PCR (Polymerase Chain Reaction) chip, application thereof and method for evaluating toxic effect of aromatic hydrocarbon pollutants on zebra fish

Abstract

The invention relates to the technical field of molecular biology and environmental biology, and discloses a PCR chip, application thereof and a method for evaluating the toxic effect of petroleum pollutants on zebra fish. The PCR chip comprises 21 pairs of target gene primers, each pair of target gene primers comprises an upstream primer and a downstream primer, and the sequences of the 21 pairs of target gene primers are as shown in SEQ ID NO. 1 to SEQ ID NO. 42. According to the invention, on the basis of AhR-mediated biotoxicity effect in aromatic hydrocarbon petroleum pollutants, 21 genes related to I-phase metabolism and II-phase metabolism influenced by mutation of aromatic hydrocarbon pollutants in zebrafish are selected in a targeted manner, and 21 pairs of gene primers are specifically designed and are integrated into a PCR chip; the genes can be used as biomarkers for accumulation of aromatic hydrocarbon pollutants in an organism, and a basis is provided for evaluating potential carcinogenic risks and risk prevention and control of the aromatic hydrocarbon petroleum pollutants.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and environmental biotechnology, and in particular to a PCR chip and application thereof, and a method for evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish. Background Art

[0002] Petroleum is an organic compound with complex components. It is called "the blood of industry" and plays an indispensable role in human life. With the increasing demand for petroleum in production and life, crude oil is being mined continuously. As for petroleum pollutants, both China and foreign countries are facing severe petroleum pollution environmental problems. Petroleum pollution mainly occurs during the process of petroleum extraction, smelting, transportation, use and disposal. Petroleum pollutants cause a certain degree of pollution to surface water, groundwater, soil and air, and are a type of pollutant with a wide range of impact.

[0003] Common aromatic petroleum pollutants are divided into two categories: monocyclic aromatic hydrocarbons and polycyclic aromatic hydrocarbons. Aromatic compounds contained in petroleum are considered to have carcinogenic risks. Among them, the metabolism of polycyclic aromatic hydrocarbons in the body first undergoes catalytic oxidation by cytochrome enzymes and hydroxylation by epoxidases. The resulting hydrophilic products become more water soluble. At the same time, the reactivity of hydroxylated polycyclic aromatic hydrocarbons with DNA and proteins is enhanced, making it easier to form adducts with the nucleophilic part of the particles. Such adducts can destroy proteins or cause DNA mutations, thereby producing carcinogenicity. With the in-depth study of the carcinogenic mechanism of aromatic petroleum pollutants, it has been found that AhR is involved in many signal transduction pathways and plays an important role in regulating cell proliferation, differentiation and apoptosis. These processes are related to the occurrence and development of tumors and ultimately lead to the formation of malignant tumors. The toxicity of aromatic petroleum pollutants is closely related to their ability to induce AhR and activate receptors to bind to AhRe. The levels of AhRmRNA in various cancer cell lines show relatively high expression. After AhR binds to the ligand, AhR forms a dimer with arnt, translocates to the nucleus, binds to the regulatory elements of exogenous chemicals, and mediates the expression of a series of exogenous metabolic enzymes, including phase I metabolic enzymes (mainly members of the cytochrome p450 superfamily, such as cyp1a1, cyp1b1, cyp1c1, etc.) and phase II metabolic enzymes (glutathione transferase gst, glucuronyl transferase ugt, etc.), thereby causing the body to produce adaptive and toxic reactions to exogenous compounds. In addition, sulfotransferase sult, as the main phase II metabolic enzyme in the liver, is also involved in the metabolic process of cancer-inducing substances.

[0004] However, there is no report in the prior art on how PCR chips can intuitively and comprehensively evaluate the toxic effects of exposure to aromatic hydrocarbon pollutants on zebrafish. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem that the existing PCR chip cannot intuitively and comprehensively evaluate the toxic effects of aromatic hydrocarbon pollutants on zebrafish, and to provide a PCR chip and its application as well as a method for evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish. The PCR chip can obtain a large amount of toxicity data through a single experiment and provide a basis for evaluating the potential carcinogenic risk prevention and control of aromatic hydrocarbon petroleum pollutants.

[0006] In order to achieve the above objectives, the present invention provides a PCR chip on one hand, which includes 21 pairs of target gene primers, each pair of target gene primers includes an upstream primer and a downstream primer, and the sequences of the 21 pairs of target gene primers are shown in SEQ ID NO.1 to SEQ ID NO.42.

[0007] Preferably, each pair of target gene primers is in solution form.

[0008] Preferably, the concentration of each pair of target gene primers is 8-12 μM.

[0009] Preferably, the PCR chip further comprises two pairs of internal reference gene primers, each pair of internal reference gene primers comprises an upstream primer and a downstream primer, and the sequences thereof are shown in SEQ ID NO.43 to SEQ ID NO.46.

[0010] Preferably, the internal reference gene primer is in the form of a solution.

[0011] Preferably, the concentration of each pair of internal reference gene primers is 8-12 μM.

[0012] Preferably, the PCR chip further comprises a vector.

[0013] Preferably, the carrier is a 96-well plate.

