CN114544846B - Resistance gene research method under influence of tide in coastal region and solid phase extraction instrument - Google Patents
Resistance gene research method under influence of tide in coastal region and solid phase extraction instrument Download PDFInfo
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Abstract
The invention relates to a method for researching resistance genes under the influence of tides in coastal areas and a solid-phase extraction instrument, wherein the method comprises the steps of arranging sampling points in coastal areas, sampling samples at two depths of a surface layer and a shallow layer at the sampling points after a tide and a tide are increased, refrigerating the samples to be taken back to a laboratory for rationalization analysis and grinding, then carrying out solid-phase extraction, and carrying out mass spectrometry to obtain antibiotic types, recovery rates and detection limits of samples at various points of a beach; and quantifying the types and the abundance of the resistance genes of the sample, and providing value data for the research of the distribution characteristics of the resistance genes under the influence of tide, the resistance genes and the mobile genetic elements. This solid-phase extraction appearance advance the kind basin and cut apart and be a plurality of sectorial compartments, be convenient for carry out multiunit sample extraction simultaneously, improve pretreatment efficiency.
Description
Technical Field
The invention relates to a method for researching resistance genes under the influence of tides in coastal areas and a solid-phase extraction apparatus, belonging to the technical field of marine science.
Background
Today, with the integration of global economy and regional economy, coastal cities which have regional advantages and take charge of precious resources become major cities for economic development, and are gathering places of population, economy and industry, so that the research on environmental problems of coastal areas is very important.
In recent years, due to the fact that the abuse of antibiotics causes the resistance genes to be widely spread in aquatic environments, marine environments are also a library of the resistance genes, the antibiotic resistance becomes a global important public safety problem threatening human health, and the research on the resistance genes in coastal regions has great significance.
At present, research on resistance genes in coastal regions focuses on the type, abundance, distribution and propagation of the resistance genes in the coastal regions, and few researches are carried out on the change of the resistance genes under the influence of tides in the coastal regions, so that a method needs to be established for researching the evolution rule of the resistance genes by the tidal change.
Antibiotics are mainly discharged by human beings, resistance genes spread from sewers, sewage treatment plants, farms and the like of hospitals can finally flow into the ocean, and the research on the distribution and change of the resistance genes under the influence of the tide in coastal areas can provide scientific basis for the research on the spread of the resistance genes of the ocean and the pollution prevention and treatment.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for researching resistance genes under the influence of tides in coastal regions and a solid-phase extractor, and the specific technical scheme is as follows:
a method for researching resistance genes under the influence of tides in coastal areas comprises the following steps:
seaside field operations
Step 1: determining the layout of the sampling points of the coastal mudflat: according to a topographic map and a water system map of a sampling area, distributing points along a coastline according to high, medium and low tide zones, distributing N points in each tide zone, wherein N is more than or equal to 5 and less than or equal to 15, and X is more than or equal to 50 and less than or equal to 150 at intervals of adjacent points;
step 2: determining a sampling period: according to a tide schedule of a national ocean information center, respectively selecting a big tide with the largest tidal range and a small tide with the smallest tidal range for sampling, and mastering the evolution rules of rising and falling tides in time and space;
and step 3: performing site survey and stationing verification: surveying the site sampling points by using a Beidou positioning system, observing whether the stationing environment is suitable for sampling, recording site conditions and beach substrate types, avoiding selecting points in a vegetation center zone, avoiding areas with various substrates distributed in a staggered manner, reasonably adjusting the sampling points, and determining a sampling method of each point;
and 4, step 4: sampling at each point: according to a field investigation and determination sampling method, sampling is carried out on each point in the time periods of heavy tide and light tide respectively, the sampling depth is two depths of a surface layer and a superficial layer, samples are collected on each point at two depths, each sampling depth of each point collects a sample Ykg, Y is more than or equal to 0.5 and less than or equal to 1.5, the samples are loaded into a sterilization plastic bag, each point is sampled at least twice, a label is attached to the outside of the sterilization plastic bag and marks the sampling name, the point, the time, the place and the information of sampling personnel, then the sterilization plastic bag is placed into a shading low-temperature box and stored by an ice bag to be used as a substrate sample, and the substrate sample is transported back to a laboratory for next treatment;
laboratory operations
And 5: determining the occurrence characteristics of the antibiotics:
step 51, pretreatment of a substrate sample: equally dividing a substrate sample in each sterilized plastic bag into two parts, wherein one part of the substrate sample is refrigerated at-4 ℃ for storage and is used for analyzing physicochemical indexes, the other part of the substrate sample is frozen at-20 ℃ for 24h, and then is dried in a 105 +/-5 ℃ blast drying oven to constant weight, removing stones and tiny animal and plant impurities in the air-dried substrate, grinding and sieving by a 2mm sieve to obtain a fine particle sample, grinding the unscreened substrate sample again, sieving by a 2mm sieve again after grinding, repeating for multiple times, uniformly mixing all the fine particle samples, putting the mixture into a self-sealing bag, sealing, and storing at-20 ℃;
step 52, solid-liquid extraction:
step 521, mixing a PBS buffer solution with acetonitrile, preparing an acetonitrile extraction solvent with a pH =3, sampling 5g of fine particle samples at multiple points prepared in step 51, respectively, adding the fine particle samples and 10mL of the acetonitrile extraction solvent into 50mL centrifuge tubes and labeling, modulating the amplitude of a shaker at 200rpm, oscillating for 10min at room temperature, ultrasonically extracting for 10min, fully mixing the fine particle samples and the acetonitrile extraction solvent to form a suspension, modulating the rotation speed of a centrifuge to 5000r/min, centrifuging for 10min, centrifuging the fine particle samples in the suspension in the centrifuge tubes to precipitate at the bottom, extracting antibiotics in the fine particle samples by the acetonitrile extraction solvent, adding the antibiotics into a supernatant, respectively collecting the supernatants in the centrifuge tubes, retaining the fine particle sample precipitates, continuously adding 10mL of the acetonitrile extraction solvent into each centrifuge tube, oscillating, ultrasonically centrifuging, taking the supernatant, adding 10mL of acidified acetonitrile into the centrifuge tube precipitates, oscillating, ultrasonically centrifuging, taking the supernatants, combining the supernatants extracted in the centrifuge tubes for three times, adding ultrapure water to 300mL, and obtaining diluted samples at each layer with constant volume;
522, assembling all components of the solid phase extraction instrument, placing no test tube or test tube rack in a vacuum tank, using the vacuum tank as a sample waste liquid cylinder, pouring the diluent of each layer of sample at each point position prepared in the step 521 into each sample introduction water tank of the solid phase extraction instrument, and labeling until the sample introduction water tanks are full of bins;
523, connecting the lower end outlet of the strong anion exchange LC-SAX column to the inlet at the upper end of the HLB column to form a series column (hereinafter referred to as series column) integrating the LC-SAX and HLB columns, installing the lower end of the HLB column, i.e., the lower end of the series column, on the liquid inlet of the solid phase extractor, connecting the vacuum pump to the vacuum tank, starting the air suction mode, opening the switches of the flow control valves, sucking gas from the upper end of the series column, activating the series column sequentially using 10mL of 100% (volume ratio) methanol solution and 10mL of ultrapure water, allowing the methanol solution and the ultrapure water to rapidly pass through the series column under the influence of gravity and negative pressure, entering the waste liquid tank of the vacuum tank 522, after activation, placing one end of the liquid transfer tube into the dilution of the sample introduction water tank, connecting one end of the liquid transfer tube to the upper end of the corresponding LC-SAX column through a closed interface, allowing the dilution of the sample introduction water tank to pass through the series column under the influence of the negative pressure of the vacuum pump, entering the waste liquid transfer tube into the waste liquid tank, forming the target object in the liquid transfer tube in the series column, adding ultrapure water to the water tank, cleaning the series column, and removing the sample transfer tube after the sample transfer tube, and removing the sample transfer tube. Placing the test tube and the test tube rack into a vacuum tank, aligning each test tube port with an HLB column above, continuously opening a vacuum pump, vacuumizing at negative pressure until the vacuum tank is dried for 10min, eluting the HLB target object with 10mL of methanol to obtain an eluent target object in the test tube, blowing the collected eluent into the vacuum tank at 40 ℃ till the eluent is nearly dry, then diluting the volume to 1mL with methanol containing 0.1% (volume ratio) formic acid, filtering with a 0.22 mu m filter membrane, storing in a brown sample sending bottle to be tested, and forming a sample to be tested solution;
performing three parallel experiments by the steps 521-524, namely taking 15g of each substrate sample, dividing the substrate sample into three groups, taking 5g of each group as a fine particle sample to perform the experiment, and setting a blank control group;
step 53, preparing an antibiotic standard stock solution: weighing an antibiotic standard substance, dissolving the antibiotic standard substance by using a methanol solution and preparing into an antibiotic standard solution;
step 54, analyzing the antibiotic occurrence characteristics of the sample solution to be detected in the step 524 by using a high performance liquid chromatography-tandem mass spectrometer, setting a gradient elution program according to the use instruction of the chromatographic separation column to obtain mass spectrum analysis parameters of the antibiotic, performing integral analysis on the off-line original data, and comparing the integrated analysis parameters with the antibiotic standard solution in the step 53 to obtain the antibiotic type, recovery rate and detection limit of each point sample of the mudflat;
step 61, DNA extraction: extracting total DNA of microorganisms in sediments by adopting a DNA kit, taking mudflat substrate samples at all points from the sterilized plastic bag, performing three repeated experiments on each sample, determining and evaluating the concentration and quality of the DNA by adopting a spectrophotometer, performing the extraction step according to the kit specification, and storing the extracted DNA samples at-20 ℃ until the extracted DNA samples are analyzed in subsequent experiments;
