PCR (Polymerase Chain Reaction) chip and kit for evaluating toxic effect of zebra fish and application of PCR chip and kit

Abstract

The invention provides a PCR chip for evaluating the toxic effect of zebra fish, a kit and application thereof, belongs to the technical field of biotoxicity detection, and can solve the problem that the existing PCR chip cannot intuitively and comprehensively evaluate the comprehensive toxic effect of petrochemical pollutant exposure on zebra fish. The PCR chip comprises 59 pairs of primers designed for target genes related to detoxification metabolism, neurodevelopment and endocrine disruption, each pair of primers respectively comprises an upstream primer and a downstream primer, and the sequences of the 59 pairs of target gene primers are respectively shown as SEQ ID NO.1-SEQ ID NO.118. According to the invention, 59 genes related to detoxication metabolism, neurodevelopment and endocrine disruption of the zebra fish are selected in a targeted manner, and a PCR chip for researching the comprehensive toxicity effect of petrochemical pollutants on the zebra fish is integrated. The method can be applied to evaluation of the toxic effect of the petrochemical pollutants on the zebra fish.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological toxicity detection, and particularly relates to a PCR chip, a kit and an application thereof for evaluating the toxicity effect of zebrafish. Background Art

[0002] With the progress of oil production technology, the types and usage amounts of oilfield chemicals are increasing. The quality certification of the standards of oilfield chemical production enterprises belongs to second-party certification, so the standards are relatively poor. In particular, many production enterprises are keen on hyping up concept products such as new terms and new codes. There are many compounded products and few innovative products. Coupled with the lack of supervision, the quality of oilfield chemical products is chaotic, which brings great risks to the hazard identification and safe use of oilfield chemicals. Since the existing regulations and standards in China do not compulsorily require the true components of oilfield chemical products to be described in the Chemical Safety Data Sheet (MSDS), many oilfield chemical manufacturers only publish the type of their products in the MSDS of their products based on the requirement of formula confidentiality, and do not publish the specific composition and component information of the products. Enterprise managers and operators cannot correctly understand the biological toxicity and health hazards of oilfield chemicals.

[0003] At the same time, as the development of each major oilfield in China has entered the middle and late stages, the water cut of oilfield produced fluids is as high as 70%, and some even exceed 90%. In order to ensure the crude oil production, the exploitation intensity is continuously increased, resulting in the increasing production of produced water year by year. According to statistics, the total amount of oilfield reinjection water treatment in China reached 2.26 billion tons in 2011. According to the data of Liaohe Oilfield, in 2019, about 100,000 tons of various heavy oil produced water was produced daily, of which about 80,000 tons were deeply treated and reused for thermal recovery injection boilers daily. Due to the imbalance between injection and production, there is still an external drainage of 20,000 tons per day, accounting for about 1 / 5.

[0004] In addition to alkanes, alcohols and ketones in oilfield produced water, there are also more than 40 chemical agent components with poor biodegradability. Since the current external discharge standards for sewage treatment in China only have requirements for COD, ammonia nitrogen, suspended solids and oil content, etc., and there is no requirement for ecological toxicity or health detection, the huge amount of oilfield produced water brings great pressure to the external sewage treatment plants, and brings non-negligible hidden dangers to the surrounding environment and personnel health. If the requirements for full wastewater toxicity testing in the United States, Canada, etc. are followed, hundreds of fish will be required for one acute and short-term test, and invertebrate tests need to be carried out weekly, rainbow trout acute toxicity tests need to be carried out monthly, and sub-lethal toxicity tests need to be carried out twice a year. The huge amount of external drainage and periodic detection will require a large number of test animals. The method using gene chips can reduce the usage amount of animals by 10%, can greatly reduce the detection cost and improve the detection efficiency. At present, most of the toxicity studies on oilfield produced water, fracturing flowback fluid and oilfield additives focus on the multi-trophic ecological toxicity, and the research on its toxicity mechanism is less.

[0005] PCR chip technology can study the expression of genes related to signal pathways or biological functions at the whole genome scale and has been widely developed and applied. PCR array chip technology can measure the differential expression of multiple genes on a set of chips and is a high-throughput toxicity testing technology. The PCR chip combines the detection characteristics of SYBR Green real-time fluorescence quantitative PCR, with advantages such as good repeatability, high sensitivity, and strong specificity. Its greatest advantage is the ability to simultaneously quantitatively detect the expression of multiple genes. The PCRArray toxicity chip is a toxicity testing method that can interpret toxicity pathways and mechanisms of action. By loading different types of primers and measuring the response to toxicity endpoints, it can then interpret the toxicity pathways activated by pollutants and reveal the toxic mechanism. However, there is no relevant report in the existing technology on how to intuitively and comprehensively evaluate the comprehensive toxicity effect of petrochemical pollutants on zebrafish using a PCR chip. Summary of the Invention

[0006] In view of the technical problem that the existing PCR chip cannot intuitively and comprehensively evaluate the comprehensive toxicity effect of petrochemical pollutants on zebrafish, the present invention provides a PCR chip, kit, and its application for evaluating the toxicity effect of zebrafish, which can comprehensively evaluate the comprehensive toxicity effect of petrochemical pollutants on zebrafish.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A PCR chip for evaluating the toxicity effect of zebrafish includes 59 pairs of primers designed for target genes related to detoxification metabolism, neurodevelopment, and endocrine disruption. Each pair of primers includes an upstream primer and a downstream primer, and the sequences of the 59 pairs of target gene primers are shown in SEQ ID NO.1 - SEQ ID NO.118 respectively.