[0014] A second aspect of the present invention provides a PCR chip kit, characterized in that the PCR chip kit comprises the PCR chip described above, a qPCR reaction premix containing a dye, and RNase-free water.

[0015] Preferably, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

[0016] A third aspect of the present invention provides an application of the PCR chip or the PCR chip kit described above in evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish.

[0017] Preferably, the aromatic hydrocarbon pollutants are selected from one or more of oilfield produced water, oilfield additives and aromatic hydrocarbons.

[0018] Preferably, the oilfield produced water is selected from one or more of oilfield produced water, oilfield produced water three-phase separator outlet water and oilfield produced water outlet water.

[0019] Preferably, the oilfield additive is selected from one or more of a heavy oil viscosity reducer, an oil-soluble demulsifier and a pressure-reducing and injection-increasing surfactant.

[0020] Preferably, the aromatic hydrocarbons contained in the aromatic hydrocarbon pollutants are selected from one or more of phenol, phenanthrene, naphthalene, benzene, toluene, xylene, trimethylbenzene and dimethylphenol.

[0021] A fourth aspect of the present invention provides a method for evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish, the method comprising the following steps:

[0022] (1) placing zebrafish embryos at the cell stage in an experimental group solution and a control group solution for incubation, collecting surviving embryos and larvae, extracting total RNA from the surviving embryos and larvae, and then reverse transcribing the total RNA as a template to obtain cDNA;

[0023] (2) mixing the cDNA with the PCR chip according to any one of claims 1 to 5 to perform RT-qPCR reaction to obtain a CT value;

[0024] (3) Calculate the relative expression fold of the target gene relative to the reference gene based on the CT value, and then use the relative expression fold of the gene for analysis.

[0025] Preferably, in step (1), the incubation conditions include: a temperature of 20 to 30° C. and a time of 90 to 100 hours.

[0026] Preferably, in step (1), the experimental group solution is obtained by diluting the aromatic hydrocarbon pollutant sample to be tested with standard dilution water; and the control group solution is standard dilution water.

[0027] Preferably, in step (1), the reverse transcription conditions include: reacting at 38-45°C for 10-20 min, and then reacting at 80-90°C for 3-8 seconds.

[0028] Preferably, step (2) specifically comprises: mixing the cDNA, the PCR chip according to any one of claims 1 to 5, a qPCR reaction premix containing a dye and RNase-free water to perform RT-qPCR reaction to obtain a CT value.

[0029] Preferably, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

[0030] Preferably, in step (2), the conditions of the RT-qPCR reaction include: pre-denaturation at 93-96°C for 28-33s, denaturation at 93-96°C for 4-6s, and then annealing and extension at 58-62°C for 14-16s.

[0031] Aromatic hydrocarbon pollutants in petroleum may have carcinogenic risks. Aromatic hydrocarbon pollutants can activate AhR receptors in the body, enter the cell nucleus and bind to aromatic hydrocarbon nuclear transfer protein (arnt) to form AhR-arnt dimers. AhR-arnt dimers can specifically bind to the dioxin response element (DRE) in the upstream regulatory region of the target gene, activate a series of auxiliary factors, and regulate the transcription and expression of downstream genes, mainly cytochrome cyp1a1, cyp1a2, and cyp1b1. The present invention focuses on the biological toxicity effects mediated by AhR in aromatic hydrocarbon petroleum pollutants, and targets the metabolic process of aromatic hydrocarbon petroleum pollutants in the body. A total of 21 genes related to phase I metabolism and phase II metabolism that are affected by the mutagenic effects of aromatic hydrocarbon pollutants in zebrafish are selected in a targeted manner, and 21 pairs of gene primers are specifically designed and assembled into PCR chips. These genes can be used as biomarkers for the accumulation of aromatic hydrocarbon pollutants in the body, providing a basis for evaluating the potential carcinogenic risk and risk prevention and control of aromatic hydrocarbon petroleum pollutants.

[0032] The present invention uses biological toxicity assessment to directly provide a basis for risk assessment of aromatic petroleum pollutants. Aromatic petroleum pollutants act on different target sites of organisms, causing damage to the molecular level of organisms; the mutagenic and carcinogenic potential of aromatic petroleum pollutants is defined based on the toxic endpoints mediated by the aromatic hydrocarbon receptor AhR. The present invention can be used to reflect the biological toxic effects mediated by AhR in aromatic petroleum pollutants, and can provide a basis for the potential risk prevention and control of aromatic petroleum pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram of the reaction system setup in a PCR experiment;

[0034] Figure 2 The changes in gene expression levels of the chip-related genes exposed to oilfield produced water in Example 1;

[0035] Figure 3 The changes in the gene expression levels of the chip-related genes exposed to the effluent of the three-phase separator of oilfield produced water in Example 2;

[0036] Figure 4 It is the change in gene expression level related to the oilfield produced water exposure chip in Example 3;

[0037] Figure 5 The changes in gene expression levels of the active additives (normal temperature heavy oil viscosity reducer) exposed to the chip in Example 4 Shengli Oilfield;

