CN108060243B - Method for detecting fixation strength of microorganisms and dirt purification carrier with high precision - Google Patents
Method for detecting fixation strength of microorganisms and dirt purification carrier with high precision Download PDFInfo
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Abstract
The invention provides a method for detecting the fixation strength of microorganisms and a dirt purification carrier with high precision, which comprises the following steps: (1) preparing a microorganism-loaded decontamination carrier; (2) setting N test groups according to sampling time; (3) carrying out running water washing on the microorganism-loaded sewage purification carrier, and sampling and retaining the water body and the sewage purification carrier; (4) extracting the DNA of the pollution cleaning carrier and the water body microorganism, and selecting a PCR amplification primer; (5) preparing a standard curve sample for detecting the abundance of the microorganisms; (6) configuring a PCR reaction system, setting a reaction program, and carrying out real-time fluorescence quantitative PCR; (7) and (4) calculating the abundance, the fixation rate and the effective fixation time of the microorganisms on the water body and the dirt purification carrier. The advantages are that: (1) the detection precision is high, and the error is small; (2) different microorganism groups can be accurately distinguished; (3) real-time and rapid detection; (4) the technical means is mature, simple and convenient.
Description
Technical Field
The invention relates to a method for detecting the fixation strength of microorganisms and a dirt purification carrier with high precision, belonging to the technical field of water environment restoration.
Background
In the technical field of water environment remediation, microbial remediation is favored in the aspects of river and lake pollution treatment, farmland water-withdrawal nitrogen and phosphorus removal, pesticide removal and the like at present due to the advantages of low energy consumption, no secondary pollution, high efficiency, simple and convenient operation, economy, practicality, wide application range and the like, and becomes an important way for removing pollutants in water environment. In actual in situ remediation applications, in order to exert the effects of indigenous microorganisms or artificially screened specific functional bacteria, microorganisms and a pollution cleaning carrier are usually coupled and attached by means of natural processes and artificially applied physical or chemical methods, and originally free microorganisms are loaded or limited to a specific surface space range of the pollution cleaning carrier, so that the self-reactivity of the microorganisms is maximally reserved and the functions of the microorganisms are exerted.
At present, people fix microorganisms on various pollution cleaning carriers (such as activated carbon, porous ecological bricks, organic glass, porous ceramsite, natural polymer carriers and the like) by utilizing an adsorption method, a crosslinking method, a covalent bonding method and the like, namely, microorganism immobilization. Compared with free microorganisms, the immobilized microorganisms have the advantages of high microorganism density, high reaction speed, easier control of the reaction process and the like, and are applied to practical production and have remarkable results. The fixation bonding strength of the microorganisms and the decontamination carrier is a key factor for controlling the repair efficiency of the microorganisms. The fixation method, strain characteristics, type of the decontamination carrier, hydrodynamic characteristics, etc. all have an effect on the fixation strength of the microorganisms and the decontamination carrier. Before practical application, according to hydrodynamic features of an area to be repaired, the fixation strength of microorganisms and a dirt purification carrier is determined to be particularly important for guaranteeing the repairing effect. However, the existing method for detecting the fixation strength of the microorganisms and the pollution cleaning carriers is very limited, and the existing method is poor in accuracy, most of the microorganism-loaded pollution cleaning carriers are applied in situ blindly without being detected by the fixation strength, and the results of serious microorganism shedding, poor repairing effect, high biological risk and the like are caused in some water areas. In chinese patent publication No. CN103940695A, a method for testing the fixation strength of microorganisms on a carbon fiber carrier is disclosed, in which a method for testing the fixation strength of microorganisms and a carbon fiber carrier is characterized by using a post-ultrasonic-oscillation weighing differential subtraction method, although the method quantitatively detects the fixation strength, ultrasonic-oscillation is not a real condition in an actual water environment, and the weighing differential subtraction method has the disadvantages of low accuracy, poor timeliness, and incapability of distinguishing different microorganism groups, and the like. The detection method has important significance for guiding people to scientifically select and optimally implement the microbial remediation technology according to local conditions.
Disclosure of Invention
The invention provides a method for detecting the fixation strength of microorganisms and a dirt purification carrier with high precision, and aims to overcome the defects of the existing method for detecting the fixation strength of the microorganisms and the dirt purification carrier, improve the accuracy and the timeliness and distinguish different microorganism groups.
