CN116970719B - Method for indirectly detecting cadmium pollution in water - Google Patents
Method for indirectly detecting cadmium pollution in water Download PDFInfo
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- CN116970719B CN116970719B CN202311237895.6A CN202311237895A CN116970719B CN 116970719 B CN116970719 B CN 116970719B CN 202311237895 A CN202311237895 A CN 202311237895A CN 116970719 B CN116970719 B CN 116970719B
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Abstract
The invention belongs to the technical field of heavy metal detection methods, and particularly relates to a method for indirectly detecting cadmium pollution in water. The invention detects the ArWz-2 content of the gene in the red Mao Fudan soft-shelled turtle of the sample water body, discovers that the ArWz-2 content is related to the cadmium content in the water body, and can detect the cadmium content in the water body through the corresponding relation of the ArWz-2 content and the cadmium content in the water body so as to monitor the water quality. Compared with the traditional direct detection method, the method avoids the harm of the strong corrosive reagent to detection personnel, and has higher sensitivity to cadmium detection in water.
Description
Technical Field
The invention belongs to the technical field of heavy metal detection methods, and particularly relates to a method for indirectly detecting cadmium pollution in water.
Background
Cadmium is a non-essential element of human body, and usually exists in a compound state in nature, and the cadmium has low content and does not influence the health of the human body in a normal environment state. However, when the environment is polluted by cadmium, the cadmium can be enriched in organisms and enter the human body through a food chain to cause chronic poisoning. It is therefore necessary to monitor the cadmium content of water.
Cadmium pollution in water is mainly from surface runoff and industrial wastewater. Cadmium particles formed by lead zinc ore in the atmosphere, nonferrous metal smelting, burning and plastic product burning can enter water; cadmium discharged by catalysts, pigments, plastic stabilizers, synthetic rubber vulcanizing agents, bactericides and the like which are used as raw materials of the pot also can pollute the water body, and in the process of urban water, the cadmium content in the drinking water can be increased due to the pollution of containers and pipelines. The discharge of industrial wastewater results in higher cadmium content in offshore seawater and plankton than in open sea.
The cadmium content in the seawater is generally 0.1-0.15 mug/L, and for the cadmium content standard in the seawater water quality detection, the method generally recognizes that: for the first type of the water treatment agent which is suitable for protecting marine biological resources and human safety utilization (including water for salt farms, food processing, sea water desalination, fishery, mariculture and the like) and offshore natural protection areas, the cadmium content is not more than 0.005mg/L; the second type is suitable for the sea bathing place, landscape sightseeing area and the third type is suitable for general industrial water, harbor water area, ocean development operation area and the like, and the cadmium content is not more than 0.01mg/L.
Various methods are disclosed in the prior art for testing the cadmium content in a water sample, and spectrophotometry, such as extraction-spectrophotometry, is most commonly used, has high technical requirements and long analysis time, and in order to improve the sensitivity of atomic absorption spectrophotometry, the sample is generally subjected to concentration treatment by an extraction method. However, the detection method involves a highly corrosive reagent in many cases, and the sensitivity is not high enough.
Water quality detection by means of biological features in water has been disclosed in the prior art. If the water pollution condition is judged according to the flora characteristic in the water body, as disclosed in the patent with the application number of CN202210983119.X, a river ecosystem health evaluation method based on the specific response of a microbial community comprises the steps of measuring 12 indexes of pH, water Temperature (WT), dissolved Oxygen (DO), COD, TN, total organic carbon, nitrate nitrogen, ammonium nitrogen, nitrite nitrogen, total phosphorus, sulfide and fluoride of the water body, and calculating a water quality grade index WQI; extracting microorganism DNA of a water body and sequencing a bacterial 16s rRNA amplicon; obtaining a bacterial genus level information table, and screening effective bacterial genus information; dividing microbial sensitivity based on the ecological niche model; and (5) evaluating reliability analysis of the result. The method can rapidly, accurately and objectively reflect the health condition of the urban river ecosystem, but on one hand, the method consumes large time and material resources for sequencing microorganisms, and on the other hand, the method has weak pertinence to specific substances in the water body, and in addition, the sensitivity is not high enough.
The prior art also discloses a method for reflecting the quality level of water body by detecting specific biomarkers in specific species, for example, patent application number CN202110227984.7 discloses a group of red midge Hb genes and application thereof in water quality biological monitoring. Comprising the following steps: five gene sequences of PaHb-3, paHb-5, paHb-13, paHb-15 and PaHb-19 show that the experimental results show that: the five Hb genes can be used as biomarkers for monitoring heavy metal copper water pollution. However, the method has a large number of genes detected, and aims at copper detection, so that the method has a poor effect on cadmium pollution detection.