[0038] Figure 6 The changes in gene expression levels of the chip-related exposure to the active additives (demulsifiers for gathering and transportation / oil-soluble demulsifiers) in Shengli Oilfield in Example 5;

[0039] Figure 7 It is the changes in gene expression levels of the active adjuvant (pressure-reducing and injection-increasing surfactant) exposed to the chip in Example 6 Shengli Oilfield;

[0040] Figure 8 It is the change in gene expression level related to the phenol exposure chip in Example 7;

[0041] Fig. 9 The changes in gene expression levels of the phenanthrene exposure chip in Example 8;

[0042] Fig.10 The changes in gene expression levels of the naphthalene exposure chip in Example 9;

[0043] Fig.11 This is the change in gene expression levels related to the benzene exposure chip in Example 10. DETAILED DESCRIPTION

[0044] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0045] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0046] Since petroleum pollutants are highly toxic to organisms, the zebrafish genome has 87% homology with human genes, and has the advantages of being easy to raise, having strong fertility, and developing quickly, the present invention has selected zebrafish as a model organism and developed a zebrafish biotoxicity PCR chip for detecting the toxic effects of aromatic petroleum pollutants.

[0047] The PCR chip provided in the first aspect of the present invention comprises 21 pairs of target gene primers, each pair of target gene primers comprises an upstream primer and a downstream primer, and the sequences of the 21 pairs of target gene primers are shown as SEQ ID NO.1 to SEQ ID NO.42.

[0048] The 21 pairs of target gene primers on the PCR chip of the present invention are related to the phase I metabolism and phase II metabolism of aromatic hydrocarbon pollutants in zebrafish. By specifically designing the relevant gene primers, a zebrafish toxicity chip for aromatic hydrocarbon petroleum pollutants is integrated; the PCR chip containing the specific target gene primers can obtain a large amount of toxicity data through one experiment, and provide a basis for evaluating the potential carcinogenic risk prevention and control of aromatic hydrocarbon petroleum pollutants. The gene and primer sequences are shown in Table 1 below.

[0049] Preferably, the PCR chip further comprises 2 pairs of internal reference gene primers, each pair of internal reference gene primers comprises an upstream primer and a downstream primer, and the sequences thereof are shown in SEQ ID NO. 43 to SEQ ID NO. 46. The gene and primer sequences are shown in Table 1 below.

[0050] Table 1

[0051]

[0052]

[0053]

[0054] In the present invention, the target gene primers can be in various forms when used in the RT-qPCR experiment. Preferably, each pair of target gene primers is in the form of a solution. More preferably, the concentration of each pair of target gene primers is 8 to 12 μM.

[0055] In a specific embodiment, the internal reference gene primers can be used for RT-qPCR reaction in the same manner and under the same conditions as the target gene primers. Preferably, the internal reference gene primers are in the form of a solution. More preferably, the concentration of each pair of internal reference gene primers is 8 to 12 μM.

[0056] According to the present invention, specifically, the PCR chip further comprises a carrier; preferably, the carrier is a 96-well plate.

[0057] When conducting the experiment, different target gene primers and internal reference gene primers are placed in different wells of the carrier to obtain the PCR chip; deionized water is placed in one of the wells as a negative control to detect whether there is template, i.e., cDNA contamination. If abnormal results appear in the negative control well, it may be that the reagent or deionized water is contaminated with template. At the same time, according to the needs of the RT-qPCR experiment, qPCR reaction premix containing dye and RNase-free water need to be added to each well to form an RT-qPCR reaction system.

[0058] A second aspect of the present invention provides a PCR chip kit, which comprises the PCR chip described above, a qPCR reaction premix containing a dye, and RNase-free water.

[0059] In a preferred embodiment, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBRGreenⅠ fluorescent dye and oxyribonucleotide triphosphate.

[0060] A third aspect of the present invention provides an application of the PCR chip or the PCR chip kit described above in evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish.

[0061] In the present invention, the source of the aromatic hydrocarbon pollutants is not limited, as long as they are petroleum-related pollutants. Specifically, the aromatic hydrocarbon pollutants are selected from one or more of oilfield produced water, oilfield additives and aromatic hydrocarbons. Herein, the oilfield produced water and oilfield additives both contain aromatic hydrocarbons.

[0062] In a specific embodiment, the oilfield produced water is selected from one or more of oilfield produced water, oilfield produced water three-phase separator water and oilfield produced water outlet water.

[0063] In a specific embodiment, the oilfield additive is selected from one or more of a heavy oil viscosity reducer, an oil-soluble demulsifier (demulsifier for gathering and transportation), and a pressure-reducing and injection-increasing surfactant.

[0064] In the present invention, preferably, the aromatic hydrocarbons contained in the aromatic hydrocarbon pollutants are selected from one or more of phenol, phenanthrene, naphthalene, benzene, toluene, xylene, trimethylbenzene and dimethylphenol.

[0065] A fourth aspect of the present invention provides a method for evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish, the method comprising the following steps:

[0066] (1) placing zebrafish embryos at the cell stage in an experimental group solution and a control group solution for incubation, collecting surviving embryos and larvae, extracting total RNA from the surviving embryos and larvae, and then reverse transcribing the total RNA as a template to obtain cDNA;

[0067] (2) mixing the cDNA with the PCR chip described above to perform RT-qPCR reaction to obtain a CT value;

[0068] (3) Calculate the relative expression fold of the target gene relative to the reference gene based on the CT value, and then use the relative expression fold of the gene for analysis.