The technical solution of the invention is as follows: a method for detecting the fixation strength of microorganisms and a dirt purification carrier with high precision specifically comprises the following steps:
(1) preparing a microorganism-loaded decontamination carrier, sampling and storing the decontamination carrier to be detected;
(2) setting N test groups (4 < N < 10) according to the sampling time, sterilizing the water body to be restored, and setting the flow rate of the water body in the water tank according to the flow rate of the water body to be restored;
(3) putting the microorganism-loaded clean carrier into a water tank, flushing the clean carrier by running water for different time, and sequentially sampling and retaining the water body and the clean carrier in the N test groups to be tested;
(4) extracting the DNA of the decontamination carrier and the water body microorganism remained in the steps (1) and (3), and selecting a PCR amplification primer according to a target gene of the detected microorganism;
(5) preparing a standard curve sample for detecting the abundance of the microorganisms;
(6) configuring a PCR reaction system, setting a reaction program, and simultaneously carrying out real-time fluorescence quantitative PCR on a sample to be detected and a manufactured marking sample;
(7) and (3) calculating the abundance of the microorganisms on the water body and the sewage purification carrier according to the standard curve, and calculating the fixation rate and the effective fixation time by analyzing the accuracy of the microbial abundance test method of the microorganisms lost into the water body, remained on the carrier and on the original carrier.
The invention has the advantages that:
1) the absolute abundance of the microorganisms is used for accurate quantification, the detection precision is high, and the error is small;
2) specific primers are utilized to carry out PCR amplification on specific gene segments, so that different microorganism groups can be distinguished;
3) the detection time is short, and the efficiency is high;
4) the detection process has mature technical means, is provided with a commercialized kit, and is simple and rapid.
Drawings
FIG. 1 is a schematic diagram of a standard curve for detecting bacterial abundance in the examples.
FIG. 2 is a graph showing the abundance of bacteria lost to the water as a function of wash time in the examples.
FIG. 3 is a graph showing the abundance of bacteria remaining on the carriers with wash time in the examples.
FIG. 4 is a graph showing the fixation rate of bacteria on the soil cleaning carrier as a function of the washing time in the examples.
Detailed Description
A method for detecting the fixation strength of microorganisms and a dirt purification carrier with high precision specifically comprises the following steps:
(1) preparing a microorganism-loaded decontamination carrier, sampling and storing the decontamination carrier to be detected;
(2) setting N test groups according to the sampling time, sterilizing the water body to be restored and setting the flow rate of the water body in the water tank according to the flow rate of the water body to be restored;
(3) putting the microorganism-loaded clean carrier into a water tank, flushing the clean carrier by running water for different time, and sequentially sampling and retaining the water body and the clean carrier in the N test groups to be tested;
(4) extracting the DNA of the decontamination carrier and the water body microorganism remained in the steps (1) and (3), and selecting a PCR amplification primer according to a target gene of the detected microorganism;
(5) preparing a standard curve sample for detecting the abundance of the microorganisms;
(6) configuring a PCR reaction system, setting a reaction program, and simultaneously carrying out real-time fluorescence quantitative PCR on a sample to be detected and a manufactured marking sample;
(7) and (3) calculating the abundance of the microorganisms on the water body and the sewage purification carrier according to the standard curve, and calculating the fixation rate and the effective fixation time by analyzing the accuracy of the microbial abundance test method of the microorganisms lost into the water body, remained on the carrier and on the original carrier.
The pollution cleaning carrier in the step (1) is preferably a porous carrier material, the loaded microorganism is a functional microorganism strain, flora or indigenous microorganism obtained by artificial screening, and when the pollution cleaning carrier loaded with the microorganism is prepared, the pollution cleaning carrier is preferably placed in a natural water body for a period of time, so that a biofilm formed by the indigenous microorganism is formed on the surface of the pollution cleaning carrier; if functional microbial strains or floras are selected, a co-culture adsorption method is preferred, so that the uniformity of the microbes loaded on the surface of the decontamination carrier is ensured as much as possible.
Setting N test groups according to the sampling time in the step (2): taking a water body to be restored, sterilizing the water body at high temperature (120 ℃, 20 minutes), adding the sterilized water body into N water tanks, uniformly setting the flow rate of the water body in the water tanks according to the flow rate of the water body to be restored, wherein the N water tanks correspond to N test groups (4 < N < 10).
