Gene primer pretreatment method of PCR (Polymerase Chain Reaction) chip, PCR chip and method for evaluating toxic effect of oilfield pollutants on zebra fish

Abstract

The invention relates to the technical field of biotoxicity detection, and discloses a gene primer pretreatment method of a PCR (Polymerase Chain Reaction) chip, the PCR chip and a method for evaluating the toxic effect of oil field pollutants on zebra fish. The method comprises the following steps: (1) respectively placing upstream and downstream primers of a target gene and upstream and downstream primers of a reference gene in different holes of a PCR (Polymerase Chain Reaction) plate, sealing the holes and pricking the holes, and then carrying out heat treatment in a metal bath; and (2) after the heat treatment is finished, sealing the orifice again, and storing for later use. The gene primers are pretreated in a heat treatment mode, the method is easy to operate, and experiments prove that the heat treatment mode has the minimum influence on the stability and amplification effect of the primers; according to the present invention, the gene primer is pretreated by using the method, and the gene primer is fixed in the PCR plate so as to obtain the PCR-Array chip beneficial to transportation and storage, such that the technical support is provided for the popularization of the zebra fish PCR-Array chip.

Description

Technical Field

[0001] The invention relates to the technical field of biological toxicity detection, and in particular to a gene primer pretreatment method for a PCR chip, a PCR chip, and a method for evaluating the toxic effects of oilfield pollutants on zebrafish. Background Art

[0002] As the oil and gas field exploitation process deepens, the difficulty of oil and gas collection gradually increases. At the same time, due to the constraints of technical equipment and other factors, the actual exploitation capacity and oil and gas quality will be affected to a certain extent, so chemical agents are needed to improve the actual exploitation efficiency. Therefore, the demand for oil and gas field additives is gradually increasing, and the functional requirements are increasing. The amount of domestic oil and gas field additives is increasing year by year, and the types and components are quite complex. A large number of chemical agents containing acids, alcohols, ethers, polymers and even heavy metals are used in oil and gas exploitation, and they inevitably enter the environmental medium during their life cycle to produce ecological risks. Therefore, it is necessary to analyze the toxicity and ecological hazards of oil and gas field chemical additives.

[0003] In addition, during the mining process, the mixed oil and gas field wastewater produced together with oil and gas generally contains oil and gas and converted or unconverted oil and gas field additives, including high-molecular-weight refractory pollutants such as surfactants and polymer oil displacement agents. The method of sewage reinjection into the formation is commonly used to treat produced water at home and abroad, and some methods are used to treat it by reinjecting most of it and discharging a small amount after treatment. The huge amount of oil field produced water carrying trace amounts of oil and a large amount of oil field additives has brought great pressure to the external sewage treatment plant, the surrounding environment and human health. There is now data proving that sewage reinjection has potential pollution risks to groundwater. Therefore, it is very necessary to conduct toxicity testing on oil and gas field produced water.

[0004] At present, the toxicity research of oilfield additives and produced water is mainly limited to ecotoxicity. The above-mentioned biological toxicity test methods can only obtain the acute toxicity data of pollutants, but the classification of specific toxic effects of pollutants, toxic endpoints, toxic pathways and explanations of toxicity mechanisms are relatively lacking. There is also a lack of corresponding health toxicity data, and it is impossible to know the existence of toxic effects such as teratogenicity, carcinogenicity and mutagenicity, which is not conducive to optimizing additive ingredients, improving pollution treatment technology and comprehensively evaluating the biological and ecotoxicity of pollutants. PCR Array toxicity chip is a toxicity test method that can interpret toxic pathways and mechanisms of action. By carrying different types of primers and measuring the response to toxic endpoints, it can interpret the toxic pathways activated by pollutants and reveal the toxicity mechanism.

[0005] Currently, there are few studies on the toxic effects and toxic mechanisms of oil and gas field additives and produced water. Only a few articles have studied the toxic effects of oil and gas field-related additives, produced water or crude oil at the molecular level. At present, my country is gradually promoting biological monitoring to supplement environmental monitoring data. PCR Array toxicity chips can systematically analyze toxic effects and mechanisms through molecular omics methods on the basis of ecological monitoring and acute toxicity research, which helps to understand pollutants and propose solutions. However, in the existing technology, there are few ways to preserve PCR chip primers for detecting the toxic effects of oil and gas field additives and produced water, and the stability is poor, the amplification effect is greatly affected, and the test results are unstable. Summary of the invention

[0006] The purpose of the present invention is to overcome the problem that the stability of PCR chip primers used in the prior art for detecting the toxic effects of oil and gas field additives and produced water is poor, the amplification effect is greatly affected, and the detection results are unstable, and a method for pretreating gene primers for a PCR chip and a PCR chip as well as a method for evaluating the toxic effects of oil field pollutants on zebrafish are provided. The present invention pretreats gene primers by heat treatment, the method is simple to operate, and it is experimentally verified that the heat treatment method has the least effect on the stability and amplification effect of the primers; the gene primers are pretreated by the method described in the present invention, and the gene primers are fixed in a PCR plate to obtain a PCR-Array chip that is convenient for transportation and storage, providing technical support for the promotion of zebrafish PCR-Array chips.

[0007] In order to achieve the above object, the present invention provides a method for pretreating gene primers of a PCR chip in a first aspect, the method comprising the following steps:

[0008] (1) placing the upstream and downstream primers of the target gene and the upstream and downstream primers of the internal reference gene in different wells of a PCR plate, sealing the wells and piercing the wells, and then heat treating them in a metal bath;

[0009] (2) After the heat treatment is completed, seal the hole again and keep it for future use.

[0010] Preferably, in step (1), the heat treatment conditions include: a temperature of 50 to 70° C. and a time of 8 to 15 minutes.

[0011] Preferably, in step (1), the target gene is selected from one or more of cyp1a1, hsp 70.3, ahrra, ugt5a4, ugt1a1 and hsp90aa 1.2.

[0012] Preferably, in step (1), the internal reference gene is β-actin and / or gadph.

[0013] Preferably, in step (1), the PCR plate is a 96-well plate or a 384-well plate.

[0014] The second aspect of the present invention provides a PCR chip obtained by the method described above.

[0015] A third 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.