[0069] In the present invention, in step (1), the experimental group solution is obtained by diluting the sample of aromatic hydrocarbon pollutants to be tested with standard dilution water; the control group solution is standard dilution water. Specifically, the sample to be tested is the aromatic hydrocarbon pollutants mentioned above.

[0070] In the method described in the present invention, the standard dilution water is the standard dilution water recorded in "Determination of Acute Toxicity of Water Quality - Zebrafish Egg Method HJ1069-2019".

[0071] According to the present invention, preferably, the zebrafish embryo at the cell stage in step (1) is a zebrafish embryo at the 4-128 cell stage.

[0072] In a specific implementation manner, the storage and processing method of the sample to be tested after collection is determined according to the specific type. For water samples related to oilfield produced water, the sample to be tested is stored at 4°C after collection, transported to the laboratory within 24 hours and frozen at -20°C. Before testing, it needs to be balanced and aerated at 26°C for 30 minutes to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L; dilute before use.

[0073] In a preferred embodiment, in step (1), the incubation conditions include: a temperature of 20 to 30° C. and a time of 90 to 100 hours.

[0074] According to the present invention, specifically, after extracting total RNA, a full-wavelength microplate reader is used to measure the concentration and quality of RNA. When the ratio of OD260 / OD280 is between 1.9 and 2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription.

[0075] In a preferred embodiment, in step (1), the reverse transcription conditions include: reacting at 38-45°C for 10-20 min, and then reacting at 80-90°C for 3-8 seconds.

[0076] Furthermore, step (2) specifically includes: mixing the cDNA, the PCR chip mentioned above, the qPCR reaction premix containing the dye and RNase-free water to perform RT-qPCR reaction to obtain a CT value.

[0077] In the present invention, preferably, the qPCR reaction premix containing the dye is a commercial kit, which contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

[0078] In the method of the present invention, specifically, in step (2), the conditions of the RT-qPCR reaction include: pre-denaturation at 93-96°C for 28-33s, denaturation at 93-96°C for 4-6s, and then annealing and extension at 58-62°C for 14-16s. Preferably, in step (2), the conditions of the RT-qPCR reaction include: pre-denaturation at 94°C for 30s, denaturation at 94°C for 5s, and then annealing and extension at 60°C for 15s.

[0079] The existing technology only detects PAHs in the environment through AhR gene promoter. The zebrafish toxicity chip for aromatic petroleum pollutants provided by the present invention involves the toxic effect pathway of aromatic hydrocarbon receptors (AhR) induced by PAHs in body cells, and a total of 21 related genes that activate the aromatic hydrocarbon receptors after PAHs enter the body, initiate a series of downstream biological effects, and thus exert toxic effects; it can not only characterize the ability of aromatic petroleum pollutants to induce AhR after entering the body, but also characterize the metabolic toxicity of aromatic petroleum pollutants in the body, which has a major reference value for evaluating their toxicity and environmental concentration.

[0080] The present invention focuses on the biological toxicity effects mediated by AhR in aromatic petroleum pollutants, selects a total of 21 genes related to phase I and phase II metabolism that are affected by aromatic pollutants in zebrafish, specifically designs relevant gene primers, and integrates a zebrafish toxicity chip for aromatic petroleum pollutants. The present invention can be used as a biomarker for the metabolism, accumulation and toxicity of aromatic pollutants in the body. At the same time, a large amount of toxicity data can be obtained through a single experiment, and it provides a basis for evaluating the potential carcinogenic risk prevention and control of aromatic petroleum pollutants.

[0081] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0082] The following examples carry out zebrafish embryo toxicity experiments according to "HJ 1069-2019 Determination of acute toxicity of water quality - zebrafish egg method", "OECD 236 Fish Embryo Acute Toxicity (FET) Test" and "GB / T 21807-2008 Short-term toxicity test of chemicals for fish embryos and yolk sac larvae".

[0083] In the following examples, the base sequences in the target gene primers and the internal reference gene primers were independently developed and designed and synthesized by Shanghai Shenggong Biotechnology Co., Ltd.; the qPCR reaction premix containing the dye was a commercial kit purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0084] Example 1

[0085] This example is used to illustrate the process of using the PCR chip described in the present invention to evaluate the toxic effects of produced water from a certain collection site in Shengli Oilfield (containing 391 mg / L of toluene, 536 mg / L of xylene, 174 mg / L of 2,6-dimethylphenol, 136 mg / L of 2,4-dimethylphenol, 172 mg / L of 2,4,6-trimethylbenzene, 451 mg / L of phenol, and 219 mg / L of naphthalene) on zebrafish.