step 62, PCR amplification and plasmid construction: setting a target gene PCR amplification system, carrying out primer, sequence, fragment size and annealing temperature on a resistance gene to obtain a PCR product, carrying out agarose gel electrophoresis on the product, tapping the single fragment with consistent size of a fragment band, purifying the fragment by using a DNA purification kit, connecting the fragment to a pMD19-T plasmid vector, transferring the fragment to an LB (lysogeny broth) flat plate, carrying out overnight culture, selecting a positive clone colony, centrifuging, collecting thalli, carrying out plasmid extraction by using a plasmid extraction kit, measuring the concentration of a plasmid solution by using a spectrophotometer, carrying out proportioning dilution by using a special diluent for a standard product, and drawing a quantitative standard curve of the resistance gene;
and 8, data analysis: the diversity and abundance of antibiotics, resistance genes and mobile genetic elements in the substrate samples of different tidal zones and different tidal periods are compared and analyzed, the differences are detected by using a minimum difference method and are analyzed by a one-factor variance analysis method, the correlation is analyzed by using a spearman correlation method, the coexistence pattern of the resistance genes and the mobile genetic elements is analyzed in a networking mode, the abundance and the composition of the resistance genes in different tidal zones and sampling points of the tidal zones are obtained, and the distribution characteristics of the resistance genes under the influence of tides and the coexistence pattern of the resistance genes and the mobile genetic elements are obtained.
Further, the geometric scale of the sampling area topography map and the water system map in the step 1 is 1:25 ten thousand, 10 dots are distributed on each tide zone, and the interval between every two dots is 100m.
Further, in the step 4, the surface layer is the surface of the substrate, the depth is represented by 0cm, the shallow layer represents that the surface layer is downward 20-30cm, and the depth is represented by 20-30 cm.
Furthermore, in the step 4, 1kg of depth of each layer of each sampling point is respectively sampled, and each point is sampled by adopting a mudflat substrate columnar collector.
The solid phase extraction instrument comprises a sample introduction water tank, a water tank bracket and a vacuum tank from top to bottom in sequence, wherein the sample introduction water tank is integrally in a cylindrical shape, M clapboards are arranged from the center line to the periphery and are divided into M +1 compartments, the sample introduction water tank is erected vertically above the vacuum tank through the water tank bracket,
a small column tray is arranged at the top of the vacuum groove, a plurality of small columns penetrate through the small column tray around the circle of the small column tray close to the edge of the small column tray, a flow control valve is arranged at the part of each small column above the small column tray, the vertical upper part of each flow control valve corresponds to one vacuum groove compartment, and a glass test tube is arranged vertically below each flow control valve;
the side wall of the vacuum groove is provided with a vacuum groove air exhaust hole and a vacuum pressure gauge, and the vacuum groove air exhaust hole is connected with an air exhaust pipe.
Furthermore, a columnar channel is reserved in the middle of the sample injection water tank to arrange a liquid conveying pipe, the liquid conveying pipe is connected with the sample injection water tank and the solid-phase extraction column, automatic sample injection is realized by means of negative pressure of the vacuum tank, and the bottom of the sample injection water tank is funnel-shaped.
Further, the glass test tube passes through the test-tube rack slope setting, the test-tube rack includes fixed upper tray and lower floor's tray through many test tube holders, reserve the interval between upper tray and the lower floor's tray, the round through-hole has all been seted up to upper tray and lower floor's tray, the through-hole of lower floor's tray encloses into the circle and is less than the circle that upper tray through-hole encloses, and every glass test tube all passes the through-hole of a set of correspondence of upper tray and lower floor's tray, and every glass test tube from the top down is inwards inclined.
The invention has the beneficial effects that:
the solid phase extraction instrument of the invention forms negative pressure through vacuum, can realize low-cost and low-energy consumption automatic sample introduction, does not need to be observed during the automatic sample introduction, and is time-saving and labor-saving.
The solid phase extraction apparatus comprises a plurality of sample introduction water tanks, and can extract a plurality of samples simultaneously, thereby greatly improving the extraction efficiency.
The test tube in the vacuum tank of the solid phase extractor is designed to be obliquely placed, an eluent target object can firstly drip to the inner side wall of the oblique test tube port and then slide into the test tube, so that the phenomenon that the liquid splashes outside due to the fact that the eluent target object directly drips into the liquid in the test tube is avoided, the loss of the eluent target object is minimized, and the eluent target object in other test tubes is prevented from being polluted.
The method provided by the invention realizes statistics of horizontal data and longitudinal data of the sampling region, is convenient for comprehensive comparison to generate high-value reference data, and can be used for more comprehensively researching the evolution rule of the resistance gene under the influence of tide.
