CN117106881A - Cadmium pollution detection method and marker - Google Patents
Cadmium pollution detection method and marker Download PDFInfo
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- CN117106881A CN117106881A CN202311370144.1A CN202311370144A CN117106881A CN 117106881 A CN117106881 A CN 117106881A CN 202311370144 A CN202311370144 A CN 202311370144A CN 117106881 A CN117106881 A CN 117106881A
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- 229910052793 cadmium Inorganic materials 0.000 title claims abstract description 55
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 239000003550 marker Substances 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 title abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 31
- 230000014509 gene expression Effects 0.000 claims description 30
- 238000003753 real-time PCR Methods 0.000 claims description 11
- 210000001142 back Anatomy 0.000 claims description 9
- 238000012544 monitoring process Methods 0.000 claims description 9
- 230000002596 correlated effect Effects 0.000 claims description 8
- 239000013535 sea water Substances 0.000 claims description 8
- 238000000636 Northern blotting Methods 0.000 claims description 4
- 238000003559 RNA-seq method Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 4
- 241001148106 Brucella melitensis Species 0.000 claims 1
- 229940038698 brucella melitensis Drugs 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 6
- 229910001385 heavy metal Inorganic materials 0.000 abstract description 6
- 235000015170 shellfish Nutrition 0.000 abstract description 6
- 239000000523 sample Substances 0.000 description 6
- 238000007400 DNA extraction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 101150081086 Msh6 gene Proteins 0.000 description 2
- 241000237536 Mytilus edulis Species 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- 241000863430 Shewanella Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000010842 industrial wastewater Substances 0.000 description 2
- 235000020638 mussel Nutrition 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003911 water pollution Methods 0.000 description 2
- 241000965254 Apostichopus japonicus Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000548230 Crassostrea angulata Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000276569 Oryzias latipes Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000237509 Patinopecten sp. Species 0.000 description 1
- GOENIMGKWNZVDA-OAMCMWGQSA-N Spinetoram Chemical compound CO[C@@H]1[C@H](OCC)[C@@H](OC)[C@H](C)O[C@H]1OC1C[C@H]2[C@@H]3C=C4C(=O)[C@H](C)[C@@H](O[C@@H]5O[C@H](C)[C@H](CC5)N(C)C)CCC[C@H](CC)OC(=O)CC4[C@@H]3CC[C@@H]2C1 GOENIMGKWNZVDA-OAMCMWGQSA-N 0.000 description 1
- 239000005929 Spinetoram Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 231100000739 chronic poisoning Toxicity 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004993 emission spectroscopy Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000009616 inductively coupled plasma Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052745 lead Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 235000020637 scallop Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000003902 seawater pollution Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000004454 trace mineral analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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Abstract
The invention belongs to the technical field of heavy metal detection methods, and particularly relates to a cadmium pollution detection method and a marker. The invention determines the cadmium content in the water body by detecting the content of the NsWz-4 of the back point shellfish in the water body, and provides a reference basis for purposefully treating cadmium pollution in the water. Compared with the traditional detection method, the method simplifies the pretreatment steps of the sample, avoids the harm of the strong corrosive reagent to detection personnel, and has the advantages of simple experimental method, high detection speed, high stability and the like.
Description
Technical Field
The invention belongs to the technical field of heavy metal detection methods, and particularly relates to a cadmium pollution detection method and a marker.
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. 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. Cadmium pollution in water is mainly from surface runoff and industrial wastewater. The discharge of industrial wastewater results in higher cadmium content in the offshore seawater and plankton than in the offshore, industrial area surface water than in the non-industrial area.
Chemical analysis of cadmium can be generally classified into 3 categories: atomic absorption spectrometry, colorimetry and inductively coupled plasma emission spectrometry. However, these methods are cumbersome to pre-treat and the reagents used are highly corrosive, which poses a threat to the safety of the operators. In addition, cadmium is widely distributed in nature, and the trace analysis accuracy of cadmium detection for low limit is low. There is a need to develop a detection method that is safe, has a low detection limit, and is not easily contaminated.
