CN109735606B - Rapid detection method for imidacloprid water pollution by fluorescent quantitative PCR - Google Patents
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Abstract
The invention relates to a method for detecting water pollutants, in particular to a method for rapidly detecting imidacloprid water pollution by fluorescent quantitative PCR, belonging to the field of molecular biology. The invention detects the Yixing widal mitochondrial gene of aquatic insectnad4LThe technology for monitoring imidacloprid pollution by response of the expression level to imidacloprid comprises the following specific implementation steps: (1) culturing Yixing wide medium of the water sample to be detected; (2) extracting total RNA; (3) detecting gel electrophoresis; (4) measuring the concentration of RNA; (5) RNA is inverted into cDNA; (6) constructing a standard product and manufacturing a standard curve; (7) measuring the expression quantity of the gene; (8) and (6) analyzing the data. The method has the advantages of high sensitivity, good timeliness, strong specificity and simple operation, and can be used for rapidly detecting whether the water body is polluted by imidacloprid.
Description
Technical Field
The invention relates to a method for detecting water pollutants, in particular to a method for rapidly detecting imidacloprid water pollution by fluorescent quantitative PCR, belonging to the field of molecular biology.
Background
Imidacloprid belongs to neonicotinoid pesticides, and is a nervous insecticidal pesticide which can keep insects excited or paralyzed and then die. The pesticide has the characteristics of high toxicity, easy use, flexibility, long-term durability and the like on invertebrates, so that the invertebrates become a pesticide with higher global market share. Such pesticides, represented by imidacloprid, have lethal or sublethal effects on terrestrial and large benthonic animals such as birds, bees, and aquatic invertebrates such as mayflies, causing a decrease in the fertility of earthworms, etc., affecting the ecological balance. Meanwhile, because the water-soluble organic fertilizer has better water solubility and high leaching rate, the organic fertilizer is easy to enter an aquatic ecosystem through a runoff and drainage system of an agricultural area, has great influence on the water environment, and threatens human health and the aquatic ecosystem. Researches show that the neonicotinoid pesticides are widely distributed and accumulated in the global environment, and water, soil, crops and animals are polluted to different degrees. Therefore, the imidacloprid water body pollution detection is made in time and is very important to be treated, and the existing imidacloprid detection mainly aims at the residues in vegetables, and relatively few imidacloprid detection methods are used for detecting imidacloprid in environmental water bodies.
Disclosure of Invention
In order to solve the problem of imidacloprid water body pollution detection, the invention provides a method for rapidly detecting imidacloprid water body pollution by fluorescent quantitative PCR, which is used for rapidly detecting whether a water body is polluted by imidacloprid by measuring the Yixing-widescreen mitochondrial gene expression level of aquatic insects by a real-time fluorescent quantitative PCR technology and has the characteristics of convenience, rapidness, high sensitivity, simple operation and the like.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for rapidly detecting imidacloprid water body pollution by fluorescent quantitative PCR comprises the following steps:
(1) cultivation of Yixing wide medium for detecting water sample
Collecting Yixing broadside larvae in a water area which is not polluted by imidacloprid by using a net kicking method, carrying out laboratory feeding, and establishing an experimental material for imidacloprid pollution research;
simultaneously collecting a water sample of a water sample plot to be detected, and culturing the Yixing wide medium in the water sample to be detected for 24 hours;
(2) extracting total RNA from Yixing wide medium cultured in a water sample to be detected for 24 h;
(3) agarose gel electrophoresis detection verifies that the total RNA of the sample is successfully extracted;
(4) determination of total RNA concentration
Using a micro ultraviolet spectrophotometer to determine the RNA concentration after the zero adjustment of the deionized water without the RNase, and A260/A280; the RNA concentration is more than 60 ng/mu L, A260/A280 is between 1.9 and 2.2, which indicates that the concentration determination data is credible and can be used for reversal experiments;