[0016] Preferably, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

[0017] A fourth 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 oilfield pollutants on zebrafish.

[0018] Preferably, the oilfield pollutants are selected from oilfield additives and / or oilfield produced 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] A fifth aspect of the present invention provides a method for evaluating the toxic effects of oilfield pollutants on zebrafish, the method comprising the following steps:

[0021] (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;

[0022] (2) mixing the cDNA and the PCR chip according to claim 5 to perform RT-qPCR reaction to obtain a CT value;

[0023] (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.

[0024] Preferably, in step (1), the incubation conditions include: a temperature of 20 to 30° C. and a time of 90 to 100 hours.

[0025] Preferably, in step (1), the experimental group solution is obtained by diluting the oilfield pollutant sample to be tested with standard dilution water; and the control group solution is standard dilution water.

[0026] 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.

[0027] Preferably, step (2) specifically comprises: mixing the cDNA, the PCR chip according to any one of claims 5, a qPCR reaction premix containing a dye and RNase-free water to perform RT-qPCR reaction to obtain a CT value.

[0028] Preferably, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

[0029] 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.

[0030] The present invention uses a heat treatment method to pre-treat the gene primers of the PCR chip, and fixes the primers in the PCR plate. Experimental demonstration shows that the pre-treatment method has little effect on the stability and amplification effect of the primers, and is conducive to the storage, transportation and use of the PCR-Array chip, providing technical support for the promotion of zebrafish PCR-Array chips. At the same time, the PCR chip obtained by heat treatment is used to detect the toxic effects of oil and gas field additives and produced water in zebrafish. Compared with the freeze-dried PCR chip, the gene expression level changes less, and the error of the test result is smaller than that of the temporary storage at 4°C.

[0031] In the prior art, gene primer pairs are separately placed in the wells of a 96-well PCR plate, the operation process is relatively complicated, and the storage method of the primers in the 96-well PCR plate is not introduced in detail; compared with the prior art, the pretreatment method described in the present invention has a simple operation process and low cost.

[0032] Preferably, the PCR chip of the present invention involves internal reference genes that can be stably expressed in zebrafish, namely β-actin and gadph. After independent research and development of the involved gene primers, the designed internal reference gene primers have good specificity, high sensitivity, and good reference effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the effect of different pretreatments on cyp1a1 expression (cyp1a1 / β-actin);

[0034] Figure 2 is the effect of different pretreatments on the standard deviation of cyp1a1 expression (cyp1a1 / β-actin);

[0035] Figure 3 is the effect of different pretreatments on the expression of cyp1a1 (cyp1a1 / gadph);

[0036] Figure 4 is the effect of different pretreatments on the standard deviation of cyp1a1 expression (cyp1a1 / gadph);

[0037] Figure 5 is the effect of different pretreatments on the expression of gadph (gadph / β-actin);

[0038] Figure 6 It is the effect of different pretreatments on the standard deviation of gadph expression (gadph / β-actin).

[0039] Figure 7 is the effect of different pretreatments on the expression of hsp 70.3 (hsp 70.3 / gadph);

[0040] Figure 8 is the effect of different pretreatments on the standard deviation of hsp 70.3 expression (hsp 70.3 / gadph);

[0041] Fig. 9 is the effect of different pretreatments on ahrra expression (ahrra / gadph);

[0042] Fig.10 is the effect of different pretreatments on the standard deviation of ahrra expression (ahrra / gadph);

[0043] Fig.11 is the effect of different pretreatments on the expression of ugt5a4 (ugt5a4 / gadph);

[0044] Fig.12 is the effect of different pretreatments on the standard deviation of ugt5a4 expression (ugt5a4 / gadph).

[0045] Fig.13 is the effect of different pretreatments on the expression of ugt1a1 (ugt1a1 / gadph);

[0046] Fig.14 is the effect of different pretreatments on the standard deviation of ugt1a1 expression (ugt1a1 / gadph);

[0047] Fig.15 is the effect of different pretreatments on the expression of hsp90aa 1.2 (hsp90aa 1.2 / gadph);

[0048] Fig.16 is the effect of different pretreatments on the standard deviation of hsp90aa 1.2 expression (hsp90aa 1.2 / gadph);

[0049] Fig.17 It is the melting curve of cyp1a1 gene primer amplification during heat treatment;

[0050] Fig.18 It is the melting curve of the primer amplification of gadph gene during heat treatment;

[0051] Fig.19 It is the melting curve of primer amplification of β-actin gene during heat treatment;

[0052] Fig. 20 is the melting curve of the primer amplification of hsp 70.3 gene during heat treatment;

[0053] Fig.21 It is the melting curve of primer amplification of ahrra gene during heat treatment;

[0054] Fig. 22 It is the melting curve of primer amplification of ugt5a4 gene during heat treatment;

[0055] Fig.23 It is the melting curve of primer amplification of ugt1a1 gene during heat treatment;

[0056] Fig.24 This is the melting curve of the hsp90aa 1.2 gene primer amplification during heat treatment. DETAILED DESCRIPTION

[0057] 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.

[0058] 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.

[0059] In one aspect, the present invention provides a method for pretreating gene primers of a PCR chip, the method comprising the following steps:

[0060] (1) placing the upstream and downstream primers of the target gene and the upstream and downstream primers of the internal reference gene in different wells of a PCR plate, sealing the wells and piercing the wells, and then heat treating them in a metal bath;

[0061] (2) After the heat treatment is completed, seal the hole again and keep it for future use.

[0062] In the method of the present invention, the type of the target gene is not limited and there are multiple options. Preferably, as long as the primers of the target gene can be used to detect and evaluate the biological toxicity of oilfield pollutants to zebrafish.

[0063] In a preferred embodiment, in step (1), the target gene can be selected from one or more of cyp1a1, hsp 70.3, ahrra, ugt5a4, ugt1a1 and hsp90aa 1.2. Specifically, when multiple target genes are used in the same experiment, the upstream and downstream primers of different target genes are placed in different wells of the PCR plate, that is, only one upstream and downstream primer of the target gene is placed in one well of the PCR plate. The gene and primer sequences are shown in Table 1.