For the detection of a cadmium pollution biomarker in seawater, the prior art also discloses a biomarker for risk assessment of marine pollutants in an aquatic ecosystem, and the toxicity mechanism of two typical marine pollutants on a marine synechococcus PCC7002 strain is examined, the biomarker for risk assessment of environmental pollutants is screened out from growth and photosynthesis parameters obtained in a toxicity test, obvious differential analysis is carried out by a variance analysis method, and a novel and efficient toxicological biomarker delta FIP is screened out by combining an IC50 value on the basis of Pearson correlation coefficient analysis. The sample pretreatment method is simple, the chlorophyll fluorescence instrument can be used for in-situ detection of the algae strain, the result stability is good, the repeatability is high, the risk of marine harmful substances can be accurately estimated and predicted, and early prediction and early warning of marine pollutant risk estimation are provided. But this approach is not very targeted.
Disclosure of Invention
The invention provides a method for indirectly measuring cadmium pollution in water, which fully considers the influence of biological enrichment effect or environmental effect generated by cadmium pollution on the metabolism of organisms, and detects the content of cadmium in the water body by detecting the content side reaction water body of gene ArWz-2 in the red stripe Mao Fudan turtle in the water body.
In one aspect, the invention provides an application of ArWz-2 in red Mao Fudan soft-shelled turtles in detecting cadmium pollution.
Specifically, the cadmium pollution in water is detected by detecting the ArWz-2 content in the red Mao Fudan soft-shelled turtles.
Specifically, the detection sample is seawater.
The ArWz-2 is a gene existing in red stripe Mao Fudan soft-shelled turtle, and the specific sequence is shown in SEQ ID NO. 1.
Sequence ID with higher similarity with SEQ ID NO.1 is XM_009066399.1 in NCBI;
on the other hand, the invention provides a method for detecting cadmium pollution in water.
Specifically, the detection method comprises the step of measuring the content of ArWz-2 in the red stripe Mao Fudan soft-shelled turtle.
Further, the content of the ArWz-2 is the expression level of the ArWz-2, and the method for detecting the expression level of the ArWz-2 can be any one of the disclosed or unpublished methods in the prior art, so that the detection method of the ArWz-2 expression level data can be embodied, and the technical scheme of the invention can be realized.
Preferably, the expression level of ArWz-2 can be detected by any one or more of Northern blot hybridization, southern blot hybridization, amplification, microarray technology, RNA sequencing, DNA sequencing, or proteomics, but is not limited to the above method.
Specifically, the microarray technology determines the expression level of ArWz-2 by analyzing the signal intensity on the chip.
Specifically, the Northern blot hybridization is to transfer RNA or DNA from agarose gel to nitrocellulose membrane to determine the expression level.
Specifically, the amplification includes, but is not limited to, polymerase chain reaction amplification, and preferably, real-time fluorescent quantitative PCR.
Specifically, the proteomics is to measure the expression level of ArWz-2 by detecting the expression level of the corresponding protein.
Further, the method for detecting the expression level of the protein includes, but is not limited to, immunoblotting, immunohistochemistry, or mass spectrometry.
In some embodiments, the amount of ArWz-2 is relative to the amount of the reference gene, as required by the different assay methods. Indeed, in some absolute quantification methods, reference to reference genes is not required, as will be appreciated by those skilled in the art.
When the level of ArWz-2 is relative, the reference genes include, but are not limited to, CYTB; if the reference gene is CYTB, when the content of ArWz-2 relative to the content of the reference gene is less than 0.5, judging that the cadmium content in water is more than 0.01mg/L; preferably, when the content of ArWz-2 relative to the content of the reference gene is < 0.2, it can be judged that the cadmium content in water is more than 0.01mg/L.
In the invention, the content of ArWz-2 in the red Mao Fudan soft-shelled turtle can be detected by a method disclosed or not disclosed in the prior art, and according to the knowledge of a person skilled in the art, the main purpose of the invention is to find out the corresponding relation between the content of ArWz-2 and cadmium pollution; the method for detecting the content of ArWz-2 is not limited.