(5) RNA inversion to cDNA
(6) Construction of standards and preparation of calibration curves
Firstly, preparing PCR reaction solution
Respectively diluting the cDNA template by using EASY Dilution with 1, 10, 100, 1000 and 10000 times of gradient;
the PCR reaction solution was prepared on ice, and 20. mu.L of the PCR reaction solution contained SYBR Premix Ex Taq II (2X) (Tli RNaseH Plus) at a final concentration of 1X, Bulk, 0.4. mu.M PCR fronard Primer and 0.4. mu.M PCR Reverse Primer;
② PCR reaction
Placing the prepared reaction solution into a real-time fluorescent quantitative PCR instrument for reaction, wherein the reaction procedure is pre-denaturation at 95 ℃ for 30s, and then entering the following circulation: denaturation at 95 ℃ for 5s, annealing at 56 ℃ for 30s, and circulating for 40 times; carrying out data statistics by using a computer, and observing the conditions of a dissolution curve and a standard curve; the dissolution curve is unimodal, the standard curve R2Values greater than 0.980, E values between 90 and 110, indicating that a standard curve is available;
(7) gene expression level measurement
Preparing fluorescent quantitative PCR reaction liquid, wherein the reaction program is pre-denaturation at 95 ℃ for 30s, and then entering the following circulation: denaturation at 95 ℃ for 5s, annealing at 56 ℃ for 30s, circulating for 40 times, and counting the relative expression quantity of nad4L genes of each sample by a computer;
(8) data analysis
And (3) carrying out t test by using SPSS or Excel, and observing whether the expression quantity of the gene is significantly different from that of Yixing broad base which is not polluted by imidacloprid, wherein if the significant difference exists, the water body is most likely to be polluted by the imidacloprid, and if the significant difference does not exist, the water body is not polluted by the imidacloprid.
The invention relates to a technology for monitoring imidacloprid pollution by detecting response of the expression level of a Yixing Gen mitochondrial gene nad4L of aquatic insects to imidacloprid, and the method is used for quickly detecting the imidacloprid water body pollution by measuring the expression level of the Yixing Gen mitochondrial gene on the basis of real-time fluorescent quantitative PCR (polymerase chain reaction).
The invention screens and cultures aquatic insect Yixing Wide base used for water quality monitoring as a research material, screens out fluorescent quantitative PCR primers highly sensitive to imidacloprid, and can quickly and highly sensitively detect the low-concentration imidacloprid pollution by carrying out fluorescent quantitative PCR by using the specific primer sequence.
Preferably, the fluorescent quantitative primer YXKJ-nad4L is designed according to the Yixing WIDTH mitochondrial nad4L gene and is YXKJ-J-nad 4L: 5'-CGTAAGCATTTATTGGGTAT-3', respectively;
YXKJ-N-nad4L：5’-AGACTGAAAGACCTAAAGCC-3’；
the fluorescent quantitative primer YXKJ-actin is designed according to the Yixing broad gene beta-actin gene and is YXKJ-J-actin: 5'-CTTCCTTCCTGGGTATGG-3', the nucleotide sequence is shown in SEQ ID No. 3;
YXKJ-N-actin: 5'-GCGGTGATTTCCTTCTGC-3', the nucleotide sequence is shown in SEQ ID No. 4.
Preferably, in the step (2), the total RNA is extracted by using TaKaRa Mini BEST Universal RNA Extraction Kit.
Preferably, the agarose gel electrophoresis detection method in the step (3) is as follows:
firstly, preparing 1% agarose gel, putting 0.4g agarose and 40mL of 0.5 × TBE solution into a conical flask, uniformly mixing, heating by a microwave oven until the agarose is completely dissolved, cooling to 50 ℃ at room temperature, adding 2 μ l Gold View nucleic acid staining agent, uniformly mixing, pouring into a gel making plate, and standing for 20 min;
secondly, respectively sucking 4 mu L of Marker and total RNA of each sample into sample application holes, placing a gel plate into an electrophoresis tank, and carrying out 120V electrophoresis for 30min from the negative electrode to the positive electrode;
thirdly, the gel is placed in a gel imaging system for detection, whether 18S and 28S rRNA bands appear in each lane is observed, and if a target band appears, the successful extraction of the total RNA of the sample is indicated.
Preferably, the RNA is inverted into cDNA in step (5), and the RNA is inverted into cDNA using TaKaRa Mini BEST Universal RNA Extraction Kit.