[0086] 1) Incubation process

[0087] The produced water sample (sample 1 to be tested) from a collection station of Shengli Oilfield was collected and stored at 4°C, transported to the laboratory and frozen at -20°C within 24 hours, balanced and aerated at 26°C for 30 minutes before testing to ensure that the initial dissolved oxygen concentration was not less than 4 mg / L, and diluted before use;

[0088] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0089] Control solution: standard dilution water;

[0090] The incubation process includes:

[0091] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in one well. Incubate at 28°C for 96 h, and collect the surviving embryos and larvae.

[0092] 2) Extraction of total RNA

[0093] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0094] 3) Reverse transcription

[0095] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0096] 4) RT-qPCR reaction

[0097] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (components are Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in the figure. Figure 2 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0098] from Figure 2 It can be seen that the expression levels of cyp1a1, cyp1b1, cyp1c1 and cyp1c2 increased significantly when exposed to oilfield produced water, indicating that pollutants may have entered the embryo and induced the embryo to start the detoxification metabolism mechanism, especially organic pollutants, which were oxidized to open the ring or expose highly reactive active sites through phase I metabolism. The increased expression of genes related to phase I metabolism indicates that there are organic components in the produced water of the oilfield and they are metabolized through the phase I metabolic pathway. Through the changes in the expression of genes related to the AhR receptor family, it can be seen that the AhR2 gene is highly expressed, and the activation of AhR induces the cytochrome enzyme cyp1a1 that can oxidize AhR ligands to promote the metabolic clearance and detoxification of AhR ligands (halogenated aromatic hydrocarbons or polycyclic aromatic hydrocarbons, etc.), which is consistent with the high expression of phase I metabolism cyp1a1, indicating that the organic pollutants in the produced water of the oilfield have aromatic components.

[0099] Example 2

[0100] This example is used to illustrate the process of using the PCR chip described in the present invention to evaluate the toxic effect of the effluent from the three-phase separator of oilfield produced water at a certain collection site of Shengli Oilfield (containing 273 mg / L of toluene, 452 mg / L of xylene, 152 mg / L of 2,6-dimethylphenol, 117 mg / L of 2,4-dimethylphenol, 111 mg / L of 2,4,6-trimethylbenzene, 343 mg / L of phenol, and 175 mg / L of naphthalene) on zebrafish.

[0101] 1) Incubation process

[0102] The effluent sample of the three-phase separator of the produced water in the oil field (sample 2 to be tested) was collected from a collection station of Shengli Oilfield and stored at 4°C, transported to the laboratory and frozen at -20°C within 24 hours, balanced and aerated at 26°C for 30 minutes before testing to ensure that the initial dissolved oxygen concentration was not less than 4 mg / L, and diluted before use;

[0103] Experimental solution: dilute 10 mL of sample 2 with 90 mL of standard dilution water;

[0104] Control solution: standard dilution water;

[0105] The incubation process includes:

[0106] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0107] 2) Extraction of total RNA

[0108] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low temperature environment. To ensure that the extracted RNA was not contaminated or dissolved, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0109] 3) Reverse transcription

[0110] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0111] 4) RT-qPCR reaction

[0112] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 3 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0113] from Figure 3 It can be seen that the expression levels of cyp1a1, cyp1b1, cyp1c1 and cyp1c2 increased significantly when the effluent of the three-phase separator of oilfield produced water was exposed, indicating that the pollutants may have entered the embryo and induced the embryo to start the detoxification metabolism mechanism, especially the organic pollutants, which were oxidized to open the ring or expose highly reactive active sites through phase I metabolism. Phase I metabolism indicates that there are organic components in the effluent of the three-phase separator of oilfield produced water. Through the changes in the expression of genes related to the AhR receptor family, it can be seen that the AhR2 gene is highly expressed, and the activation of AhR induces the cytochrome enzyme cyp1a1 that can oxidize AhR ligands to promote the metabolic clearance and detoxification of AhR ligands (halogenated aromatic hydrocarbons or polycyclic aromatic hydrocarbons, etc.), which is consistent with the high expression of cyp1a1 in the first phase metabolism, indicating that the organic pollutants in the effluent of the three-phase separator of oilfield produced water have aromatic components.

[0114] Example 3

[0115] This example is used to illustrate the process of using the PCR chip described in the present invention to evaluate the toxic effects of produced water from a certain collection site in Shengli Oilfield (containing 165 mg / L of toluene, 367 mg / L of xylene, 116 mg / L of 2,6-dimethylphenol, 83 mg / L of 2,4-dimethylphenol, 75 mg / L of 2,4,6-trimethylbenzene, 129 mg / L of phenol, and 96 mg / L of naphthalene) on zebrafish.