Drawings
FIG. 1 is an exploded view of the solid phase extractor of the present invention,
FIG. 2 is a schematic diagram of the assembly of the solid phase extractor of the present invention,
in the figure: 1. a sample introduction water tank; 2. a sink bracket; 3. a flow control valve; 4. a pillar interface; 5. a vacuum tank; 6. a glass test tube; 7. a pillar; 8. a vacuum pressure gauge; 9. a vacuum groove air exhaust hole; 10. a test tube rack; 101. a test tube holder; 102. an upper tray; 103. a lower tray; 11. a pillar tray.
Detailed Description
The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic views illustrating only the basic structure of the present invention in a schematic manner, and thus show only the constitution related to the present invention.
As shown in fig. 1-2, the solid phase extractor of the present invention has the following specific structure: combine figure 1, mainly include from the top down and in proper order include advance a kind basin 1, basin support 2 and vacuum chamber 5, advance a kind basin 1 whole and be the cylinder shape, be provided with M baffle from the central line orientation all around, cut apart into M +1 compartments with it, the demonstration roll-off is provided with 17 baffles in the figure, cuts apart into 18 compartments, can test 18 samples simultaneously. Advance kind basin 1 and erect the perpendicular top at vacuum tank 5 through basin support 2, the basin support is formed by two wooden system rings and six post connections for take the basin on vacuum tank, reduce area, make things convenient for the advance kind operation of solid phase extraction.
A columnar channel is reserved in the middle of the sample injection water tank 1 to arrange a liquid conveying pipe, the liquid conveying pipe is connected with the sample injection water tank 1 and the solid-phase extraction column, automatic sample injection is realized by means of negative pressure of the vacuum tank, the bottom of the sample injection water tank 1 is funnel-shaped, the middle of the sample injection water tank is lower, and efficiency of extracting diluent by the liquid conveying pipe is improved.
The sample introduction water tank is used as a container for containing sample diluent, the material of the sample introduction water tank is plastic, the sample introduction water tank is light and corrosion resistant, the sample introduction water tank is easy to wash, the sample introduction water tank is in a cake shape, and each compartment corresponds to one flow control valve downwards. The flow control valve is used as a valve for controlling the sample injection flow of the diluent, a switch is opened during sample injection, and the valve is closed after sample injection is finished, so that air leakage is avoided.
A small column tray 11 is arranged at the top of the vacuum groove 5, a plurality of small columns 7 are arranged on the small column tray 11 in a penetrating manner around the edge of the small column tray 11, a flow control valve 3 is arranged on the part, above the small column tray 11, of each small column 7, a vacuum groove 5 compartment is arranged vertically above each flow control valve 3, and a glass test tube 6 is arranged vertically below each flow control valve 3; glass test tube 6 is used for adorning the container of liquid after the extraction, and glass test tube 6 inclines obliquely in the test-tube rack to be placed, and the glass test tube mouth of pipe just corresponds the pillar lower extreme export, and liquid drippage can not have the spill on the glass test tube inside wall, avoids leading to the risk of other medicament pollutions, and pillar interface 4 connects solid-phase extraction post and vacuum tank, and the diluent drops in the vacuum tank after solid-phase extraction post, pillar 11.
The side wall of the vacuum groove 5 is provided with a vacuum groove air suction hole and a vacuum pressure gauge 8, and the vacuum groove air suction hole is connected with an air suction pipe 9. The vacuum pressure gauge 8 is used for observing the negative pressure in the vacuum groove. The vacuum groove air exhaust hole is a point for extracting air in the vacuum groove, and an air exhaust pipe 9 (which can be a rubber hose) is connected with the air exhaust hole to a vacuum pump, and the air in the vacuum groove is extracted through the vacuum pump.
The test tube holder 101 is a part of the test tube rack, and is composed of three columns for connecting two trays of the test tube rack to ensure correct placement of the test tubes.