Patent application CN106191247a discloses a method for detecting cadmium pollution in water by using a repair protein 6 (MSH 6) gene, wherein fish is used as a biological sample for monitoring water pollution, the MSH6 gene is used as a sensitive biomarker for monitoring cadmium pollution in water, and the relative expression level of the MSH6 gene is tested to monitor the water pollution degree. The method can sensitively reflect whether the water body is polluted or not and the pollution degree, and even sensitively measure the trace pollution source; meanwhile, the hazard and the hazard degree of heavy metal cadmium pollution can be sensitively monitored from the biological point of view, and an accurate and rapid scientific method is provided for monitoring, toxicological diagnosis, regulation and control of the ecological environment of the water body. However, the used organisms are medaka, which are mainly found in fresh water and are favored by dense aquatic plants, clear water and still water or slow flow, so that the method has a certain limit in detecting seawater pollution.
The Sharp back shellfish of Shi is widely distributed along the coast of each island, and is a conventional species. Generally living on high and medium tide belt rocks and on stone ventral surfaces. Researches show that (Liu Yang, xiang Jieyou, zhang Fanshun) the heavy metal content and evaluation [ J ] of the Qingdao offshore intertidal zone organisms are great in heavy metal content difference among the Qingdao offshore intertidal zone investigation organisms, different organisms have preference on heavy metal element enrichment, the content of Cu and Zn in crassostrea gigas is highest, the content of Pb, as, hg and Cr in the conch is highest, and the content of Cd in the Shandong back scallop is highest. However, the relation between the spinetoram gene and the cadmium content in water is not reported in the prior art.
Disclosure of Invention
In order to solve the problems, the invention provides a cadmium pollution detection method and a marker. The marker is an NsWz-4 gene in the dorsum of the Shih, the expression quantity of the gene is positively correlated with the cadmium content, and the cadmium content in the water body is determined by detecting the expression quantity of the NsWz-4 gene in the dorsum of the Shih.
As used herein, "amount of NsWz-4" may refer to the amount of NsWz-4 expressed, and the method of measuring the amount of NsWz-4 expressed may be selected from the group consisting of real-time fluorescent quantitative PCR, RNA sequencing and Northern blot, but is not limited to these detection methods.
In one aspect, the invention provides a marker for detecting cadmium pollution in water, wherein the marker is a BsWz-4 gene of BsWz, and the sequence of the BsWz-4 gene is shown as SEQ ID NO. 1.
In another aspect, the invention provides a method for detecting cadmium pollution by the marker, wherein the method can be used for measuring the expression quantity of the NsWz-4 gene.
Specifically, the expression level of the NsWz-4 gene is positively correlated with the cadmium content in water.
Specifically, the method may comprise the steps of:
(1) Extracting the gene of the dorsum schneiderianum;
(2) Determination of the above-mentioned NsWz-4 gene expression level.
Specifically, the method for extracting the dorsum of the Stichopus japonicus gene described in the step (1) is not limited to the use of a DNA extraction kit.
Specifically, the assay method described in step (2) includes, but is not limited to: one of real-time fluorescent quantitative PCR, RNA sequencing or Northern blot.
Preferably, the assay method of step (2) is real-time fluorescent quantitative PCR; the reverse transcription primer in the real-time fluorescence quantitative PCR can be Oligo dT and Random 6 mers, and the amplification primer can be
SEQ ID NO.2-SEQ ID NO.3。
In yet another aspect, the invention provides the use of the aforementioned marker or method in water quality monitoring.
Specifically, the water quality monitoring can be to measure the cadmium content in water.
More specifically, the cadmium content can be determined by the expression level of the NsWz-4 gene.
Specifically, the cadmium content is positively correlated with the expression level of the NsWz-4 gene.
Specifically, the method for measuring the expression level of the NsWz-4 gene comprises the following steps: one of real-time fluorescent quantitative PCR, RNA sequencing or Northern blot.
Preferably, the assay method is real-time fluorescent quantitative PCR; reverse transcription primers in the real-time fluorescent quantitative PCR can be Oligo dT and Random 6 mers, and amplification primers can be SEQ ID NO.2-SEQ ID NO.3.