[0064] In a preferred embodiment, in step (1), the internal reference gene is β-actin and / or gadph. In the present invention, the internal reference gene can be used alone in an experiment or used simultaneously in an experiment. When two internal reference genes are used simultaneously in the same experiment, the upstream and downstream primers of the two internal reference genes are placed in different wells of the PCR plate, that is, only one upstream and downstream primer of the internal reference gene is placed in one well of the PCR plate. The gene and primer sequences are shown in Table 1.

[0065] Table 1

[0066]

[0067] The internal reference genes (β-actin and gadph) of the PCR chip of the present invention are used to monitor the quality control of the real-time fluorescence quantitative PCR reaction of the sample to prevent false negative results caused by the failure of amplification of the reaction reagents.

[0068] Preferably, in step (1), the temperature of the heat treatment can be 50-70°C, and the time can be 8-15 minutes. Under this condition, the heat treatment has the least effect on the primer stability and amplification effect. In a more preferred embodiment, in step (1), the temperature of the heat treatment is 60°C, and the time can be 10 minutes.

[0069] In the method of the present invention, the PCR plate can be a conventional choice in the art. Preferably, in step (1), the PCR plate is a 96-well plate or a 384-well plate.

[0070] In a preferred embodiment, the gene primer pretreatment method of the PCR chip includes: placing a total of 0.8 μL of upstream and downstream primers of the target gene and a total of 0.8 μL of upstream and downstream primers of the internal reference gene in different wells of the PCR plate, sealing the wells with a sealing film, and piercing holes with a needle to facilitate the volatilization of water vapor, and then heat treating at 50-70°C in a metal bath for 8-15 minutes, and then re-sealing the wells with a new sealing film, and storing at 4°C for use.

[0071] The present invention has found through research that, compared with freeze-drying and temporary storage at 4°C, the PCR chip obtained by treating the gene primers of the PCR chip by the heat treatment method of the present invention has the smallest error and the best stability in detecting the toxic effects of oil and gas field additives and produced water in zebrafish.

[0072] In the present invention, the step of storing at 4°C comprises: placing 0.8 μL of upstream and downstream primers of the target gene and 0.8 μL of upstream and downstream primers of the internal reference gene in different wells of the PCR plate, sealing the tube openings with sealing film, and placing at 4°C for standby use.

[0073] In the present invention, the freeze-drying treatment method comprises the following steps: placing a total of 0.8 μL of upstream and downstream primers of the target gene and a total of 0.8 μL of upstream and downstream primers of the internal reference gene in different wells of a PCR plate, precooling at 2 to 5°C for 20 to 45 minutes, and then prefreezing at -10 to -27°C for 1 to 3 hours, followed by freezing at -72 to -85°C for more than 2 hours, and finally vacuum-freezing for at least 4 hours.

[0074] The second aspect of the present invention provides a PCR chip obtained by the method described above.

[0075] A third 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.

[0076] Preferably, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

[0077] A fourth 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 oilfield pollutants on zebrafish.

[0078] In the present invention, the type of the oilfield pollutants is not limited, as long as they are petroleum-related pollutants. In a preferred embodiment, the oilfield pollutants are selected from oilfield additives and / or oilfield produced water.

[0079] In a more preferred embodiment, 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.

[0080] A fifth aspect of the present invention provides a method for evaluating the toxic effects of oilfield pollutants on zebrafish, the method comprising the following steps:

[0081] (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;

[0082] (2) mixing the cDNA and the PCR chip according to claim 5 to perform RT-qPCR reaction to obtain a CT value;

[0083] (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.

[0084] In the present invention, in step (1), the experimental group solution is obtained by diluting the oilfield pollutant sample to be tested with standard dilution water; and the control group solution is standard dilution water.

[0085] 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".

[0086] 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.

[0087] In a specific implementation manner, the storage and treatment method of the oilfield pollutant samples to be tested after collection is determined according to the specific type. For water samples related to oilfield produced water, the samples to be tested are stored at 4°C after collection, transported to the laboratory within 24 hours and frozen at -20°C. Before testing, they need 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In a preferred embodiment, 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. More 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.

[0094] Ecotoxicity chips are suitable for comprehensive toxicity assessment of oilfield chemicals and oilfield produced water. As an emerging molecular toxicity detection method, there are still many problems in its preparation, storage and transportation. Starting from the gene primer pretreatment of zebrafish toxicity chips, the present invention can simply and effectively solve the above problems, effectively promote PCR ecotoxicity chip technology, and thus provide direction for the subsequent establishment of a specific indicator system for the supervision and management of different oilfield chemicals and petrochemical industry wastewater (oilfield produced water); and can use toxicity information to perform toxicity inter-species extrapolation and achieve early warning.

[0095] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0096] In the present invention, the zebrafish embryo toxicity experiment was carried out 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 Chemical short-term toxicity test for fish embryos and yolk sac larvae".

[0097] In the present invention, the base sequences in the target gene primers and the internal reference gene primers were independently developed and designed and synthesized by Shanghai Shenggong Bioengineering Co., Ltd.; the qPCR reaction premix containing the dye was a commercial kit purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0098] Example 1

[0099] This example is used to illustrate the process of pre-treating the gene primers of the PCR chip by a heat treatment method, which specifically includes the following steps:

[0100] 0.4 μL of each of the upstream and downstream primers of the target gene and 0.4 μL of each of the upstream and downstream primers of the internal reference gene were placed in different wells of a 96-well plate, the wells were sealed with sealing film, and holes were pierced with a needle to facilitate the evaporation of water vapor. The wells were then heat treated at 60°C in a metal bath for 10 min, and the wells were re-sealed with a new sealing film. The resulting PCR chip was stored at 4°C for later use.

[0101] Comparative Example 1

[0102] This comparative example is used to illustrate the process of pre-treating the gene primers of the PCR chip by using the method of low temperature storage at 4°C, which specifically includes the following steps:

[0103] Place 0.4 μL of each of the upstream and downstream primers of the target gene and 0.4 μL of each of the upstream and downstream primers of the internal reference gene in different wells of a 96-well plate, seal the tube openings with sealing film, and store the resulting PCR chip at 4°C for later use.

