate at 28°C for 3-5 days;

(2) get the liquid after the culture and drop it on the plate, and adopt the dilution coating plate method to separate;

(3) Cultivated at 28°C for 2 days, the strains falling on the slope are the samples of strains that rapidly degrade nitrogen in sewage.

Further, the strain standard product for rapidly degrading nitrogen in sewage comprises recombinant plasmids or recombinant cells of the strain genes.

Further, the preparation method of the bacterial standard product for rapidly degrading nitrogen in sewage is as follows:

(1) Design specific primers for the genes of the strains that can degrade nitrogen in the sewage;

(2) taking a sample of the bacterial strain that rapidly degrades nitrogen in sewage, using a DNA extraction kit to extract and purify total DNA to obtain a DNA sample, and using the DNA sample as a template for PCR amplification;

(3) purifying the PCR amplification product, and linking the purified PCR amplification product with the plasmid vector to obtain a white colony transformed with the plasmid;

(4) Select the full colonies to inoculate into the culture medium, cultivate overnight, and extract the plasmids by using a plasmid extraction kit to obtain a strain standard that can rapidly degrade nitrogen in sewage.

Further, the method for PCR amplification is:

DNA extraction from samples of strains that rapidly degrade nitrogen in sewage;

Carry out the construction of PCR amplification system;

The PCR reaction was carried out at 95°C and deformed for 3 min, then denatured at 93°C for 30s, annealed at 55°C for 30s, and extended at 72°C for 3 min; 45 cycles, and finally extended at 70°C for 4 min.

Further, the method for the construction of the PCR amplification system is:

1) carrying out several times of gradient dilution of the plasmid standard, as quantitative PCR template DNA, and carrying out real-time quantitative PCR amplification;

2) Follow the instructions of the kit, add the reagents required for real-time quantitative PCR, the PCR reaction system is 20 μL, add 2 μL of plasmid standard as the reaction template, and carry out the real-time quantitative PCR reaction;

3) Take deionized water as the negative control, take the logarithmic value of the standard dilution gradient as the abscissa, and take the critical cycle number as the ordinate to establish the standard curve of real-time quantitative PCR.

Further, the PCR reaction system is:

3 μL of glycerol with a mass ratio of 50%, 1 μL of dimethyl sulfoxide, 1 μL of deoxyribonucleoside triphosphate with a concentration of 2.5 mol/L, 2 μL of Tris-HCl with a concentration of 25 mmol/L, 2 μL of MgCl with a concentration of 25 mmol/L _2. 0.5μL Taq enzyme and two primers with a concentration of 2U/L.

Further, the PCR amplification product is confirmed by electrophoresis on an agarose gel electrophoresis apparatus.

Another object of the present invention is to provide an application method for the detection method of a bacterial strain that rapidly degrades nitrogen in sewage, and the application method of the detection method for a bacterial strain that rapidly degrades nitrogen in sewage is:

Cultivate the bacterial strains that rapidly degrade nitrogen in sewage to obtain bacterial cells, collect the bacterial cells, and inoculate them into sewage;

Fermentation was carried out at 35°C and shaking at 150rpm for 40h.

Further, the operating conditions applied to sewage treatment are:

Dissolved oxygen is 0.5mg/L～5.0mg/L, carbon-nitrogen mass ratio is 15～25, temperature is 35℃, pH=7.0.

To sum up, the advantages and positive effects of the present invention are as follows: the detection standard product of the bacterial strain that rapidly degrades nitrogen in sewage provided by the present invention has the characteristics of a combination of spectral recognition and specificity, and has high sensitivity; the detection method is simple, only The test results can be obtained by analyzing the standard curve corresponding to the concentration of the samples of the strains that rapidly degrade nitrogen in sewage and the critical cycle number; the linear detection range is wide, and the strains that degrade nitrogen in the surface sewage can be rapidly and quantitatively detected, and the realization of sewage Reduction in denitrification costs.

Description of drawings

FIG. 1 is a flow chart of a method for detecting a strain that rapidly degrades nitrogen in sewage according to an embodiment of the present invention.

FIG. 2 is a flowchart of a method for obtaining a sample of a strain that rapidly degrades nitrogen in sewage according to an embodiment of the present invention.

3 is a flow chart of a method for preparing a bacterial standard product for rapidly degrading nitrogen in sewage provided in an embodiment of the present invention.