In the step (3), the clean carrier is placed in a water tank, then the flowing water flushing time is recorded as t =0 min, the water body and the clean carrier in the N test groups are respectively sampled at different flushing time (0 < t <180 min), the water body in the water tank is filtered by a 0.22 mu m filter membrane to be used for collecting the microorganisms in the water body, and the filter membrane and the clean carrier sample of each test group are placed in a refrigerator at the temperature of-20 ℃ for storage and are used for extracting the microorganism DNA sample in the step (4).
Extracting the microorganism DNA in the decontamination carrier and the water body remained in the step (4) by using a soil DNA Extraction kit and a water body DNA Extraction kit (PowerSoil and PowerWater DNA Extraction kit are recommended); selecting different amplification genes according to different detection target microorganisms, and selecting corresponding amplification primers.
Sample preparation for detecting the microbial abundance standard curve in the step (5): the concentration of the DNA template is firstly measured, and then the DNA template is diluted by 10 times of gradient to form at least 6 gradients.
The total volume of the PCR reaction system in the step (6) is preferably 20 mu l, the reaction system comprises 8.4 mu l of sterile water, 10 mu l of fluorescent quantitative reagent (SYBR Green real is recommended), 0.6 mu l of forward primer (20 mu M), 0.6 mu l of reverse primer (20 mu M) and 0.4 mu l of DNA sample, wherein the DNA sample comprises a standard curve sample, a water body to be detected and a DNA sample in a decontamination carrier; after the PCR reaction system is prepared, the PCR reaction system is placed in a real-time fluorescence quantitative PCR instrument, a PCR reaction program is set, usually at 94 ℃ for 1 minute and at 40 cycles (denaturation at 94 ℃ for 30 seconds, annealing at 50-62 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds), and the specific annealing temperature is selected according to the detection target gene.
The step (7) is to draw a standard curve by taking the Ct value of the standard curve sample as an X coordinate and the known microbial abundance of the standard curve sample as a Y coordinate according to the fluorescent quantitative PCR result; according to the standard curve, calculating the abundance of microorganisms in the sample to be detected by using Ct values obtained by detecting the DNA samples of the water body and the clean carrier; the accuracy test method of the microorganism abundance detection method is that the sum of the abundance of the microorganisms in the water body and the microorganism remained on the carrier at a certain moment is calculated and compared with the abundance of the microorganisms on the original carrier, if the deviation of the two is within +/-5 percent, the accuracy of the detection method is higher; fixation rate (%) = (abundance of microorganism remaining on carrier at a certain time/abundance of microorganism on original carrier) × 100%; the effective fixation time is the time when the fixation rate begins to stabilize along with the flowing water scouring.
The following detailed description of the implementation steps of the invention is provided by specific examples for the purpose of further understanding the invention by those skilled in the art, but not for the purpose of limiting the invention in any way; it should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Examples
Method for detecting fixation strength of microorganisms and dirt purification carrier with high precision
The invention is utilized to degrade the organophosphorus pesticide chlorpyrifosSphingomonasDetecting the fixation strength of sp, Dsp-2 and activated carbon particles, comprising the following steps:
(1) preparation of the load by Co-culture adsorptionSphingomonasActivated carbon particles of sp, Dsp-2: will be provided withSphingomonasInoculating sp, Dsp-2 strain to 4L LB culture medium (LB culture medium containing tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, pH 7.0) and culturing in shaking table at 200 rmp at 30 deg.C for 12 hr, and adsorbing the degrading bacteria on the porous surface of active carbon to form loadSphingomonasActivated carbon particles of sp, Dsp-2; taking 10 g of activated carbon particles loaded with degrading bacteria, placing at-20 ℃, and reserving for later use;