[0104] Comparative Example 2

[0105] This comparative example is used to illustrate the process of pre-treating the gene primers of the PCR chip by freeze-drying method, which specifically includes the following steps:

[0106] Place 0.4 μL of each of the upstream and downstream primers of the target gene and 0.4 μL of each of the upstream and downstream primers of the internal reference gene in different wells of a 96-well plate, precool at 3°C ​​for 30 min, then prefreeze at -18°C for 2 h, then freeze at -78°C for 3 h, and finally freeze-dry under vacuum for 5 h. Store the resulting PCR chip at 4°C for later use.

[0107] Application Example 1

[0108] This application example is used to illustrate the process of using the PCR chip obtained in Example 1 to detect the toxic effects of oilfield produced water (containing Fe4.283mg / L, Ba2.224mg / L, B5.569mg / L, Li2.362mg / L, toluene423.67mg / L, phenol581.76mg / L and 2-methylphenol319.92mg / L) on zebrafish. Among them, the target gene is cyp1a1 and the internal reference gene is β-actin.

[0109] 1) Incubation process

[0110] Collect produced water from the oil field (sample 1 to be tested) and store it at 4°C, transport it to the laboratory and freeze it at -20°C within 24 hours, balance it at 26°C and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it before use;

[0111] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0112] Control solution: standard dilution water;

[0113] The incubation process includes:

[0114] 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.

[0115] 2) Extraction of total RNA

[0116] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0117] 3) Reverse transcription

[0118] 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 gDNA Remover 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.

[0119] 4) RT-qPCR reaction

[0120] The RT-qPCR experiment was performed using the PCR chip obtained by the treatment in Example 1, and repeated 3 times. Different wells of the 96-well plate contained a pair of cyp1a1 target gene primers and a pair of β-actin internal reference gene primers, and the primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of cyp1a1 target gene primers and a pair of β-actin internal reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0121] Record the CT value obtained in the reaction, and calculate the relative expression multiple of the cyp1a1 target gene relative to the β-actin reference gene based on the CT value. The results are as follows Figure 1 The relative fold expression of genes was then used for analysis.

[0122] The standard deviation of cyp1a1 expression was calculated. Figure 2 shown.

[0123] Application Comparative Example 1

[0124] The method of Application Example 1 is used, except that the comparative example uses a PCR chip obtained by the method described in Comparative Example 1 to detect the toxic effect of oilfield produced water on zebrafish.

[0125] Record the CT value obtained in the reaction, and calculate the relative expression multiple of the cyp1a1 target gene relative to the β-actin reference gene based on the CT value. The results are as follows Figure 1 The relative fold expression of genes was then used for analysis.

[0126] The standard deviation of cyp1a1 expression was calculated. Figure 2 shown.

[0127] Application Comparative Example 2

[0128] The method of Application Example 1 is used, except that the present application comparative example uses the PCR chip obtained by the method described in Comparative Example 2 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0129] Record the CT value obtained in the reaction, and calculate the relative expression multiple of the cyp1a1 target gene relative to the β-actin reference gene based on the CT value. The results are as follows Figure 1 The relative fold expression of genes was then used for analysis.

[0130] The standard deviation of cyp1a1 expression was calculated. Figure 2 shown.

[0131] The following conclusions can be drawn from Application Example 1, Application Comparative Example 1 and Application Comparative Example 2:

[0132] from Figure 1 It can be seen that the expression level of cyp1a1 in Application Comparative Example 1 is 139.2, the expression level of cyp1a1 in Application Example 1 is 138.2, and the expression level of Application Comparative Example 2 is as high as 179.5. Compared with Application Comparative Example 2, the gene expression levels of Application Example 1 and Application Comparative Example 1 are closer, and the change in gene expression level in Application Example 1 is less affected than that in Application Comparative Example 1;

[0133] At the same time from Figure 2 It can be seen that the SD value (standard deviation) within the group of Application Example 1 is much smaller than the SD value of 13.19 within the group of Application Comparative Example 1, so the SD value within the group of Application Example 1 is the smallest;

[0134] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable. The heat treatment method only uses a metal bath for short-term heat treatment, which is simple to operate and suitable for large-scale preparation.

[0135] Application Example 2

[0136] This application example is used to illustrate the process of using the PCR chip obtained in Example 1 to detect the toxic effect of oilfield produced water (the composition of the oilfield produced water in Application Example 1) on zebrafish. Among them, the target gene is cyp1a1 and the internal reference gene is gadph.

[0137] 1) Incubation process

[0138] Collect produced water from the oil field (sample 1 to be tested) and store it at 4°C, transport it to the laboratory and freeze it at -20°C within 24 hours, balance it at 26°C and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it before use;

[0139] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0140] Control solution: standard dilution water;

[0141] The incubation process includes:

[0142] 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.

[0143] 2) Extraction of total RNA

[0144] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0145] 3) Reverse transcription

[0146] 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 gDNA Remover 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.

[0147] 4) RT-qPCR reaction

[0148] The PCR chip obtained by the treatment in Example 1 was used to perform RT-qPCR experiments, which were repeated 3 times. Different wells of the 96-well plate contained a pair of cyp1a1 target gene primers and a pair of gadph internal reference gene primers, respectively. The primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of cyp1a1 target gene primers and a pair of gadph internal reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0149] Record the CT value obtained in the reaction, and calculate the relative expression multiple of the cyp1a1 target gene relative to the gadph reference gene based on the CT value. The results are as follows Figure 3 The relative fold expression of genes was then used for analysis.

[0150] The standard deviation of cyp1a1 expression was calculated. Figure 4 shown.

[0151] Application Comparative Example 3

[0152] The method of Application Example 2 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 1 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0153] Record the CT value obtained in the reaction, and calculate the relative expression multiple of the cyp1a1 target gene relative to the gadph reference gene based on the CT value. The results are as follows Figure 3 The relative fold expression of genes was then used for analysis.

[0154] The standard deviation of cyp1a1 expression was calculated. Figure 4 shown.

[0155] Application Comparative Example 4

[0156] The method of Application Example 2 is used, except that the comparative example uses the PCR chip obtained by the method described in Comparative Example 2 to detect the process of toxic effects of oilfield produced water on zebrafish.