The specific method for researching the resistance gene under the influence of the tide in the coastal region comprises the following steps:
seaside field operations
Step 1: determining the layout of the sampling points of the coastal mudflat: according to 1: distributing points along a coastline according to high, medium and low tidal zones of a topographic map and a water system map of a sampling area with 25 ten thousand equal scales, distributing 10 points in each tidal zone, and drawing a sampling point location distribution map at the interval of 100m between adjacent points;
step 2: determining a sampling period: according to a tide schedule of a national ocean information center, respectively selecting a big tide with the largest tidal range and a small tide with the smallest tidal range for sampling, and mastering the evolution rules of rising and falling tides in time and space;
and step 3: and (3) carrying out stationing verification by site survey: surveying the site sampling point positions by using a Beidou positioning system, observing whether the stationing environment is suitable for sampling, recording the site condition and the beach substrate type, avoiding selecting points in the center zone of vegetation, avoiding the areas with various substrates distributed in a staggered manner, reasonably adjusting the sampling point positions and determining the sampling method of each point position;
and 4, step 4: sampling at each point: according to a field survey and determination sampling method, sampling is carried out on each point in the time period of a large tide and a small tide respectively, the sampling depth is 20-30cm from the surface layer to the bottom, each point collects samples at two depths, 10kg of sample is collected at each sampling depth of each point, each point is placed into a sterilization plastic bag, each point is sampled at least twice, a label is attached to the outside of the sterilization plastic bag, the sampling name, the point, the time, the place and the information of sampling personnel are marked, then the sterilization plastic bag is placed into a shading low-temperature box, stored by using an ice bag and taken as a substrate sample, and the substrate sample is transported back to a laboratory for next processing;
laboratory operations
Step 51, pretreatment of a substrate sample: equally dividing the substrate sample in each sterilized plastic bag into two parts, wherein one part of the substrate sample is refrigerated at-4 ℃ for storage and used for analyzing physicochemical indexes, the other part of the substrate sample is frozen at-20 ℃ for 24h, and then is dried in a 105 +/-5 ℃ blast drying oven to constant weight, stone blocks and tiny animal and plant impurities in the air-dried substrate are removed, the grinding is carried out and the grinding is carried out by a 2mm sieve, so as to obtain a fine particle sample, the unscreened substrate sample is ground again, the grinding is carried out by a 2mm sieve again, after repeated times, all the fine particle samples are uniformly mixed, put into a self-sealing bag for sealing, and stored at-20 ℃;
step 52, solid-liquid extraction:
step 521, mixing PBS buffer solution with acetonitrile, preparing acetonitrile extraction solvent with pH =3, sampling 5g of fine particle samples at multiple points prepared in step 51, respectively, loading the samples with 10mL of acetonitrile extraction solvent into 50mL centrifuge tubes and labeling, adjusting the amplitude of a shaker to 200rpm, oscillating for 10min at room temperature, ultrasonically extracting for 10min, fully mixing the fine particle samples with the acetonitrile extraction solvent to form suspension, adjusting the rotation speed of a centrifuge to 5000r/min for 10min, centrifugally precipitating the fine particle samples in the suspension in the centrifuge tubes at the bottom, extracting antibiotics in the fine particle samples by the acetonitrile extraction solvent, entering into supernatant, collecting supernatant in each centrifuge tube, retaining the fine particle sample precipitate, continuously adding 10mL of acetonitrile extraction solvent into each centrifuge tube, oscillating, ultrasonically centrifuging, taking supernatant, adding 10mL of acidified acetonitrile into the centrifuge tube precipitate again, oscillating, ultrasonically centrifuging, taking supernatant, combining supernatants extracted by each centrifuge tube three times, adding diluted ultrapure water to 300mL, and obtaining diluted ultrapure water of each layer of sample;
522, assembling all components of the solid phase extraction instrument, placing no test tube or test tube rack in a vacuum tank, using the vacuum tank as a sample waste liquid cylinder, pouring the diluent of each layer of sample at each point position prepared in the step 521 into each sample introduction water tank of the solid phase extraction instrument, and labeling until the sample introduction water tanks are full of bins;
523, connecting the lower end outlet of the strong anion exchange LC-SAX column to the inlet at the upper end of the HLB column to form a series column (hereinafter referred to as series column) integrating the LC-SAX and HLB columns, connecting the lower end of the HLB column, i.e., the lower end of the series column, to the liquid inlet of the solid phase extractor (i.e., the column port 4 at the upper end of the column 11 in fig. 1 and 2), connecting a vacuum pump to the vacuum tank, starting an air suction mode, opening switches of the flow control valves, sucking air from the upper end of the series column, activating the series column with 10mL of 100% (volume ratio) methanol solution and 10mL of ultrapure water in sequence, allowing the methanol solution and the ultrapure water to rapidly pass through the series column under the influence of gravity and negative pressure, entering the waste liquid cylinder of the vacuum tank 522, after activation, placing one end of the liquid transport tube into the diluent of the water tank, connecting one end of the liquid transport tube to the upper end of the corresponding LC-SAX column through a sealed port, allowing the sample diluent to flow through the series column under the influence of the vacuum pump and negative pressure, entering the waste liquid transport tube, flowing through the series column, removing the sample introduction diluent into the waste liquid transport tube, and removing the sample injection sample, after all the sample injection sample transport tube, removing the sample. Placing the test tube and the test tube rack into a vacuum tank, aligning each test tube port to an upper HLB column, continuously opening a vacuum pump, vacuumizing at negative pressure until the vacuum tank is dried for 10min, eluting the HLB target object by using 10mL of methanol, obtaining an eluent target object in the test tube, blowing the collected eluent to be nearly dry at 40 ℃ by using nitrogen, then diluting the volume of the eluent to 1mL by using methanol containing 0.1 percent (volume ratio) formic acid, filtering by using a 0.22 mu m filter membrane, and storing in a brown sample sending bottle to be detected to form a sample liquid to be detected;