(2) set up 6 tests according to the sampling timeThe method comprises the steps of taking a water body in a drainage ditch of a certain paddy field in suburbs of Nanjing, sterilizing the water body at high temperature (120 ℃, 20 minutes), adding the sterilized water body into N water tanks, uniformly setting the flow rate of the water body in the water tanks according to the flow rate of the water body to be repaired, and enabling the N water tanks to correspond to N test groups (4)<N<10) Setting the water flow velocity in the water tank to be 4.2 cm s according to the water flow velocity in the drainage ditch of the suburb certain rice field to be repaired-1；
(3) Loading activated carbon particles loaded with degrading bacteria into a net frame (the aperture of the net frame is smaller than that of the activated carbon particles), placing the net frame into a water tank, recording the flowing water scouring time to be t =0 min, respectively sampling water and activated carbon in 6 test groups when the scouring time is 0, 15, 30, 60, 120 and 180 min, filtering the water in the water tank through a 0.22 mu m filter membrane to collect microorganisms in the water, and placing the filter membrane and the activated carbon (about 5 g) sample of each test group into a refrigerator at-20 ℃ for storage for extracting a microorganism DNA sample in the step (4);
(4) extracting microorganism DNA in the active carbon sample and the filter membrane sample remained in the steps (1) and (3) by using PowerSoil DNA Extraction kit and PowerWater DNA Extraction kit, wherein the specific steps refer to kit instructions, becauseSphingomonassp, Dsp-2 is bacteria, and can be used16SrRNAThe gene is used as a target gene for detection, and the primer selection is as follows: 5'-ACTCCTACGGGAGGCAGCAG-3' (forward primer), 5'-ATTACCGCGGCTGCTGG-3' (reverse primer);
(5) preparation of standard curve DNA samples: the DNA concentration of the activated carbon sample at t =0 was measured, and the sample was diluted with a 10-fold gradient to form 1 and 10-1、10-2、10-3、10-4、10-5A total of 6 concentration gradients;
(6) configuring a PCR reaction system and setting a PCR reaction program: the total volume of the PCR reaction system is 20 mul, the reaction system comprises 8.4 mul of sterile water, 10 mul of fluorescent quantitative reagent (SYBR Green real is recommended), 0.6 mul of forward primer (20 mul), 0.6 mul of reverse primer (20 mul) and 0.4 mul of DNA sample, wherein the DNA sample comprises a standard sample, a water body to be detected and a DNA sample in active carbon; after the PCR reaction system is prepared, placing the PCR reaction system in a real-time fluorescent quantitative PCR instrument, and setting a PCR reaction program, wherein the PCR reaction program is usually 94 ℃ for 1 minute and 40 cycles (denaturation 94 ℃ for 30 seconds, annealing 55 ℃ for 30 seconds, and extension 72 ℃ for 30 seconds);
(7) according to the fluorescent quantitative PCR result, taking the Ct value of the standard curve sample as an X coordinate, and taking the known bacterial abundance of the standard curve sample as a Y coordinate to draw a standard curve (see attached figure 1); according to a standard curve, Ct values obtained by detecting water and activated carbon DNA samples are used for calculating the abundance of bacteria in the sample to be detected, the abundance of the bacteria lost into the water changes with the washing time as shown in figure 2, and the abundance of the bacteria remained on the activated carbon changes with the washing time as shown in figure 3; fixation rate (%) = (abundance of bacteria remaining on carrier/abundance of bacteria on original carrier at a certain time) × 100%, and fixation rate of degraded bacteria on activated carbon at different times is shown in figure 4; the effective fixation time is the time when the fixation rate begins to stabilize along with flowing water scouring, and the effective fixation time in the embodiment is 60 minutes as can be seen from the attached figure 4;
(8) when the method is used for checking the accuracy, taking 60 minutes as an example, the sum of the abundance of bacteria remaining on the activated carbon and the water body at the moment is calculated to be 23.1 multiplied by 108 Number of copies g-1Activated carbon, bacterial abundance (23.3X 10) compared to the original activated carbon8 Number of copies g-1Activated carbon), both<And 5 percent, the detection method has higher accuracy.