FIG. 4 is a flowchart of a method for constructing a PCR amplification system provided in an embodiment of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

In view of the problems existing in the prior art, the present invention provides a method for detecting a strain that rapidly degrades nitrogen in sewage and its application. The present invention is described in detail below with reference to the accompanying drawings.

As shown in Figure 1, the detection method of the bacterial strain that rapidly degrades nitrogen in sewage provided by the embodiment of the present invention comprises the following steps:

S101, obtaining a sample of a strain that rapidly degrades nitrogen in sewage, and obtaining a standard product of a strain that rapidly degrades nitrogen in sewage;

S102, establishing a standard curve corresponding to the concentration of the strain standard product that rapidly degrades nitrogen in sewage and the critical cycle number;

S103, performing PCR amplification and electrophoresis confirmation on the samples of the strains that rapidly degrade nitrogen in sewage in S101, and establishing a standard curve corresponding to the concentration of the samples of strains that rapidly degrade nitrogen in sewage and the critical cycle number;

S104, according to the standard curve corresponding to the concentration of the sample of the strain that rapidly degrades nitrogen in the sewage and the critical cycle number, obtain the copy number of the gene of the strain in the sample of the strain that rapidly degrades nitrogen in the sewage, that is, to obtain the Number of strains in a sample of strains that rapidly degrade nitrogen in sewage.

As shown in FIG. 2 , the method for obtaining a sample of a strain that rapidly degrades nitrogen in sewage provided by the embodiment of the present invention is as follows:

S201, collect 5 mL of sewage, drop it into a conical flask containing 30 mL of enriched culture solution, and cultivate at 28°C for 3-5 days;

S202, take the cultured liquid and drop it on the plate, and separate by the dilution coating plate method;

S203, cultured at 28°C for 2 days, the strains falling on the slope are the samples of strains that rapidly degrade nitrogen in sewage.

As shown in Figure 3, the preparation method of the bacterial standard product for rapidly degrading nitrogen in sewage provided by the embodiment of the present invention is as follows:

S301, design specific primers for the genes of the strains that can degrade nitrogen in the sewage;

S302, taking a sample of the strain that rapidly degrades nitrogen in sewage, extracting and purifying total DNA using a DNA extraction kit to obtain a DNA sample, and performing PCR amplification with the DNA sample as a template;

S303, purifying the PCR amplification product, and linking the purified PCR amplification product with a plasmid vector to obtain a plasmid-transformed white colony;

S304, selecting the full colony to inoculate in the culture medium, culturing overnight, extracting the plasmid by using a plasmid extraction kit, to obtain a strain standard product for rapidly degrading nitrogen in sewage.

As shown in Figure 4, the method for constructing the PCR amplification system provided in the embodiment of the present invention is:

S401, carrying out several fold gradient dilutions on the plasmid standard, as quantitative PCR template DNA, and performing real-time quantitative PCR amplification;

S402, operate according to the kit instructions, add reagents required for real-time quantitative PCR, the PCR reaction system is 20 μL, add 2 μL of plasmid standard as a reaction template, and perform real-time quantitative PCR reaction;

S403, taking deionized water as a negative control, taking the logarithmic value of the dilution gradient of the standard product as the abscissa, and taking the critical cycle number as the ordinate to establish a standard curve of real-time quantitative PCR.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

<110> Taizhou Vocational and Technical College

<120> A detection method and application of strains that rapidly degrade nitrogen in sewage

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Claims (11)

1. A method for detecting a bacterial strain capable of rapidly degrading nitrogen in sewage is characterized by comprising the following steps:

acquiring a sample of a strain for rapidly degrading nitrogen in sewage to obtain a standard product of the strain for rapidly degrading the nitrogen in the sewage;

establishing a standard curve corresponding to the concentration of a bacterial strain standard substance for rapidly degrading nitrogen in the sewage and the critical cycle number;

performing PCR amplification and electrophoresis confirmation on the sample of the strain for rapidly degrading the nitrogen in the sewage in the step one, and establishing a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number;

and step four, obtaining the copy number of the strain gene in the sample of the strain for rapidly degrading the nitrogen in the sewage according to a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number, and obtaining the number of the strains in the sample of the strain for rapidly degrading the nitrogen in the sewage.