[0116] 1) Incubation process

[0117] The produced water sample (sample 3 to be tested) from a collection station of Shengli Oilfield was collected and stored at 4°C, transported to the laboratory and frozen at -20°C within 24 hours, balanced and aerated at 26°C for 30 minutes before testing to ensure that the initial dissolved oxygen concentration was not less than 4 mg / L, and diluted before use;

[0118] Experimental group solution: dilute 10 mL of sample 3 with 90 mL of standard dilution water;

[0119] Control solution: standard dilution water;

[0120] The incubation process includes:

[0121] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0122] 2) Extraction of total RNA

[0123] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low temperature environment. To ensure that the extracted RNA was not contaminated or dissolved, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0124] 3) Reverse transcription

[0125] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0126] 4) RT-qPCR reaction

[0127] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 4 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0128] from Figure 4 It can be seen that the expression levels of cyp1a1, cyp1b1, cyp1c1 and cyp1c2 increased significantly when exposed to oilfield produced water, indicating that pollutants may have entered the embryo and induced the embryo to start the detoxification metabolism mechanism, especially organic pollutants, which were oxidized to open the ring or expose highly reactive active sites through phase I metabolism. Phase I metabolism indicates that there are organic components in the oilfield produced water. Through the changes in the expression of genes related to the AhR receptor family, it can be seen that the AhR2 gene is highly expressed, and the activation of AhR induces the cytochrome enzyme cyp1a1 that can oxidize AhR ligands to promote the metabolic clearance and detoxification of AhR ligands (halogenated aromatic hydrocarbons or polycyclic aromatic hydrocarbons, etc.), which is consistent with the high expression of cyp1a1 in phase I metabolism, indicating that there are aromatic hydrocarbon components in the organic pollutants in the oilfield produced water.

[0129] Example 4

[0130] This example is used to illustrate the process of using the PCR chip described in the present invention to evaluate the toxic effects of the current additives in Shengli Oilfield (room temperature heavy oil viscosity reducer, purchased from Huabin Chemical, 50% by mass of heavy diesel; 25% by mass of polyoxyethylene alkyl alcohol ether; 25% by mass of polyoxyethylene polyoxypropylene glycol ether) on zebrafish.

[0131] 1) Incubation process

[0132] The room temperature heavy oil viscosity reducer sample (sample 4 to be tested) was stored at room temperature 25°C, transported to the laboratory within 24 hours and stored at room temperature and away from light, and diluted before testing;

[0133] Experimental group solution: dilute 3 mL of sample 4 with 97 mL of standard dilution water;

[0134] Control solution: standard dilution water;

[0135] The incubation process includes:

[0136] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0137] 2) Extraction of total RNA

[0138] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0139] 3) Reverse transcription

[0140] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0141] 4) RT-qPCR reaction

[0142] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 5 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0143] from Figure 5 It can be seen that the expression levels of the first-phase metabolism-related genes cyp1a1, cyp1b1, cyp1c1 and cyp1c2 increased when exposed to oilfield additives (normal temperature heavy oil viscosity reducer), especially the expression level of cyp1c1 increased significantly. And the three types of second-phase metabolism-related genes screened in the study, namely sulfotransferases, UDP-glucuronosyltransferases and glutathione S-transferases, all increased in expression to varying degrees, and they can add different polar groups to the first-phase metabolites to increase the polarity of pollutants through second-phase metabolism and promote metabolic excretion of the body.

[0144] Example 5

[0145] This example is used to illustrate the process of using the PCR chip described in the present invention to evaluate the toxic effects of the current auxiliary agent in Shengli Oilfield (demulsifier / oil-soluble demulsifier for gathering and transportation, purchased from Dongying Lifeng, containing 40% by mass of polyoxypropylene polyoxyethylene block polymer and 60% by mass of aromatic solvent oil) on zebrafish.

[0146] 1) Incubation process

[0147] Store the demulsifier / oil-soluble demulsifier sample for gathering and transportation (sample 5 to be tested) at room temperature of 25°C, transport it to the laboratory within 24 hours and store it at room temperature and away from light, and dilute it before testing;

[0148] Experimental group solution: dilute 3 mL of sample 5 with 97 mL of standard dilution water;

[0149] Control solution: standard dilution water;

[0150] The incubation process includes:

[0151] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0152] 2) Extraction of total RNA

[0153] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0154] 3) Reverse transcription

[0155] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0156] 4) RT-qPCR reaction

[0157] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 6 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0158] from Figure 6 It can be seen that the expression levels of the first-phase metabolism-related genes cyp1a1, cyp1b1, cyp1c1 and cyp1c2 increased when exposed to oilfield additives (demulsifiers for gathering and transportation / oil-soluble demulsifiers), especially the expression level of cyp1c1 increased significantly. The three types of genes related to second-phase metabolism screened in the study, namely sulfotransferases, UDP-glucuronosyltransferases and glutathione S-transferases, all increased in expression to varying degrees, and they can add different polar groups to the first-phase metabolites to increase the polarity of pollutants through second-phase metabolism and promote metabolic excretion of the body. At the same time, the expression of the aromatic hydrocarbon receptor AhR family genes was upregulated, indicating the presence of aromatic hydrocarbon solvent oil as an additive component.

[0159] Example 6

[0160] This example is used to illustrate the process of using the PCR chip described in the present invention to evaluate the toxic effects of the current adjuvant in Shengli Oilfield (a pressure-reducing and injection-increasing surfactant purchased from Dongying Lifeng, containing 75% by mass of white oil and 25% by mass of polyacrylamide) on zebrafish.