Claims (5)
1. A method for detecting the fixation strength of microorganisms and a dirt purification carrier with high precision specifically comprises the following steps:
(1) preparing a microorganism-loaded decontamination carrier; the dirt purifying carrier is a porous carrier material; the loaded microorganism is a functional microorganism strain, flora or indigenous microorganism obtained by artificial screening, and is sampled and stored to be tested;
(2) setting N test groups according to the sampling time, sterilizing the water body to be restored and setting the flow rate of the water body in the water tank according to the flow rate of the water body to be restored;
(3) carrying out running water washing on the microorganism-loaded clean carrier, recording the running water washing time as t =0 min, and respectively sampling and retaining the water body and the clean carrier in the N test groups to be tested in different washing times, namely 0< t <180 min;
(4) extracting the DNA of the pollution cleaning carrier and the water body microorganism, and selecting a PCR amplification primer;
(5) preparing a standard curve sample for detecting the abundance of the microorganisms;
(6) configuring a PCR reaction system, setting a reaction program, and carrying out real-time fluorescence quantitative PCR;
(7) calculating the abundance, fixation rate and effective fixation time of microorganisms on the water body and the sewage purification carrier, and drawing a standard curve by taking the Ct value of the standard curve sample as an X coordinate and the known abundance of the microorganisms of the standard curve sample as a Y coordinate according to the fluorescent quantitative PCR result; according to the standard curve, calculating the abundance of microorganisms in the sample to be detected by using Ct values obtained by detecting the DNA samples of the water body and the clean carrier; the accuracy of the microorganism abundance detection method is determined by calculating the sum of the abundance of the microorganisms in the water body and the abundance of the microorganisms remaining on the carrier at a certain moment and comparing the sum with the abundance of the microorganisms on the original carrier; and then, calculating the fixation rate and the effective fixation time, wherein the method for calculating the fixation rate comprises the following steps: fixation rate = abundance of microorganisms remaining on the carrier at a certain time/abundance of microorganisms on the original carrier × 100%; the effective fixation time is the time when the fixation rate begins to be stable along with flowing water scouring;
in the step (3), the clean carrier is placed in a water tank, then the flowing water scouring time is recorded as t =0 min, the water body and the clean carrier in the N test groups are respectively sampled at different scouring times, namely 0< t <180 min, the water body in the water tank is filtered by a 0.22 mu m filter membrane to be used for collecting the microorganisms in the water body, and the filter membrane and the clean carrier sample of each test group are placed in a refrigerator at the temperature of-20 ℃ for storage and are used for extracting the microorganism DNA sample in the step (4);
extracting the microbial DNA in the dirt-removing carrier and the water body remained in the step (4) by using a soil DNA extraction kit and a water body DNA extraction kit respectively; selecting different amplification genes according to different detection target microorganisms, and selecting corresponding amplification primers.
2. The method according to claim 1, wherein the microorganism-loaded pollution cleaning carrier in step (1) is prepared by placing the pollution cleaning carrier in a natural water body for a period of time to form a biofilm formed by indigenous microorganisms on the surface of the pollution cleaning carrier; if functional microbial strains or floras are selected, a co-culture adsorption method is selected to ensure the uniformity of the microorganisms loaded on the surface of the dirt-removing carrier.
3. The method for detecting the anchorage strength of microorganisms and carriers with high precision according to claim 1, wherein 4< N < 10; sterilizing at 120 deg.C for 20 min; and adding the sterilized water body into N water tanks, wherein the flow rates of the water body in the water tanks are uniformly set according to the flow rate of the water body to be restored.
4. The method for detecting the anchorage strength of microorganisms and clean carriers with high accuracy as claimed in claim 1, wherein the step (5) of sample preparation of the standard curve for detecting the abundance of microorganisms comprises: the concentration of the DNA template is firstly measured, and then the DNA template is diluted by 10 times of gradient to form at least 6 gradients.
5. The method for detecting the anchorage strength of microorganisms and clean carriers with high precision as claimed in claim 1, wherein the total volume of the PCR reaction system in step (6) is 20 μ l, the reaction system comprises 8.4 μ l of sterile water, 10 μ l of fluorescent quantitative reagent, 0.6 μ l of forward primer, 0.6 μ l of reverse primer, and 0.4 μ l of DNA sample, wherein the DNA sample comprises the standard curve sample, the water to be detected and the DNA sample in the clean carriers; after the PCR reaction system is prepared, the PCR reaction system is placed in a real-time fluorescence quantitative PCR instrument, and a PCR reaction program is set, wherein the PCR reaction program is generally 94 ℃ for 1 minute, and the PCR reaction program is 40 cycles: denaturation at 94 ℃ for 30 seconds, annealing at 50-62 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds, wherein the specific annealing temperature is selected according to the detected target gene.
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