performing parallel experiments for three times by the steps 521-524, namely taking 15g of each substrate sample, dividing the substrate sample into three groups, taking 5g of each group as a fine particle sample to perform the experiment, and setting a blank control group;
step 53, preparing an antibiotic standard stock solution: weighing an antibiotic standard substance, dissolving the antibiotic standard substance by using a methanol solution and preparing into an antibiotic standard solution;
step 54, analyzing the antibiotic occurrence characteristics of the sample solution to be detected in the step 524 by using a high performance liquid chromatography-tandem mass spectrometer, setting a gradient elution program according to the use instruction of the chromatographic separation column to obtain mass spectrum analysis parameters of the antibiotic, performing integral analysis on the off-line original data, and comparing the integrated analysis parameters with the antibiotic standard solution in the step 53 to obtain the antibiotic type, recovery rate and detection limit of each point sample of the mudflat;
step 61, DNA extraction: extracting total DNA of microorganisms in sediments by adopting a DNA kit, taking mudflat substrate samples at all points from the sterilized plastic bag, performing three repeated experiments on each sample, determining and evaluating the concentration and quality of the DNA by adopting a spectrophotometer, performing the extraction step according to the kit specification, and storing the extracted DNA samples at-20 ℃ until the extracted DNA samples are analyzed in subsequent experiments;
step 62, PCR amplification, plasmid construction: setting a target gene PCR amplification system, carrying out primer, sequence, fragment size and annealing temperature on a resistance gene to obtain a PCR product, carrying out agarose gel electrophoresis on the product, tapping on fragments with single fragment bands and consistent sizes, purifying the fragments by using a DNA purification kit, connecting the fragments to a pMD19-T plasmid vector, transferring the fragments to an LB (lysogeny broth) flat plate, carrying out overnight culture, selecting a positive clone colony, centrifuging the colony, collecting thalli, carrying out plasmid extraction by using a plasmid extraction kit, measuring the concentration of a plasmid solution by using a spectrophotometer, carrying out proportioning dilution by using a special diluent for a standard product, and drawing a quantitative standard curve of the resistance gene;
and 8, analyzing data: the diversity and abundance of antibiotics, resistance genes and mobile genetic elements in the substrate samples of different tidal zones and different tidal periods are compared and analyzed, the differences are detected by using a minimum difference method and are analyzed by a one-factor variance analysis method, the correlation is analyzed by using a spearman correlation method, the coexistence pattern of the resistance genes and the mobile genetic elements is analyzed in a networking mode, the abundance and the composition of the resistance genes in different tidal zones and sampling points of the tidal zones are obtained, the distribution characteristics of the resistance genes under the influence of tides and the coexistence pattern of the resistance genes and the mobile genetic elements provide important high-value data for the research of the resistance genes in coastal waters.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (6)
1. A method for researching resistance genes under the influence of tides in coastal areas is characterized in that: the method comprises the following steps:
seaside field operations
Step 1: determining the layout of the sampling points of the coastal mudflat: according to a topographic map and a water system map of a sampling area, distributing points along a coastline according to high, medium and low tide zones, distributing N points in each tide zone, wherein N is more than or equal to 5 and less than or equal to 15, and X is more than or equal to 50 and less than or equal to 150 at intervals of adjacent points;
step 2: determining a sampling period: according to a tide schedule of a national ocean information center, respectively selecting a big tide with the largest tidal range and a small tide with the smallest tidal range for sampling, and mastering the evolution rules of rising and falling tides in time and space;
and 3, step 3: and (3) carrying out stationing verification by site survey: surveying the site sampling point positions by using a Beidou positioning system, observing whether the stationing environment is suitable for sampling, recording the site condition and the beach substrate type, avoiding selecting points in the center zone of vegetation, avoiding the areas with various substrates distributed in a staggered manner, reasonably adjusting the sampling point positions and determining the sampling method of each point position;
and 4, step 4: sampling at each point: according to a field survey and determination sampling method, sampling is carried out on each point in the time period of a large tide and a small tide respectively, the sampling depth is two depths of a surface layer and a superficial layer, each point collects samples at the two depths, each sampling depth of each point collects the sample Ykg, Y is more than or equal to 0.5 and less than or equal to 1.5, each point is arranged in a sterilization plastic bag, each point is sampled at least twice, a label is attached to the outside of the sterilization plastic bag and marks the sampling name, the point, the time, the place and the information of a sampling person, then the sterilization plastic bag is arranged in a shading low-temperature box and stored by an ice bag to be used as a substrate sample, and the substrate sample is transported back to a laboratory for next processing;