[0157] Record the CT value obtained in the reaction, and calculate the relative expression multiple of the cyp1a1 target gene relative to the gadph reference gene based on the CT value. The results are as follows Figure 3 The relative fold expression of genes was then used for analysis.

[0158] The standard deviation of cyp1a1 expression was calculated. Figure 4 shown.

[0159] The following conclusions can be drawn from Application Example 2, Application Comparative Example 3 and Application Comparative Example 4:

[0160] from Figure 3 It can be seen that the cyp1a1 expression levels of Application Comparative Example 3 and Application Example 2 are both 114, while the expression level of Application Comparative Example 4 is as high as 147.86. Compared with Application Comparative Example 4, the gene expression levels of Application Example 2 and Application Comparative Example 3 are closer, and the change in gene expression level of Application Example 2 is less affected than that of Application Comparative Example 3.

[0161] At the same time from Figure 4 It can be seen that the SD value (standard deviation) within the group of Application Example 2 is much smaller than the SD value of 15.71 within the group of Application Comparative Example 3, so the SD value within the group of Application Example 2 is the smallest;

[0162] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable. The heat treatment method only uses a metal bath for short-term heat treatment, which is simple to operate and suitable for large-scale preparation.

[0163] Example 2

[0164] This example is used to illustrate the process of pretreating the primers of two internal reference genes (β-actin and gadph) of the PCR chip by a heat treatment method, which specifically includes the following steps:

[0165] 0.4 μL of the upstream and downstream primers of the β-actin internal reference gene and 0.4 μL of the upstream and downstream primers of the gadph internal reference gene were placed in different wells of a 96-well plate, the wells were sealed with sealing film, and holes were pierced with a needle to facilitate the evaporation of water vapor. The wells were then heat treated at 60°C in a metal bath for 10 min, and then the wells were re-sealed with a new sealing film. The resulting PCR chip was stored at 4°C for later use.

[0166] Comparative Example 3

[0167] This comparative example is used to illustrate the process of pre-treating the gene primers of the PCR chip by using the method of low temperature storage at 4°C, which specifically includes the following steps:

[0168] 0.4 μL of the upstream and downstream primers of the β-actin internal reference gene and 0.4 μL of the upstream and downstream primers of the gadph internal reference gene were placed in different wells of a 96-well plate, the tube openings were sealed with sealing film, and the obtained PCR chip was stored at 4°C for future use.

[0169] Comparative Example 4

[0170] This comparative example is used to illustrate the process of pre-treating the gene primers of the PCR chip by freeze-drying method, which specifically includes the following steps:

[0171] 0.4 μL of the upstream and downstream primers of the β-actin internal reference gene and 0.4 μL of the upstream and downstream primers of the gadph internal reference gene were placed in different wells of a 96-well plate, pre-cooled at 3°C ​​for 30 min, then pre-frozen at -18°C for 2 h, then frozen at -78°C for 3 h, and finally vacuum-freezed for 5 h. The obtained PCR chip was stored at 4°C for later use.

[0172] Application Example 3

[0173] This application example is used to illustrate the process of using the PCR chip obtained in Example 2 to detect the toxic effects of oilfield produced water (with the same composition as the oilfield produced water in Application Example 1) on zebrafish.

[0174] 1) Incubation process

[0175] Collect produced water from the oil field (sample 1 to be tested) and store it at 4°C, transport it to the laboratory and freeze it at -20°C within 24 hours, balance it at 26°C and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it before use;

[0176] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0177] Control solution: standard dilution water;

[0178] The incubation process includes:

[0179] 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.

[0180] 2) Extraction of total RNA

[0181] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0182] 3) Reverse transcription

[0183] 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 gDNA Remover 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.

[0184] 4) RT-qPCR reaction

[0185] The RT-qPCR experiment was performed using the PCR chip obtained by the treatment in Example 1, and repeated 3 times. Different wells of the 96-well plate contained a pair of β-actin reference gene primers and a pair of gadph reference gene primers, respectively. The primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of β-actin reference gene primers and a pair of gadph reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0186] Record the CT value obtained by the reaction, and calculate the relative expression multiple of gadph relative to β-actin based on the CT value. The results are as follows Figure 5 The relative fold expression of genes was then used for analysis.

[0187] Calculate the standard deviation of gadph expression, the result is as follows Figure 6 shown.

[0188] Application Comparative Example 5

[0189] The method of Application Example 3 is used, except that the comparative example uses the PCR chip obtained by the method described in Comparative Example 3 to detect the process of toxic effects of oilfield produced water on zebrafish.

[0190] Record the CT value obtained by the reaction, and calculate the relative expression multiple of gadph relative to β-actin based on the CT value. The results are as follows Figure 5 The relative fold expression of genes was then used for analysis.

[0191] Calculate the standard deviation of gadph expression, the result is as follows Figure 6 shown.

[0192] Application Comparative Example 6

[0193] The method of Application Example 3 is used, except that the present application comparative example uses the PCR chip obtained by the method described in Comparative Example 4 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0194] Record the CT value obtained by the reaction, and calculate the relative expression multiple of gadph relative to β-actin based on the CT value. The results are as follows Figure 5 The relative fold expression of genes was then used for analysis.

[0195] Calculate the standard deviation of gadph expression, the result is as follows Figure 6 shown.

[0196] The following conclusions can be drawn from Application Example 3, Application Comparative Example 5 and Application Comparative Example 6:

[0197] from Figure 5 It can be seen that the gadph expression levels of Application Comparative Example 5 and Application Example 3 are 1.33 and 1.37 respectively, while the expression level of Application Comparative Example 6 is 1.24. Compared with Application Comparative Example 6, the gene expression levels of Application Example 3 and Application Comparative Example 5 are closer, and the change in gene expression level of Application Example 3 is less affected than that of Application Comparative Example 5.

[0198] At the same time from Figure 6 It can be seen that the SD value (standard deviation) within the group of Application Example 3 is smaller than the SD value of 13.87 within the group of Application Comparative Example 5, so the SD value within the group of Application Example 2 is the smallest;

[0199] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable. In this application example, the independently designed internal reference gene primers have high specificity and stable expression, and are excellent internal reference gene primers.