2. The method for detecting the bacterial strain capable of rapidly degrading the nitrogen in the sewage according to claim 1, wherein the method for obtaining the sample of the bacterial strain capable of rapidly degrading the nitrogen in the sewage comprises the following steps:

(1) collecting sewage of 5m L, adding dropwise into a triangular flask containing 30m L of enrichment culture solution, and culturing at 28 deg.C for 3-5 days;

(2) dripping the cultured liquid onto a flat plate, and separating by adopting a dilution coating flat plate method;

(3) culturing for 2 days at 28 ℃, wherein the strain falling into the inclined plane is a sample of the strain for rapidly degrading nitrogen in the sewage.

3. The method for detecting a strain capable of rapidly degrading nitrogen in sewage according to claim 1, wherein the standard substance of the strain capable of rapidly degrading nitrogen in sewage comprises a recombinant plasmid or a recombinant cell of a gene of the strain.

4. The method for detecting the strain capable of rapidly degrading the nitrogen in the sewage according to claim 1, wherein the preparation method of the standard strain capable of rapidly degrading the nitrogen in the sewage comprises the following steps:

(1) designing specific primers aiming at the genes of the bacterial strains capable of degrading nitrogen in sewage;

(2) taking a sample of the strain for rapidly degrading nitrogen in the sewage, extracting and purifying total DNA by using a DNA extraction kit to obtain a DNA sample, and performing PCR amplification by using the DNA sample as a template;

(3) purifying the PCR amplification product, and linking the purified PCR amplification product with a plasmid vector to obtain a white colony transformed with the plasmid;

(4) and (3) selecting a full colony to inoculate in a culture solution, culturing overnight, and extracting plasmids by using a plasmid extraction kit to obtain a bacterial strain standard substance for rapidly degrading nitrogen in sewage.

5. The method for detecting the bacterial strain for rapidly degrading the nitrogen in the sewage according to claim 1, wherein the PCR amplification method comprises the following steps:

extracting DNA of a sample of a strain for rapidly degrading nitrogen in sewage;

constructing a PCR amplification system;

PCR reaction was performed.

6. The method according to claim 4, wherein the PCR reaction is performed at 95 ℃ for 3min, followed by denaturation at 93 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 3 min; 45 cycles, and final extension at 70 ℃ for 4 min.

7. The method for detecting the bacterial strain capable of rapidly degrading the nitrogen in the sewage according to claim 4, wherein the method for constructing the PCR amplification system comprises the following steps:

1) carrying out gradient dilution on the plasmid standard substance by a plurality of times to be used as a quantitative PCR template DNA, and carrying out real-time quantitative PCR amplification;

2) operating according to the kit instructions, adding reagents required by real-time quantitative PCR, wherein the PCR reaction system is 20 mu L, and adding 2 mu L plasmid standard substance as a reaction template to perform real-time quantitative PCR reaction;

3) and (3) establishing a real-time quantitative PCR standard curve by taking deionized water as a negative control, taking the logarithm value of the dilution gradient of the standard substance as an abscissa and taking the critical cycle number as an ordinate.

8. The method according to claim 6, wherein the PCR reaction system comprises 50% by mass of glycerol 3 μ L, dimethyl sulfoxide 1 μ L, deoxyribonucleoside triphosphate 1 μ L at 2.5 mol/L, Tris-HCl 2 μ L at 25 mmol/L, MgCl 2 μ L at 25 mmol/L₂Taq enzyme with concentration of 0.5 mu L of 2U/L and two primers.

9. The method according to claim 1, wherein the PCR amplification product is confirmed by electrophoresis on an agarose gel electrophoresis apparatus.

10. An application method of the strain for rapidly degrading nitrogen in sewage according to any one of claims 1 to 8 is characterized in that the application method of the strain for rapidly degrading nitrogen in sewage comprises the following steps: culturing a strain capable of rapidly degrading nitrogen in sewage to obtain thalli, collecting the thalli, and inoculating the thalli into the sewage; fermenting at 35 deg.C under 150rpm shaking for 40 h.

11. The method for detecting bacterial strains capable of rapidly degrading nitrogen in sewage as claimed in claim 9, wherein the operating conditions for sewage treatment are that the dissolved oxygen is 0.5 mg/L-5.0 mg/L, the carbon-nitrogen mass ratio is 15-25, the temperature is 35 ℃, and the pH is 7.0.

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