laboratory operations
And 5: determining the occurrence characteristics of the antibiotics:
step 51, pretreatment of a substrate sample: equally dividing the substrate sample in each sterilized plastic bag into two parts, wherein one part of the substrate sample is refrigerated at-4 ℃ for storage and used for analyzing physicochemical indexes, the other part of the substrate sample is frozen at-20 ℃ for 24h, and then is dried in a 105 +/-5 ℃ blast drying oven to constant weight, stone blocks and tiny animal and plant impurities in the air-dried substrate are removed, the grinding is carried out and the grinding is carried out by a 2mm sieve, so as to obtain a fine particle sample, the unscreened substrate sample is ground again, the grinding is carried out by a 2mm sieve again, after repeated times, all the fine particle samples are uniformly mixed, put into a self-sealing bag for sealing, and stored at-20 ℃;
step 52, solid-liquid extraction:
step 521, mixing PBS buffer solution with acetonitrile, preparing acetonitrile extraction solvent with pH =3, sampling 5g of fine particle samples at multiple points prepared in step 51, respectively, loading the samples with 10mL of acetonitrile extraction solvent into 50mL centrifuge tubes and labeling, adjusting the amplitude of a shaker to 200rpm, oscillating for 10min at room temperature, ultrasonically extracting for 10min, fully mixing the fine particle samples with the acetonitrile extraction solvent to form suspension, adjusting the rotation speed of a centrifuge to 5000r/min for 10min, centrifugally precipitating the fine particle samples in the suspension in the centrifuge tubes at the bottom, extracting antibiotics in the fine particle samples by the acetonitrile extraction solvent, entering into supernatant, collecting supernatant in each centrifuge tube, retaining the fine particle sample precipitate, continuously adding 10mL of acetonitrile extraction solvent into each centrifuge tube, oscillating, ultrasonically centrifuging, taking supernatant, adding 10mL of acidified acetonitrile into the centrifuge tube precipitate again, oscillating, ultrasonically centrifuging, taking supernatant, combining supernatants extracted by each centrifuge tube three times, adding diluted ultrapure water to 300mL, and obtaining diluted ultrapure water of each layer of sample;
522, assembling all components of a solid phase extraction instrument, wherein the solid phase extraction instrument sequentially comprises a sample injection water tank (1), a water tank bracket (2) and a vacuum tank (5) from top to bottom, the sample injection water tank (1) is integrally cylindrical, M partition plates are arranged from the center line to the periphery of the sample injection water tank and are divided into M +1 compartments, the sample injection water tank (1) is erected above the vacuum tank (5) through the water tank bracket (2),
a small column tray (11) is arranged at the top of the vacuum groove (5), a plurality of small columns (7) penetrate through the small column tray (11) around the edge of the small column tray (11), a flow control valve (3) is arranged on the part, above the small column tray (11), of each small column (7), a vacuum groove (5) compartment is vertically arranged above each flow control valve (3), and a glass test tube (6) is arranged below each flow control valve (3);
the side wall of the vacuum groove (5) is provided with a vacuum groove air exhaust hole and a vacuum pressure gauge (8), and the vacuum groove air exhaust hole is connected with an air exhaust pipe (9);
placing no test tube and test tube rack in the vacuum tank, using as a sample waste liquid cylinder, pouring the diluent of each layer of samples at each point position prepared in the step 521 into each sample introduction water tank of the solid phase extraction instrument, and labeling until the sample introduction water tanks are full;
523, connecting an outlet at the lower end of a strong anion exchange LC-SAX column to an inlet at the upper end of an HLB column to form an LC-SAX and HLB integrated series column, arranging the lower end of the HLB column, namely the lower end of the series column, on a liquid inlet hole of a solid phase extractor, connecting a vacuum pump to a vacuum tank, starting an air suction mode, opening switches of flow control valves, sucking gas from the upper end of the series column, activating the series column by using 10mL of methanol solution with the volume ratio of 100% and 10mL of ultrapure water in sequence, allowing the methanol solution and the ultrapure water to rapidly pass through the series column under the influence of gravity and negative pressure, entering a waste liquid cylinder of the vacuum tank 522, after activation, putting one end of a liquid conveying pipe into a sample conveying trough diluent, connecting one end of the liquid conveying pipe to the upper end of the corresponding LC-SAX column through a closed interface, allowing the sample conveying pipe diluent to flow through the series column under the influence of negative pressure of the vacuum pump, entering the waste liquid cylinder of the vacuum tank, forming a target object in the series column after all the sample conveying pipe passes through the series column, cleaning the liquid conveying pipe to the water trough, removing all the ultrapure water in the sample conveying pipe, and removing the sample conveying pipe from the sample conveying pipe in the sample conveying pipe, and pouring the sample conveying pipe into the sample conveying pipe in the sample conveying chamber;
placing the test tube and the test tube rack into a vacuum tank, aligning each test tube port to an upper HLB column, continuously opening a vacuum pump, vacuumizing at negative pressure until the vacuum tank is dried for 10min, eluting the HLB target object by using 10mL of methanol, obtaining an eluent target object in the test tube, blowing the collected eluent to be nearly dry at 40 ℃ by using nitrogen, then diluting the eluent to 1mL by using methanol containing 0.1% formic acid in volume ratio, filtering by using a 0.22 mu m filter membrane, and storing in a brown sample sending bottle to be detected to form a sample liquid to be detected;