[0200] Application Example 4

[0201] This application example is used to illustrate the process of using the PCR chip obtained in Example 1 to detect the toxic effect of oilfield produced water (the composition of the oilfield produced water in Application Example 1) on zebrafish. Among them, the target gene is hsp70.3 and the internal reference gene is gadph.

[0202] 1) Incubation process

[0203] Collect produced water from the oil field (sample 1 to be tested) and store it at 4°C, transport it to the laboratory and freeze it at -20°C within 24 hours, balance it at 26°C and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it before use;

[0204] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0205] Control solution: standard dilution water;

[0206] The incubation process includes:

[0207] 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.

[0208] 2) Extraction of total RNA

[0209] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0210] 3) Reverse transcription

[0211] 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 gDNA Remover 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.

[0212] 4) RT-qPCR reaction

[0213] The RT-qPCR experiment was performed using the PCR chip obtained by the treatment in Example 1, and repeated 3 times. Different wells of the 96-well plate contained a pair of hsp70.3 target gene primers and a pair of gadph internal reference gene primers, and the primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of hsp70.3 target gene primers and a pair of gadph internal reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0214] Record the CT value obtained in the reaction, and calculate the relative expression multiple of hsp70.3 relative to gadph based on the CT value. The results are as follows: Figure 7 The relative fold expression of genes was then used for analysis.

[0215] Calculate the standard deviation of hsp70.3 expression, the result is as follows Figure 8 shown.

[0216] Application Comparative Example 7

[0217] The method of Application Example 4 is used, except that the present application comparative example uses the PCR chip obtained by the method described in Comparative Example 1 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0218] Record the CT value obtained in the reaction, and calculate the relative expression multiple of hsp70.3 relative to gadph based on the CT value. The results are as follows: Figure 7 The relative fold expression of genes was then used for analysis.

[0219] Calculate the standard deviation of hsp70.3 expression, the result is as follows Figure 8 shown.

[0220] Application Comparative Example 8

[0221] The method of Application Example 4 is used, except that the present Application Example 2 uses the PCR chip obtained by the method described in Comparative Example 2 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0222] Record the CT value obtained in the reaction, and calculate the relative expression multiple of hsp70.3 relative to gadph based on the CT value. The results are as follows: Figure 7 The relative fold expression of genes was then used for analysis.

[0223] Calculate the standard deviation of hsp70.3 expression, the result is as follows Figure 8 shown.

[0224] The following conclusions can be drawn from Application Example 4, Application Comparative Example 7 and Application Comparative Example 8:

[0225] from Figure 7 It can be seen that the hsp70.3 expression levels of Application Comparative Example 7 and Application Example 4 are 121.71 and 120.28 respectively, while the expression level of Application Comparative Example 8 is as high as 160.58. Compared with Application Comparative Example 8, the gene expression levels of Application Example 4 and Application Comparative Example 7 are closer, and the change in gene expression level of Application Example 4 is less affected than that of Application Comparative Example 7;

[0226] At the same time from Figure 8 It can be seen that the SD value (standard deviation) within the group of Application Example 4 is smaller than the SD value of 11.37 within the group of Application Comparative Example 7, so the SD value within the group of Application Example 4 is the smallest;

[0227] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable.

[0228] Application Example 5

[0229] This application example is used to illustrate the process of using the PCR chip obtained in Example 1 to detect the toxic effect of oilfield produced water (the composition of the oilfield produced water in Application Example 1) on zebrafish. Among them, the target gene is ahrra and the internal reference gene is gadph.

[0230] 1) Incubation process

[0231] Collect produced water from the oil field (sample 1 to be tested) and store it at 4°C, transport it to the laboratory and freeze it at -20°C within 24 hours, balance it at 26°C and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it before use;

[0232] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0233] Control solution: standard dilution water;

[0234] The incubation process includes:

[0235] 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.

[0236] 2) Extraction of total RNA

[0237] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0238] 3) Reverse transcription

[0239] 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 gDNA Remover 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.

[0240] 4) RT-qPCR reaction

[0241] The RT-qPCR experiment was performed using the PCR chip obtained by the treatment in Example 1, and repeated 3 times. Different wells of the 96-well plate contained a pair of ahrra target gene primers and a pair of gadph internal reference gene primers, respectively. The primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of ahrra target gene primers and a pair of gadph internal reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0242] Record the CT value obtained by the reaction, and calculate the relative expression multiple of ahrra relative to gadph based on the CT value. The result is as follows Fig. 9 The relative fold expression of genes was then used for analysis.

[0243] Calculate the standard deviation of ahrra expression, the result is as follows Fig.10 shown.

[0244] Application Comparative Example 9

[0245] The method of Application Example 5 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 1 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0246] Record the CT value obtained by the reaction, and calculate the relative expression multiple of ahrra relative to gadph based on the CT value. The result is as follows Fig. 9 The relative fold expression of genes was then used for analysis.

[0247] Calculate the standard deviation of ahrra expression, the result is as follows Fig.10 shown.

[0248] Application Comparative Example 10

[0249] The method of Application Example 5 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 2 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0250] Record the CT value obtained by the reaction, and calculate the relative expression multiple of ahrra relative to gadph based on the CT value. The result is as follows Fig. 9 The relative fold expression of genes was then used for analysis.

[0251] Calculate the standard deviation of ahrra expression, the result is as follows Fig.10 shown.

[0252] The following conclusions can be drawn from Application Example 5, Application Comparative Example 9 and Application Comparative Example 10:

[0253] from Fig. 9 It can be seen that the ahrra expression levels of application comparative example 9 and application example 5 are 103.19 and 102.71 respectively, while the expression level of application comparative example 10 is as high as 131.58. Compared with application comparative example 10, the gene expression levels of application example 5 and application comparative example 9 are closer, and the change of gene expression level in application example 5 is less affected than that in application comparative example 9.

[0254] At the same time from Fig.10 It can be seen that the SD value (standard deviation) within the group of Application Example 5 is smaller than the SD value of 12.46 within the group of Application Comparative Example 9, so the SD value within the group of Application Example 5 is the smallest;

[0255] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable.

[0256] Application Example 6

[0257] This application example is used to illustrate the process of using the PCR chip obtained in Example 1 to detect the toxic effect of oilfield produced water (the composition of the oilfield produced water in Application Example 1) on zebrafish. Among them, the target gene is ugt5a4 and the internal reference gene is gadph.