More specifically, when the expression level of the NsWz-4 gene is more than 5, cadmium pollution exists.
In particular, the water may be sea water.
The invention has the technical effects that:
(1) The expression level of the NsWz-4 gene is positively correlated with the cadmium content in water, the fitting equation is y= 20.288x-95.358, R 2 = 0.9846. When the expression level is>5, indicating the presence of cadmium contamination.
(2) Compared with the detection method in the prior art, the method simplifies the pretreatment step of the sample, avoids the harm of the strong corrosive reagent to the detection personnel, and has the advantages of simple experimental method, high detection speed, high stability and the like.
Drawings
FIG. 1 shows the expression level of the NsWz-4 gene of dormitory mussel with different cadmium concentrations.
FIG. 2 is a graph showing the fit of the cadmium content in water to the expression level of the Shewanella schneiderian point NsWz-4.
FIG. 3 shows the NsWz-4 gene expression levels of the dorsum of Stirling with 5 different additive concentrations.
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
1.1 feeding of dorsum schneiderianum
The Shih back shellfish is divided into 6 groups by using the Shih back shellfish collected from the Taijin ditch village sea area (east longitude 121 DEG 43', north latitude 37 DEG 52') as a biological sample for monitoring cadmium pollution in water, and the Shih back shellfish is respectively fed in the configured seawater with different cadmium concentrations, wherein each group comprises 20, and each group comprises 3 parallel 360. The cadmium concentration in each group of seawater is shown in table 1, wherein the cadmium concentration is 0 as a control group. After 3 days of feeding, each group of 7 parallel dorsi spinosa was randomly collected for measuring the expression level of NsWz-4, and the average value of 3 parallel detection results was obtained.
TABLE 1
1.2 reagents
Fish tissue DNA extraction kit, shanghai-associated bioengineering limited, cat No.: LM-181.
Real-time fluorescent quantitative PCR kit, shanghai-Mei bioengineering Co., ltd., product number: LM86028C.
1.3 Determination of the amount of expression of NsWz-4
The sequence of the NsWz-4 gene is SEQ ID NO.1, and a real-time fluorescent quantitative PCR technology is adopted for the method for measuring the expression level of the NsWz-4 gene.
Extracting the gene of the dorsum schneiderianum according to the specification of a fish tissue DNA extraction kit, and quantifying according to the specification of a real-time fluorescence quantitative PCR kit, wherein reverse transcription primers are Oligo dT and Random 6 mers, and PCR amplification primers are F:5'-AGGAGAGTCGACATATGGCA-3' (SEQ ID NO. 2);
R：5’-GGCAAGAGAACAGCAAGAGT-3’（SEQ ID NO.3）。
1.4 results and analysis
The result of the expression level of NsWz-4 of the back-sharp shellfish is shown in Table 2, and it is known from Table 2 that the expression level of the NsWz-4 gene is positively correlated with the cadmium content in water, when the expression level is>5, indicating the presence of cadmium contamination. The expression level of the NsWz-4 gene is positively correlated with the cadmium content in water, the fitting equation is y= 20.288x-95.358, and R 2 = 0.9846, see fig. 2. Therefore, the cadmium content in water can be indirectly detected according to the expression level of the Shewanella schneideriana NsWz-4 in water.
TABLE 2
Example 2 detection method verification
2.1 determination of the content of NsWz-4
Researchers randomly prepare 5 (A-E) seawater with different cadmium concentrations, and the feeding method and the detection method are the same as in example 1. The dorsum spinosa in 5 seawater were noted as samples a-E, respectively; the 5 seawater samples are respectively marked as water samples A-E.
The expression level of the NsWz-4 gene is shown in Table 3, and the corresponding cadmium concentration was calculated according to the fitting equation of example 1.
TABLE 3 Table 3
2.2 atomic absorption spectrometry for measuring cadmium in water sample
2.2.1 Water sample
The same water sample as that of the dormitory mussel of Steriluz in 2.1.1 (water sample 1-5).