step 521-523, performing three parallel experiments, namely taking 15g of each substrate sample, dividing the substrate sample into three groups, taking 5g of each group as a fine particle sample to perform the experiment, and simultaneously setting a blank control group;
step 53, preparing an antibiotic standard stock solution: weighing an antibiotic standard substance, dissolving the antibiotic standard substance by using a methanol solution and preparing into an antibiotic standard solution;
step 54, analyzing the antibiotic occurrence characteristics of the sample solution to be detected by using a high performance liquid chromatography-tandem mass spectrometer, setting a gradient elution program according to the use instruction of the chromatographic separation column to obtain mass spectrum analysis parameters of the antibiotic, performing integral analysis on the original data of the next machine, and comparing the integrated analysis parameters with the antibiotic standard solution in the step 53 to obtain the antibiotic type, the recovery rate and the detection limit of each point sample of the mudflat;
step 6, quantifying the types and abundances of the resistance genes:
step 61, DNA extraction: extracting total DNA of microorganisms in sediments by adopting a DNA kit, taking mudflat substrate samples at all points from the sterilized plastic bag, performing three repeated experiments on each sample, determining and evaluating the concentration and quality of the DNA by adopting a spectrophotometer, performing the extraction step according to the kit specification, and storing the extracted DNA samples at-20 ℃ until the extracted DNA samples are analyzed in subsequent experiments;
step 62, PCR amplification, plasmid construction: setting a target gene PCR amplification system, carrying out primer, sequence, fragment size and annealing temperature on a resistance gene to obtain a PCR product, carrying out agarose gel electrophoresis on the product, tapping on fragments with single fragment bands and consistent sizes, purifying the fragments by using a DNA purification kit, connecting the fragments to a pMD19-T plasmid vector, transferring the fragments to an LB (lysogeny broth) flat plate, carrying out overnight culture, selecting a positive clone colony, centrifuging the colony, collecting thalli, carrying out plasmid extraction by using a plasmid extraction kit, measuring the concentration of a plasmid solution by using a spectrophotometer, carrying out proportioning dilution by using a special diluent for a standard product, and drawing a quantitative standard curve of the resistance gene;
step 7, the mobile genetic element is determined using high throughput PCR technology: the method is completed on a high-throughput fluorescent quantitative reaction platform, three technical repeats are set for all samples, blank negative controls are set for each pair of primers, and a PCR program is adjusted according to a specification to quantify the abundance of resistance genes and mobile genetic elements;
and 8, analyzing data: the diversity and abundance of antibiotics, resistance genes and mobile genetic elements in the substrate samples of different tidal zones and different tidal periods are compared and analyzed, the differences are detected by using a minimum difference method and are analyzed by a one-factor variance analysis method, the correlation is analyzed by using a spearman correlation method, the coexistence pattern of the resistance genes and the mobile genetic elements is analyzed in a networking mode, the abundance and the composition of the resistance genes in different tidal zones and sampling points of the tidal zones are obtained, and the distribution characteristics of the resistance genes under the influence of tides and the coexistence pattern of the resistance genes and the mobile genetic elements are obtained.
2. The method for researching a resistance gene under the influence of the tide in the coastal region according to claim 1, wherein: the equal scale of the topographic map and the water system map of the sampling area in the step 1 is 1:25 ten thousand, 10 dots are distributed on each tide zone, and the interval between every two dots is 100m.
3. The method for studying resistance gene under the influence of tide in coastal areas according to claim 1, wherein: and in the step 4, the surface layer is the surface of the substrate, the depth is represented by 0cm, the shallow layer represents that the surface layer is downward 20-30cm, and the depth is represented by 20-30 cm.
4. The method for studying resistance gene under the influence of tide in coastal areas according to claim 1, wherein: and 4, sampling 1kg of each sampling point at each layer depth in the step 4, and sampling each point by adopting a mudflat substrate columnar collector.
5. The method for studying resistance gene under the influence of tide in coastal areas according to claim 1, wherein: a columnar channel is reserved in the middle of the sample injection water tank (1) to arrange a liquid conveying pipe, the liquid conveying pipe is connected with the sample injection water tank (1) and the solid phase extraction column, automatic sample injection is realized by means of negative pressure of the vacuum tank, and the bottom of the sample injection water tank (1) is funnel-shaped.
6. The method for studying resistance gene under the influence of tide in coastal areas according to claim 1, wherein: glass test tube (6) set up through the test-tube rack slope, the test-tube rack includes fixed upper tray and lower floor's tray through many test tube holders, reserve the interval between upper tray and the lower floor's tray, the round through-hole has all been seted up to upper tray and lower floor's tray, the through-hole of lower floor's tray encloses into the circle and is less than the circle that upper tray through-hole encloses, and every glass test tube (6) all pass a set of corresponding through-hole of upper tray and lower floor's tray, and every glass test tube (6) from the top down inclines inwards.
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