Preferably, the 20. mu.l PCR reaction components in steps (6) and (7) are:
the invention has the advantages that:
(1) the sensitivity is high: sensitivity detection is carried out by imidacloprid standard concentration gradient dilution, and the significant differential expression (P <0.05) of the nad4L gene can be still detected under the condition of the low-concentration imidacloprid solution concentration of 5 mu g/L.
(2) High specificity and good accuracy: by adopting specific primers and carrying out real-time fluorescent quantitative PCR amplification on the nad4L gene, the expression level of the gene can be effectively and accurately measured.
(2) The aging is fast: after 24h of imidacloprid stress treatment, the nad4L gene can generate a significant response (P < 0.05).
(3) The operation is simple: the related data can be obtained only by completing the operation according to the indication steps of the kit, and the data analysis operation is simple. The detection method is simple, the detection speed is high, and the quantitative result is accurate and reliable.
Drawings
FIG. 1 is a dissolution curve of beta-actin gene standard;
FIG. 2 is a dissolution curve of nad4L gene standard;
FIG. 3 is a standard curve of beta-actin gene standard;
FIG. 4 shows a standard curve of nad4L gene standard;
FIG. 5 is a graph showing the relative expression levels of nad4L gene treated at different concentrations.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The TaKaRa Mini BEST Universal RNA Extraction Kit adopts Baozi physician's technology (Beijing) Co., Ltd.
A method for rapidly detecting imidacloprid water body pollution by fluorescent quantitative PCR comprises the following specific implementation steps:
(1) imidacloprid stress culture of Yixing broad base of water sample to be detected
Collecting Yixing broadside larvae in a water area which is not polluted by imidacloprid by using a net kicking method, and feeding in a laboratory to obtain a research material for carrying out an imidacloprid pollution experiment.
Different imidacloprid concentrations (0 mug/L, 5 mug/L, 10 mug/L, 20 mug/L and 40 mug/L) are prepared by standard products to culture the Yixing broad base juvenile insects for 24h under stress, each concentration is subjected to parallel experiments for 3 groups, 10 samples are counted in each group, three Yixing broad bases are adopted for each concentration to carry out gene expression quantity detection, and the rest samples are stored in a refrigerator at the temperature of-80 ℃ for later use.
(2) Extraction of Total RNA
RNA Extraction is carried out on the Yixing broad larvae after 24h stress culture by adopting a TaKaRa Mini BEST Universal RNA Extraction Kit, and the specific steps refer to product use instructions.
(3) Agarose gel electrophoresis detection
Firstly, preparing 1% agarose gel, putting 0.4g agarose and 40mL of 0.5 × TBE solution into a conical flask, uniformly mixing, heating by a microwave oven until the agarose is completely dissolved, cooling to 50 ℃ at room temperature, adding 2 μ L Gold View nucleic acid staining agent, uniformly mixing, pouring into a gel making plate, and standing for 20 min;
secondly, respectively sucking 4 mu L of Marker and total RNA of each sample into sample application holes, placing a gel plate into an electrophoresis tank, and carrying out 120V electrophoresis for 30min from the negative electrode to the positive electrode;
thirdly, the gel is placed in a gel imaging system for detection, whether 18S and 28S rRNA bands appear in each lane is observed, and if a target band appears, the successful extraction of the total RNA of the sample is indicated.
(4) Determination of total RNA concentration
RNA concentration was determined after zeroing with RNase-free deionized water using a micro UV spectrophotometer, and A260/A280. The RNA concentration is more than 60 ng/. mu.L, and A260/A280 is between 1.9 and 2.2, which indicates that the concentration determination data is credible and can be used for reversal experiments. Successfully extracted RNA was stored in a-80 ℃ refrigerator.
(5) RNA inversion to cDNA
RNA inversion is carried out to cDNA by adopting a TaKaRa Mini BEST Universal RNA Extraction Kit, the specific steps refer to a product use instruction, and the successfully inverted cDNA is stored in a refrigerator at the temperature of-20 ℃.
(6) Construction of standards and preparation of calibration curves
Firstly, preparing PCR reaction solution
1. Respectively diluting the cDNA template by using EASY Dilution with 1, 10, 100, 1000 and 10000 times of gradient;
2. PCR reaction solutions (Table 1) were prepared in the following composition, and the reaction solutions were prepared on ice and repeated three times for each concentration.