[0161] 1) Incubation process

[0162] The pressure-reducing and injection-enhancing surfactant sample (sample 6 to be tested) was stored at room temperature of 25°C, transported to the laboratory within 24 hours and stored at room temperature and away from light, and diluted before testing;

[0163] Experimental group solution: dilute 3 mL of sample 6 with 97 mL of standard dilution water;

[0164] Control solution: standard dilution water;

[0165] The incubation process includes:

[0166] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0167] 2) Extraction of total RNA

[0168] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0169] 3) Reverse transcription

[0170] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0171] 4) RT-qPCR reaction

[0172] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 7 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0173] from Figure 7 It can be seen that the expression levels of the first-phase metabolism-related genes cyp1a1, cyp1b1, cyp1c1 and cyp1c2 increased when exposed to oilfield adjuvants (pressure-reducing and injection-enhancing surfactants), especially the expression level of cyp1c1 increased significantly. The three types of second-phase metabolism-related genes screened in the study were sulfotransferases, UDP-glucuronosyltransferases and glutathione S-transferases, and their expression levels increased to varying degrees. They can add different polar groups to the first-phase metabolites to increase the polarity of pollutants through second-phase metabolism and promote metabolic excretion in the body.

[0174] Example 7

[0175] This example is used to illustrate the process of evaluating the toxic effect of phenol on zebrafish using the PCR chip described in the present invention.

[0176] 1) Incubation process

[0177] Store phenol (sample 7) in a filter-type reagent cabinet in the reagent library and dilute before testing;

[0178] Experimental group solution: dilute 5 mg of the sample 7 with 10 mL of standard dilution water;

[0179] Control solution: standard dilution water;

[0180] The incubation process includes:

[0181] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0182] 2) Extraction of total RNA

[0183] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0184] 3) Reverse transcription

[0185] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0186] 4) RT-qPCR reaction

[0187] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 8 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0188] from Figure 8 As can be seen, the high expression of phase I metabolism, phase II metabolism and AhR receptor-related genes in zebrafish embryos after phenol exposure proves that phenol can be metabolized through these metabolic pathways.

[0189] Example 8

[0190] This example is used to illustrate the process of evaluating the toxic effects of phenanthrene on zebrafish using the PCR chip described in the present invention.

[0191] 1) Incubation process

[0192] Store phenanthrene (sample 8) in a reagent storage filter reagent cabinet and dilute before testing;

[0193] Experimental group solution: dilute 5 mg of the sample 8 to be tested with 10 mL of standard dilution water;

[0194] Control solution: standard dilution water;

[0195] The incubation process includes:

[0196] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0197] 2) Extraction of total RNA

[0198] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0199] 3) Reverse transcription

[0200] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0201] 4) RT-qPCR reaction

[0202] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Fig. 9 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0203] from Fig. 9 As can be seen in the figure, phenanthrene enters the cell nucleus and binds to the aromatic hydrocarbon nuclear transfer protein (arnt) to form an AhR-arnt dimer. The AhR-arnt dimer can specifically bind to the upstream regulatory region of the target gene, activate a series of auxiliary factors, and regulate the transcription and expression of genes, mainly cytochrome cyp1a1, cyp1b1, cyp1c1 and cyp1c2 and phase II metabolism-related genes.

[0204] Example 9

[0205] This example is used to illustrate the process of evaluating the toxic effect of naphthalene on zebrafish using the PCR chip described in the present invention.

[0206] 1) Incubation process

[0207] Naphthalene (sample 9) was stored in a reagent storage filter reagent cabinet and diluted before testing;

[0208] Experimental group solution: dilute 5 mg of the sample 9 to be tested with 10 mL of standard dilution water;

[0209] Control solution: standard dilution water;

[0210] The incubation process includes:

[0211] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0212] 2) Extraction of total RNA

[0213] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0214] 3) Reverse transcription

[0215] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0216] 4) RT-qPCR reaction

[0217] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Fig.10 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0218] from Fig.10 It can be seen that the increased expression of cyp family-related genes suggests that naphthalene undergoes oxidative ring opening or exposes highly reactive active sites through phase one metabolism, including the naphthalene ring addition site in the complex structure of CYP1b1. Naphthalene may induce a comprehensive and complex metabolic network to complete metabolism and thus produce carcinogenicity.

[0219] Example 10

[0220] This example is used to illustrate the process of evaluating the toxic effect of benzene on zebrafish using the PCR chip described in the present invention.

[0221] 1) Incubation process

[0222] Store benzene (sample 10 to be tested) in a filter reagent cabinet in the reagent store and dilute before testing;

[0223] Experimental group solution: dilute 5 mg of the sample 10 to be tested with 10 mL of standard dilution water;

[0224] Control solution: standard dilution water;

[0225] The incubation process includes:

[0226] In a 24-well plate, place one zebrafish embryo at the 4-128 cell stage and 2 mL of the experimental solution in 23 wells, and place one zebrafish embryo at the 4-128 cell stage and the control solution in 1 well. Incubate at 20°C for 96 h, and collect the surviving embryos and larvae.

[0227] 2) Extraction of total RNA

[0228] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0229] 3) Reverse transcription

[0230] The extracted total RNA was used as a template for reverse transcription according to the operation steps of the All-Gold AT341-01 reverse transcription kit. In a 20 μL reaction system, there was 1 μg of total RNA template, 4 μL of All-in-one SuperMix for qPCR, 1 μL of gDNARemover and RNase-free water. The reaction conditions are incubation at 42°C for 15 min and heating at 85°C for 5 seconds. The 20 μL cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water and can then be used for RT-qPCR experiments.