In addition, the present invention is not limited to specific detection standards, since the skilled person can adjust the corresponding detection standards by replacing reference genes or detection methods based on the relationship between the content of ArWz-2 and cadmium pollution disclosed in the present invention, and the replacement of the technical means is conventional and cannot limit the protection scope of the present invention according to such replacement. Technical improvements, conventional adaptations, and modifications, all of which are within the scope of the invention, are intended to be within the limits of the invention.
The invention has the beneficial effects that:
the method detects the ArWz-2 content in the sample water body red Mao Fudan soft-shelled turtle, discovers that the ArWz-2 content is related to the cadmium content in the water body, and can detect the cadmium content in the water through the corresponding relation of the ArWz-2 content and the cadmium content in the water body, so as to monitor the water quality. Compared with the traditional direct detection method, the method avoids the harm of the strong corrosive reagent to detection personnel, and has higher sensitivity to cadmium detection in water.
Drawings
FIG. 1 is a gel electrophoresis chart of the amplified product of ArWz-2 of Trionyx sinensis Wiegmann Mao Fudan.
FIG. 2 shows the PCR quantification of ArWz-2 of red Mao Fudan soft-shelled turtles.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1
Red stripe Mao Fudan turtleAcanthochiton rubrolineatus) After 15 days of culture in manually prepared seawater (without cadmium), the culture is carried out on the manually prepared seawater with the cadmium concentration of 0 mg/L, 0.01mg/L, 1mg/L and 7.5mg/L for 3 days.
The red stripe Mao Fudan turtle in the artificially prepared seawater with the cadmium concentration of 0 mg/L, 0.01mg/L, 1mg/L and 7.5mg/L is randomly selectedAcanthochiton rubrolineatus) 3 replicates were selected for each group and the level of ArWz-2 was validated to determine the relationship of ArWz content to cadmium concentration in the water.
PCR is a method for conveniently detecting gene expression in the prior art, and the PCR method is taken as an example for detecting the ArWz-2 content in the red stripe Mao Fudan soft-shelled turtle. In this example, unless otherwise specified, the experimental conditions were conventional, and RNA extraction, reverse transcription, etc. were performed using conventional commercial kits, and in this example, trizol extraction was used for RNA extraction, and reverse transcription kit (PrimeScript ™ RT reagent Kit with gDNA Eraser (Perfect Real Time), available from Takara doctor technologies Co., ltd., cat# RR 047A) was used.
The extraction and purification of total RNA comprises the following steps:
(1) The samples were snap frozen with liquid nitrogen, quickly transferred to a liquid nitrogen pre-cooled mortar, and ground with a pestle, with liquid nitrogen added continuously until ground to a powder (no visible particles apparent).
(2) Transferring 50mg of the ground sample into a centrifuge tube, adding 1mL of RNAisolater, and standing; after the sample is completely melted, the sample is continuously blown until the lysate is transparent.
(3) The lysate was transferred to a centrifuge tube and centrifuged at 11200rpm at 4℃for 5min, and the supernatant was aspirated.
(4) 1/5 volume of chloroform was added to the above lysate. Shaking vigorously for 15 sec to form emulsion, standing at 4 ℃ for 5min, and centrifuging at 11200rpm for 15 min at 4 ℃. Carefully remove the centrifuge tube. The solution was then divided into three layers: a colorless aqueous phase (upper layer), a white middle layer, and a red organic layer (lower layer), carefully aspirate the upper aqueous phase into a new centrifuge tube.
(5) Adding the isopropyl alcohol precooled in equal volume, mixing the mixture upside down, and standing the mixture at the temperature of 4 ℃ for 10 min. Centrifugal at 11200rpm,4℃for 10min, a white precipitate was seen.
(6) The supernatant was carefully discarded, and 1mL of 75% ethanol (RNase-free ddH was added 2 And (3) O preparation). The bottom of the tube is flicked to suspend the sediment, and the sediment is turned upside down for several times and kept stand for 3-5min at room temperature.
(7) Centrifuge at 11200rpm at 4℃for 5min, discard supernatant.
(8) The pellet was left to dry in a clean environment at room temperature for 2-5 min, taking care that it could not be dried too much, otherwise it would lead to RNA dissolution difficulties.
(9) Adding appropriate amount of RNase-free ddH 2 O is dissolved and precipitated, after the O is completely dissolved, a small amount of O is taken and detected, and the rest O is preserved at the temperature of between-85 and 65 ℃.