TABLE 120 μ L PCR System
nad4L gene-specific fluorescent quantitative PCR primer YXKJ-nad 4L:
J：5’-CGTAAGCATTTATTGGGTAT-3’(SEQ ID No.1)；
N：5’-AGACTGAAAGACCTAAAGCC-3”(SEQ ID No.2)；
beta-actin gene (reference gene) specific fluorescent quantitative PCR primer YXKJ-actin:
J：5’-CTTCCTTCCTGGGTATGG-3’(SEQ ID No.3)；
N：5’-GCGGTGATTTCCTTCTGC-3’(SEQ ID No.4)。
fluorescent quantitative PCR reaction
Placing the prepared reaction solution into a real-time fluorescent quantitative PCR instrument for reaction, wherein the reaction procedure is 95 DEG CPre-denatured for 30s, then entered the following cycle: denaturation at 95 ℃ for 5s, annealing at 56 ℃ for 30s, and circulation for 40 times. The computer was used for data statistics and the dissolution curves and the standard curve conditions were observed, and the data obtained are shown in FIGS. 1-4. The dissolution curve is unimodal, the standard curve R2Values greater than 0.980 and E values between 90 and 110 indicate that a standard curve is available.
(7) Gene expression level measurement
Fluorescent quantitative PCR reaction solution was prepared according to the system of Table 1, and the technique was repeated three times for each sample. The reaction program was pre-denatured at 95 ℃ for 30s, then entered the following cycle: the relative expression of nad4L gene in each sample is calculated by computer after denaturation at 95 ℃ for 5s and annealing at 56 ℃ for 30s for 40 cycles, and the result is shown in FIG. 5.
(8) Data analysis
And (3) carrying out t test by using SPSS or Excel, and observing whether the expression quantity of the gene is significantly different from that of the Yixing broad gene which is not polluted by the imidacloprid. The results in FIG. 5 show that the level of expression of mitochondrial nad4L gene of different concentrations of imidacloprid-treated Yixing WIP larvae was significantly lower than that of the control group, wherein the 10. mu.g/L treated group was significantly different (P <0.05), and the 5. mu.g/L and 20. mu.g/L treated groups were significantly different (P < 0.01). Under the stress culture of imidacloprid with different concentrations, the nad4L gene has significant or extremely significant difference, which is consistent with the fact.
The result shows that the method can sensitively detect whether the water body is polluted by imidacloprid.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Sequence listing
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<120> method for rapidly detecting imidacloprid water body pollution by fluorescent quantitative PCR
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gcggtgattt ccttctgc 18
Claims (5)
1. A method for detecting imidacloprid water body pollution by fluorescent quantitative PCR is characterized by comprising the following steps:
(1) cultivation of Yixing wide medium for detecting water sample
Collecting Yixing broadside larvae in a water area which is not polluted by imidacloprid by using a net kicking method, carrying out laboratory feeding, and establishing an experimental material for imidacloprid pollution research;
simultaneously collecting a water sample of a water sample plot to be detected, and culturing the Yixing wide medium in the water sample to be detected for 24 hours;
(2) extracting total RNA from Yixing wide medium cultured in a water sample to be detected for 24 h;
(3) agarose gel electrophoresis detection verifies that the total RNA of the sample is successfully extracted;
(4) determination of total RNA concentration
Using a micro ultraviolet spectrophotometer to determine the RNA concentration after the zero adjustment of the deionized water without the RNase, and A260/A280; the RNA concentration is more than 60 ng/mu L, A260/A280 is between 1.9 and 2.2, which indicates that the concentration determination data is credible and can be used for reversal experiments;
(5) RNA inversion to cDNA
(6) Construction of standards and preparation of calibration curves
Firstly, preparing PCR reaction solution
Respectively diluting the cDNA template by using EASY Dilution with 1, 10, 100, 1000 and 10000 times of gradient;
the PCR reaction solution was prepared on ice, and 20. mu.L of the PCR reaction solution contained SYBR Premix Ex Taq II (2X) (Tli RNaseH Plus) at a final concentration of 1X, Bulk, 0.4. mu.M PCR fronard Primer and 0.4. mu.M PCR Reverse Primer;