[0258] 1) Incubation process

[0259] Collect produced water from the oil field (sample 1 to be tested) and store it at 4°C, transport it to the laboratory and freeze it at -20°C within 24 hours, balance it at 26°C and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it before use;

[0260] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0261] Control solution: standard dilution water;

[0262] The incubation process includes:

[0263] 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.

[0264] 2) Extraction of total RNA

[0265] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0266] 3) Reverse transcription

[0267] 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 gDNA Remover 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.

[0268] 4) RT-qPCR reaction

[0269] The PCR chip obtained by the treatment in Example 1 was used to perform RT-qPCR experiments, which were repeated 3 times. Different wells of the 96-well plate contained a pair of ugt5a4 target gene primers and a pair of gadph internal reference gene primers, respectively. The primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of ugt5a4 target gene primers and a pair of gadph internal reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0270] Record the CT value obtained in the reaction, and calculate the relative expression multiple of UGT5A4 relative to GADPH based on the CT value. The results are shown in Fig.11 The relative fold expression of genes was then used for analysis.

[0271] The standard deviation of ugt5a4 expression was calculated. Fig.12 shown.

[0272] Application Comparative Example 11

[0273] The method of Application Example 6 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 1 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0274] Record the CT value obtained in the reaction, and calculate the relative expression multiple of UGT5A4 relative to GADPH based on the CT value. The results are shown in Fig.11 The relative fold expression of genes was then used for analysis.

[0275] The standard deviation of ugt5a4 expression was calculated. Fig.12 shown.

[0276] Application Comparative Example 12

[0277] The method of Application Example 6 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 2 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0278] Record the CT value obtained in the reaction, and calculate the relative expression multiple of UGT5A4 relative to GADPH based on the CT value. The results are shown in Fig.11 The relative fold expression of genes was then used for analysis.

[0279] The standard deviation of ugt5a4 expression was calculated. Fig.12 shown.

[0280] The following conclusions can be drawn from Application Example 6, Application Comparative Example 11 and Application Comparative Example 12:

[0281] from Fig.11 It can be seen that the expression levels of ugt5a4 in Application Comparative Example 11 and Application Example 6 are 85.21 and 87.67 respectively, while the expression level of Application Comparative Example 12 is as high as 120.35. Compared with Application Comparative Example 12, the gene expression levels of Application Example 6 and Application Comparative Example 11 are closer, and the change in gene expression level in Application Example 6 is less affected than that in Application Comparative Example 11;

[0282] At the same time from Fig.12 It can be seen that the SD value (standard deviation) within the group of Application Example 6 is smaller than the SD value of 10.14 within the group of Application Comparative Example 11, so the SD value within the group of Application Example 6 is the smallest;

[0283] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable.

[0284] Application Example 7

[0285] This application example is used to illustrate the process of using the PCR chip obtained in Example 1 to detect the toxic effect of oilfield produced water (the composition of the oilfield produced water in Application Example 1) on zebrafish. Among them, the target gene is ugt1a1 and the internal reference gene is gadph.

[0286] 1) Incubation process

[0287] Collect produced water from the oil field (sample 1 to be tested) and store it at 4°C, transport it to the laboratory and freeze it at -20°C within 24 hours, balance it at 26°C and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it before use;

[0288] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0289] Control solution: standard dilution water;

[0290] The incubation process includes:

[0291] 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.

[0292] 2) Extraction of total RNA

[0293] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0294] 3) Reverse transcription

[0295] 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 gDNA Remover 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.

[0296] 4) RT-qPCR reaction

[0297] The PCR chip obtained by the treatment in Example 1 was used to perform RT-qPCR experiments, which were repeated 3 times. Different wells of the 96-well plate contained a pair of ugt1a1 target gene primers and a pair of gadph internal reference gene primers, respectively. The primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of ugt1a1 target gene primers and a pair of gadph internal reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components are Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0298] The CT value obtained by the reaction was recorded, and the relative expression multiple of ugt1a1 relative to gadph was calculated based on the CT value. The results are shown in Fig.13 The relative fold expression of genes was then used for analysis.

[0299] The standard deviation of ugt1a1 expression was calculated. Fig.14 shown.

[0300] Application Comparative Example 13

[0301] The method of Application Example 7 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 1 to detect the process of toxic effects of oilfield produced water on zebrafish.

[0302] The CT value obtained by the reaction was recorded, and the relative expression multiple of ugt1a1 relative to gadph was calculated based on the CT value. The results are shown in Fig.13 The relative fold expression of genes was then used for analysis.

[0303] The standard deviation of ugt1a1 expression was calculated. Fig.14 shown.

[0304] Application Comparative Example 14

[0305] The method of Application Example 7 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 2 to detect the process of toxic effects of oilfield produced water on zebrafish.

[0306] The CT value obtained by the reaction was recorded, and the relative expression multiple of ugt1a1 relative to gadph was calculated based on the CT value. The results are shown in Fig.13 The relative fold expression of genes was then used for analysis.

[0307] The standard deviation of ugt1a1 expression was calculated. Fig.14 shown.

[0308] The following conclusions can be drawn from Application Example 7, Application Comparative Example 13 and Application Comparative Example 14:

[0309] from Fig.13 It can be seen that the expression levels of ugt1a1 in Application Comparative Example 13 and Application Example 7 are 96.52 and 95.76 respectively, while the expression level of Application Comparative Example 14 is as high as 128.53. Compared with Application Comparative Example 14, the gene expression levels of Application Example 7 and Application Comparative Example 13 are closer, and the change in gene expression level in Application Example 7 is less affected than that in Application Comparative Example 13.

[0310] At the same time from Fig.14 It can be seen that the SD value (standard deviation) within the group of Application Example 7 is smaller than the SD value of 14.17 within the group of Application Comparative Example 14, so the SD value within the group of Application Example 7 is the smallest;

[0311] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable.

[0312] Application Example 8

[0313] This application example is used to illustrate the process of using the PCR chip obtained in Example 1 to detect the toxic effect of oilfield produced water (the composition of the oilfield produced water in Application Example 1) on zebrafish. Among them, the target gene is hsp90aa1.2, and the internal reference gene is gadph.