Reverse transcription of RNA into cDNA involves the following steps:
(1) Genomic DNA removal reaction
TABLE 1
(2) Reverse transcription reaction
TABLE 2
The ArWz-2 quantitative primers in this example were:
F: TCGTGCTCATCAGTTAGTT（SEQ ID NO.2）；
R:GTAGTCTGGCTGTGGCTTC（SEQ ID NO.3）；
the primer was quantified using the reference gene CYTB commonly used in the art as reference:
F: GGAACAGGATTATTCTCAGCCAT（SEQ ID NO.4）；
R: GGATTAGTCAGCCGTGATTTACA（SEQ ID NO.5）。
the amplification system is as follows: 20. Mu.L of the reaction system including 2. Mu.L of cDNA, 0.4. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), SYBR qPCR Master Mix. Mu.L, 7.2. Mu.L of ddH 2 O。。
The amplification procedure was: pre-denaturation at 95 ℃ for 30 min; denaturation at 95℃for 10 s, extension at 60℃for 30 s for 40 cycles; melting curve procedure: 95℃for 15 s,60℃for 60 s,95℃for 15 s.
The detection result of gel electrophoresis is shown in figure 1, the quantification result is shown in figure 2, and the quantification result is the expression level of relative internal reference and has no unit.
As can be seen from FIG. 2, when the method in this embodiment is adopted for measurement, when the relative expression level of ArWz-2 in red stripe Mao Fudan soft-shelled turtles is less than 0.2, the cadmium content in water body is more than 0.01mg/L, and the cadmium pollution can be judged by exceeding the cadmium content standard in the water quality detection of the first, second and third kinds of seawater.
Example 2
Referring to the red bar Mao Fudan turtle culture method in example 1, the ArWz-2 content was determined by microarray technology, specifically: the culture is carried out in artificially prepared seawater with the cadmium concentration of 0.5mg/L for 3 days.
Randomly selecting red bars Mao Fudan soft-shelled turtles in manually prepared seawater with cadmium concentration of 0.5mg/L, selecting 3 parallel soft-shelled turtles in each group, and verifying the content of ArWz-2 to determine the relationship between the content of ArWz-2 and the cadmium concentration in water.
As a result, the results of comparison of the amounts of ArWz-2 expression were consistent with those of example 1.
Example 3
Referring to the red bar Mao Fudan turtle culture method in example 1, the ArWz-2 content was determined by Northern blot hybridization, specifically: randomly selecting red bars Mao Fudan soft-shelled turtles in manually prepared seawater with cadmium concentration of 1mg/L, selecting 3 parallel soft-shelled turtles in each group, and verifying the content of ArWz-2 to determine the relationship between the content of ArWz-2 and the cadmium concentration in water.
As a result, the results of comparison of the amounts of ArWz-2 expression were consistent with those of example 1.
Example 4 anti-interference experiment/specificity
Experiments were performed in accordance with the method of example 1, except that other common contamination factors were added to the 0 mg/L and 0.01mg/L cadmium concentration groups, respectively, and then the relative amounts of ArWz-2 expression were measured, and the amounts of contaminants and additions were as follows:
copper content (μg/L): 4.8 and 9.2;
lead content (μg/L): 0.6 and 2.2;
chromium content (μg/L): 2.0 and 3.9;
phosphorus content (μg/L): 0.6 and 2.6;
acidic pH:6, preparing a base material;
a blank (no interferents but cadmium only) was set up for a total of 20 groups, each group being set up in 3 replicates.
Results: 2 groups of copper-containing pollutants are added, and the expression quantity is not obviously different; 2 groups of lead-containing pollutants are added, and the expression quantity is not obviously different; 2 groups of chromium-containing pollutants are added, and the expression quantity is not obviously different; 2 groups of phosphorus-containing pollutants are added, and the expression quantity is not obviously different; the expression level of the acidified group is not obviously different.
The results show that the pollution factors have no influence on the result judgment.
Claims (3)
1. The application of the primer pair in detecting the water cadmium pollution is characterized in that the primer pair is used for amplifying red stripes Mao Fudan soft-shelled turtles, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3.
2. A method for detecting cadmium pollution in water is characterized in that the primer pair in claim 1 is adopted to carry out relative quantitative detection on red bars Mao Fudan soft-shelled turtles.
3. The method according to claim 2, wherein the relatively quantitative reference gene is CYTB, and the cadmium content in water is determined to be greater than 0.01mg/L when the expression level of the amplified product is less than 0.2 relative to the expression level of the reference gene.
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| CN101904310A (en) * | 2010-07-26 | 2010-12-08 | 宁波大学 | Biological purifying method for seafood bivalve shellfishes |
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