2.2.2 instruments and reagents
GGX-6A type Zeeman flame atomic absorption spectrophotometer (Beijing Kokai sea light instrument Co., ltd.) cadmium hollow cathode lamp.
Nitric acid (analytically pure) Shenzhen chemical industry Co., ltd.
Nitric acid (top grade purity), guangzhou Panhong trade Co., ltd., product number: 65873-2.5L.
Perchloric acid (superior purity) Guangzhou Green hundred grass science instruments Co., ltd., brand: race-default, cargo number: a469-500.
Cadmium element standard solution for water quality monitoring, shanghai screening quasi-commerce technology Co., ltd., product number: 06062027.
2.2.3 Experimental methods
The cadmium in water is detected by referring to the first part of the direct method in the national standard GB/T7475-1987.
Evaporating and concentrating 1-5 water samples by 100 times respectively, carrying out digestion treatment on the concentrated water samples to prepare water samples to be tested, and carrying out 3 repeated experiments on each water sample. The instrument parameter settings are shown in table 4.
TABLE 4 Table 4
2.2.4 results and analysis
The results of atomic absorption spectrometry are shown in Table 5:
TABLE 5
From Table 5 in combination with Table 3, it is known that the cadmium calculated from the expression level of the NsWz-4 gene has a tendency equivalent to the cadmium concentration detected according to the national standard, and it is further verified that the method for detecting the cadmium concentration of the present invention is accurate and feasible.
Claims (10)
1. The marker is a Brucella melitensis NsWz-4 gene in water, and the sequence of the marker is shown as SEQ ID NO. 1.
2. The method for detecting cadmium pollution by using the marker according to claim 1, wherein the method is used for measuring the expression quantity of the NsWz-4 gene.
3. The method of claim 2, wherein the NsWz-4 gene expression is positively correlated with the cadmium content of water.
4. A method according to claim 2 or 3, characterized in that the method comprises the steps of:
(1) Extracting the gene of the dorsum schneiderianum;
(2) The method for measuring the expression level of the NsWz-4 gene according to claim 1.
5. The method of claim 4, wherein the assay of step (2) is one of real-time fluorescent quantitative PCR, RNA sequencing or Northern blot.
6. Use of a marker according to claim 1 or a method according to any one of claims 2 to 5 in water quality monitoring.
7. The use of claim 6, wherein the water quality monitoring is to determine the cadmium content of water.
8. The use according to claim 7, wherein the cadmium content is determined by the expression level of the NsWz-4 gene.
9. The use according to claim 8, wherein the cadmium content is positively correlated with the expression level of the NsWz-4 gene.
10. The use of claim 7, wherein the water is seawater.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311370144.1A CN117106881A (en) | 2023-10-23 | 2023-10-23 | Cadmium pollution detection method and marker |
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| CN202311370144.1A CN117106881A (en) | 2023-10-23 | 2023-10-23 | Cadmium pollution detection method and marker |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102808036A (en) * | 2012-08-31 | 2012-12-05 | 天津师范大学 | Biological sensitive detecting method for heavy metal cadmium in seawater |
| CN106226487A (en) * | 2016-07-12 | 2016-12-14 | 广西大学 | The method utilizing the metallothionein detection degree of water pollution of fish |
| CN108823219A (en) * | 2018-07-06 | 2018-11-16 | 浙江海洋大学 | A kind of blood clam catalase -- novel sea biological pollution detection marker |
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- 2023-10-23 CN CN202311370144.1A patent/CN117106881A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102808036A (en) * | 2012-08-31 | 2012-12-05 | 天津师范大学 | Biological sensitive detecting method for heavy metal cadmium in seawater |
| CN106226487A (en) * | 2016-07-12 | 2016-12-14 | 广西大学 | The method utilizing the metallothionein detection degree of water pollution of fish |
| CN108823219A (en) * | 2018-07-06 | 2018-11-16 | 浙江海洋大学 | A kind of blood clam catalase -- novel sea biological pollution detection marker |
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