② PCR reaction
Placing the prepared reaction solution into a real-time fluorescent quantitative PCR instrument for reaction, wherein the reaction procedure is pre-denaturation at 95 ℃ for 30s, and then entering the following circulation: denaturation at 95 ℃ for 5s, annealing at 56 ℃ for 30s, and circulating for 40 times; performing data statistics by using a computer, and observing the conditions of a melting curve and a standard curve; the melting curve is unimodal, the standard curve R2Values greater than 0.980, E values between 90 and 110, indicating that a standard curve is available;
(7) gene expression level measurement
Preparing fluorescent quantitative PCR reaction liquid, wherein the reaction program is pre-denaturation at 95 ℃ for 30s, and then entering the following circulation: denaturation at 95 ℃ for 5s, annealing at 56 ℃ for 30s, circulation for 40 times, and computer statistics of each samplenad4LRelative expression level of the gene;
(8) data analysis
Performing t test by using SPSS or Excel, and observing whether the expression quantity of the gene has a significant difference with Yixing broad base which is not polluted by imidacloprid, wherein if the significant difference exists, the water body is most likely to be polluted by the imidacloprid, and if the significant difference does not exist, the water body is not polluted by the imidacloprid;
according to Yixing W mitochondrianad4LThe fluorescent quantitative primer YXKJ-nad4L designed by the gene is
YXKJ-J-nad4L：5’-CGTAAGCATTTATTGGGTAT-3’；
YXKJ-N-nad4L：5’-AGACTGAAAGACCTAAAGCC-3’；
According to Yixing wide base β-actinThe fluorescent quantitative primer YXKJ-actin is designed by gene and is YXKJ-J-actin: 5'-CTTCCTTCCTGGGTATGG-3', the nucleotide sequence is shown in SEQ ID No. 3;
YXKJ-N-actin: 5'-GCGGTGATTTCCTTCTGC-3', the nucleotide sequence is shown in SEQ ID No. 4.
2. The detection method according to claim 1, wherein the total RNA in step (2) is extracted using TaKaRa Mini BEST Universal RNA Extraction Kit.
3. The detection method according to claim 1, wherein the agarose gel electrophoresis detection method of step (3) is:
firstly, preparing 1% agarose gel, putting 0.4g agarose and 40mL of 0.5 × TBE solution into a conical flask, uniformly mixing, heating by a microwave oven until the agarose is completely dissolved, cooling to 50 ℃ at room temperature, adding 2 μ L Gold View nucleic acid staining agent, uniformly mixing, pouring into a gel making plate, and standing for 20 min;
secondly, respectively sucking 4 mu L of Marker and total RNA of each sample into sample application holes, placing a gel plate into an electrophoresis tank, and carrying out 120V electrophoresis for 30min from the negative electrode to the positive electrode;
thirdly, the gel is placed in a gel imaging system for detection, whether 18S and 28S rRNA bands appear in each lane is observed, and if a target band appears, the successful extraction of the total RNA of the sample is indicated.
4. The detection method according to claim 1, characterized in that: and (5) reversing the RNA into cDNA by adopting a TaKaRa Mini BEST Universal RNA Extraction Kit.
5. The detection method according to claim 1, characterized in that: the PCR reaction solution in the steps (6) and (7) comprises the following components:
SYBR Premix Ex Taq II(2x) (Tli RNaseH Plus),Bulk 10 μL
PCR Froward Primer 10 μM 0.8 μL
PCR Reverse Primer 10 μM 0.8 μL
ROX Reference Dye or Dye II 0.4 μL
cDNA template <100 ng 2. mu.L
Sterilized water 6. mu.L.
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| CN101460608A (en) * | 2006-05-29 | 2009-06-17 | 大金工业株式会社 | Improved method of determining chemical |
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| CN107384882A (en) * | 2017-08-22 | 2017-11-24 | 西南大学 | The molecular cloning and authentication method of a kind of domestic silkworm gene |
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| AU2005206899A1 (en) * | 2004-01-13 | 2005-08-04 | University Of Utah Research Foundation | Compositions and methods for modulating DHR96 |
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| CN106086202A (en) * | 2016-07-12 | 2016-11-09 | 广西大学 | The method polluted with CYP1A genetic surveillance water China and Philippines |
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