[0231] 4) RT-qPCR reaction

[0232] RT-qPCR experiments were performed in 96-well plates using 21 pairs of target gene primers and 2 pairs of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. Columns 1-9 are experimental groups, and columns 10-12 are blank control groups. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Fig.11 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of exposure to aromatic petroleum pollutants on zebrafish metabolism-related genes.

[0233] from Fig.11 It can be seen that after benzene enters the human body, the expression of the first-phase metabolism-related genes, namely cyp1a1, cyp1b1, cyp1c1 and cyp1c2, is upregulated, indicating that under the action of the cytochrome P450 family, epoxybenzene is formed, and the final stable aromatic structure is broken, which can induce the aromatic hydrocarbon receptor (AhR) in the body's cells, namely Fig.11 The toxic effect pathway where the genes ahr2, ahrra, ahrb and arnt are located affects cell function and leads to the occurrence of tumors.

[0234] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A PCR chip, characterized in that: The PCR chip comprises 21 pairs of target gene primers, each pair of target gene primers comprises an upstream primer and a downstream primer, and the sequences of the 21 pairs of target gene primers are shown as SEQ ID NO.1 to SEQ ID NO.

42.

2. The PCR chip according to claim 1, characterized in that: Each pair of target gene primers is in solution form; Preferably, the concentration of each pair of target gene primers is 8-12 μM.

3. The PCR chip according to claim 1 or 2, characterized in that: The PCR chip also includes two pairs of internal reference gene primers, each pair of internal reference gene primers includes an upstream primer and a downstream primer, and the sequences thereof are shown in SEQ ID NO.43 to SEQ ID NO.

46.

4. The PCR chip according to claim 3, characterized in that: The internal reference gene primer is in the form of a solution; Preferably, the concentration of each pair of internal reference gene primers is 8-12 μM.

5. The PCR chip according to any one of claims 1 to 4, characterized in that: The PCR chip also includes a carrier; Preferably, the carrier is a 96-well plate.

6. A PCR chip kit, characterized in that: The PCR chip kit comprises the PCR chip according to any one of claims 1 to 5, a qPCR reaction premix containing a dye, and RNase-free water; Preferably, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

7. Use of the PCR chip according to any one of claims 1 to 5 or the PCR chip kit according to claim 6 in evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish.

8. The use according to claim 7, characterized in that: The aromatic hydrocarbon pollutants are selected from one or more of oilfield produced water, oilfield additives and aromatic hydrocarbons; Preferably, the oilfield produced water is selected from one or more of oilfield produced water, oilfield produced water three-phase separator effluent and oilfield produced water effluent; Preferably, the oilfield additive is selected from one or more of a heavy oil viscosity reducer, an oil-soluble demulsifier and a pressure-reducing and injection-increasing surfactant.

9. The use according to claim 7 or 8, characterized in that: The aromatic hydrocarbons contained in the aromatic hydrocarbon pollutants are selected from one or more of phenol, phenanthrene, naphthalene, benzene, toluene, xylene, trimethylbenzene and dimethylphenol.

10. A method for evaluating the toxic effects of aromatic hydrocarbon pollutants on zebrafish, characterized in that: The method comprises the following steps: (1) placing zebrafish embryos at the cell stage in an experimental group solution and a control group solution for incubation, collecting surviving embryos and larvae, extracting total RNA from the surviving embryos and larvae, and then reverse transcribing the total RNA as a template to obtain cDNA; (2) mixing the cDNA and the PCR chip according to any one of claims 1 to 5 to perform RT-qPCR reaction to obtain a CT value; (3) Calculate the relative expression fold of the target gene relative to the reference gene based on the CT value, and then use the relative expression fold of the gene for analysis.

11. The method according to claim 10, characterized in that In step (1), the incubation conditions include: a temperature of 20 to 30° C. and a time of 90 to 100 hours.

12. The method according to claim 10 or 11, characterized in that: In step (1), the experimental group solution is obtained by diluting the aromatic hydrocarbon pollutant sample to be tested with standard dilution water; the control group solution is standard dilution water.

13. The method according to any one of claims 10 to 12, characterized in that: In step (1), the reverse transcription conditions include: reacting at 38-45°C for 10-20 minutes, and then reacting at 80-90°C for 3-8 seconds.

14. The method according to any one of claims 10 to 13, characterized in that: Step (2) specifically comprises: mixing the cDNA, the PCR chip according to any one of claims 1 to 5, a qPCR reaction premix containing a dye and RNase-free water to perform RT-qPCR reaction to obtain a CT value.

15. The method according to claim 14, characterized in that The qPCR reaction premix containing dye contains Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and oxyribonucleotide triphosphate.

16. The method according to any one of claims 10 to 15, characterized in that: In step (2), the conditions of the RT-qPCR reaction include: pre-denaturation at 93-96°C for 28-33s, denaturation at 93-96°C for 4-6s, and then annealing and extension at 58-62°C for 14-16s.

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