[0314] 1) Incubation process

[0315] Collect the produced water from the oil field (sample to be tested) and store it at 4℃, transport it to the laboratory and freeze it at -20℃ within 24 hours, balance it at 26℃ and aerate it for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4mg / L, and dilute it before use;

[0316] Experimental group solution: dilute 10 mL of the sample to be tested with 90 mL of standard dilution water;

[0317] Control solution: standard dilution water;

[0318] The incubation process includes:

[0319] 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.

[0320] 2) Extraction of total RNA

[0321] The total RNA of zebrafish embryos and larvae 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, and RNase-free pipette tips and EP tubes were used to ensure that the extracted RNA was not contaminated or degraded. The exposure solution in the 1.5mL RNase-free EP tube was aspirated, the embryos were rinsed twice with pre-cooled distilled water, and the embryos were crushed with a 1mL RNase-free pipette tip. Then 500μL 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, 500μL 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.

[0322] 3) Reverse transcription

[0323] 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 gDNA Remover 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.

[0324] 4) RT-qPCR reaction

[0325] The PCR chip obtained by the treatment in Example 1 was used to perform RT-qPCR experiments, which were repeated 3 times. Different wells of the 96-well plate contained a pair of hsp90aa1.2 target gene primers and a pair of gadph internal reference gene primers, respectively. The primer sequences and annealing temperatures are shown in Table 1. Reverse transcribed cDNA, a qPCR reaction premix containing a dye (components include Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water were added to the wells containing a pair of hsp90aa1.2 target gene primers and a pair of gadph internal reference gene primers, respectively. The total reaction system was 20 μL, and the total reaction system contained 200 ng of reverse transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of a qPCR reaction premix containing a dye (components include Taq hot start enzyme, SYBR Green I fluorescent dye and oxyribonucleotide triphosphate) and RNase-free water. The reaction conditions were: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and then annealing and extension at 60°C for 15 s.

[0326] The CT value obtained by the reaction was recorded, and the relative expression multiple of hsp90aa1.2 relative to gadph was calculated based on the CT value. The results are shown in Fig.15 The relative fold expression of genes was then used for analysis.

[0327] The standard deviation of hsp90aa1.2 expression was calculated. Fig.16 shown.

[0328] Application Comparative Example 15

[0329] The method of Application Example 8 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 1 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0330] The CT value obtained by the reaction was recorded, and the relative expression multiple of hsp90aa1.2 relative to gadph was calculated based on the CT value. The results are shown in Fig.15 The relative fold expression of genes was then used for analysis.

[0331] The standard deviation of hsp90aa1.2 expression was calculated. Fig.16 shown.

[0332] Application Comparative Example 16

[0333] The method of Application Example 8 is used, except that this application comparative example uses the PCR chip obtained by the method described in Comparative Example 2 to detect the process of the toxic effect of oilfield produced water on zebrafish.

[0334] The CT value obtained by the reaction was recorded, and the relative expression multiple of hsp90aa1.2 relative to gadph was calculated based on the CT value. The results are shown in Fig.15 The relative fold expression of genes was then used for analysis.

[0335] The standard deviation of hsp90aa1.2 expression was calculated. Fig.16 shown.

[0336] The following conclusions can be drawn from Application Example 8, Application Comparative Example 15 and Application Comparative Example 16:

[0337] from Fig.15 It can be seen that the expression levels of hsp90aa1.2 in Application Comparative Example 15 and Application Example 8 are 64.82 and 62.27 respectively, while the expression level of Application Comparative Example 16 is as high as 83.45. Compared with Application Comparative Example 16, the gene expression levels of Application Example 8 and Application Comparative Example 15 are closer, and the change in gene expression level in Application Example 8 is less affected than that in Application Comparative Example 15.

[0338] At the same time from Fig.16 It can be seen that the SD value (standard deviation) within the group of Application Example 8 is smaller than the SD value of 11.76 within the group of Application Comparative Example 16, so the SD value within the group of Application Example 8 is the smallest;

[0339] It can be seen that the heat treatment method has the least effect on the stability of the primers, and the heat treatment method has the best effect on pre-treating the gene primers, and the detection results are more stable.

[0340] Figure 17-24 These are the melting curves of cyp1a1, gadph, β-actin, hsp 70.3, ahrra, ugt5a4, ugt1a1 and hsp90aa 1.2 gene primers amplified when heat treatment was used for pretreatment. Figure 17-24 It can be seen that the melting curves produced by the primer amplification of the 8 genes are all sharp single peaks, indicating that during heat treatment, the primer amplification of the 8 genes has only one product, with excellent performance, meeting the experimental requirements.

[0341] 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 method for pretreating gene primers for a PCR chip, characterized in that: The method comprises the following steps: (1) placing the upstream and downstream primers of the target gene and the upstream and downstream primers of the internal reference gene in different wells of a PCR plate, sealing the wells and piercing the wells, and then heat treating them in a metal bath; (2) After the heat treatment is completed, seal the hole again and keep it for future use.

2. The method according to claim 1, characterized in that In step (1), the heat treatment conditions include: temperature of 50 to 70° C. and time of 8 to 15 minutes.

3. The method according to claim 1 or 2, characterized in that: In step (1), the target gene is selected from one or more of cyp1a1, hsp 70.3, ahrra, ugt5a4, ugt1a1 and hsp90aa 1.2; Preferably, in step (1), the internal reference gene is β-actin and / or gadph.

4. The method according to any one of claims 1 to 3, characterized in that: In step (1), the PCR plate is a 96-well plate or a 384-well plate.

5. A PCR chip obtained by the method according to any one of claims 1 to 4.

6. A PCR chip kit, characterized in that: The PCR chip kit comprises the PCR chip according to claim 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 claim 5 or the PCR chip kit according to claim 6 in evaluating the toxic effects of oilfield pollutants on zebrafish.

8. The use according to claim 7, characterized in that: The oilfield pollutants are selected from oilfield additives and / or oilfield produced water.

9. The use according to claim 7 or 8, characterized in that: 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.

10. A method for evaluating the toxic effects of oilfield 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 claim 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 oilfield 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 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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