[0009] In one embodiment, the target genes related to detoxification metabolism, neurodevelopment, and endocrine disruption include 59 genes including cyp1a1, cyp1b1, cyp1c1, cyp1c2, ahr2, ahrra, ahrrb, ugt1a1, ugt1b5, ugt5a1, ugt5a4, ugt5c2, ugt5c3, sult1st5, sult1st7, sult3st2, inhbaa, rpl8, igf1ra, igf1rb, igf2r, crh, ttr, ghra, igf1a, slc5a5, tg, dio1, dio2, trh, gnrhr2, gnrhr3, gnrhr4, fshr, lhb, lhcgr, vtg1, vtg3, esr1, esr2a, erβ, ar, star, 3β-hsd, 17β-hsd, cyp19a1b, cyp17a1, cyp19a1a, hmgcra, glsa, grin1b, gad1b, gad2, gabrg2, abat, th1, drd2a, drd2b, and slc6a3.

[0010] In one embodiment, in the PCR chip, the use concentration of the 59 pairs of primers shown in SEQ ID NO.1 - SEQ ID NO.118 is 10 μM;

[0011] The product of the target gene cyp1a1 related to detoxification metabolism, neurodevelopment, and endocrine disruption is 224 bp in size.The product size of cyp1b1 is 159bp, the product size of cyp1c1 is 160bp, the product size of cyp1c2 is 338bp, the product size of ahr2 is 454bp, the product size of ahrra is 187bp, the product size of ahrrb is 339bp, the product size of ugt1a1 is 153bp, the product size of ugt1b5 is 358bp, the product size of ugt5a1 is 379bp, the product size of ugt5a4 is 259bp, the product size of ugt5c2 is 153bp, the product size of ugt5c3 is 266bp, the product size of sult1st5 is 246bp, the product size of sult1st7 is 336bp, the product size of sult3st2 is 131bp, the product size of inhbaa is 127bp, the product size of rpl8 is 195bp, the product size of igf1ra is 365bp, the product size of igf1rb is 207bp, the product size of igf2r is 466bp, the product size of crh is 150bp, the product size of ttr is 166bp, the product size of ghra is 299bp, the product size of igf1a is 300bp, the product size of slc5a5 is 443bp, the product size of tg is 436bp, the product size of dio1 is 142bp, the product size of dio2 is 414bp, the product size of trh is 371bp, the product size of gnrhr2 is 308bp, the product size of gnrhr3 is 372bp, the product size of gnrhr4 is 229bp, the product size of fshr is 373bp, the product size of lhb is 108bp, the product size of lhcgr is 398bp, the product size of vtg1 is 101bp, the product size of vtg3 is 286bp, the product size of esr1 is 452bp, the product size of esr2a is 265bp, the product size of erβ is 353bp, the product size of ar is 370bp, the product size of star is 148bp, the product size of 3β-hsd is 150bp, the product size of 17β-hsd is 138bp, the product size of cyp19a1b is 477bp, the product size of cyp17a1 is 212bp, the product size of cyp19a1a is 117bp, the product size of hmgcra is 406bp, the product size of glsa is 181bp, the product size of grin1b is 242bp, the product size of gad1b is 402bp, the product size of gad2 is 137bp, the product size of gabrg2 is 117bp, the product size of abat is 114bp, the product size of th1 is 390bp, the product size of drd2a is 145bp, the product size of drd2b is 130bp and the product size of slc6a3 is 195bp.,

[0012] In one embodiment, the PCR chip further includes two pairs of internal reference gene primers designed for gadph and β-actin. Each pair of the internal reference gene primers includes an upstream primer and a downstream primer, and their sequences are shown in SEQ ID NO.119-SEQ ID NO.122.

[0013] In one embodiment, the PCR chip further includes a carrier, and the carrier is a 384-well plate.

[0014] The present invention also provides a PCR chip kit, including the PCR chip according to any one of the above embodiments, a qPCR reaction premix, and RNase-free water;

[0015] Wherein, the qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye, and deoxyribonucleotide triphosphate.

[0016] The present invention also provides an application of the PCR chip or the PCR chip kit in evaluating the toxic effects of petrochemical pollutants on zebrafish, and the toxic effects include zebrafish detoxification metabolism, neurodevelopment, and endocrine disruption.

[0017] In one embodiment, the petrochemical pollutants are selected from oilfield produced water or oilfield auxiliaries;

[0018] Wherein, the oilfield produced water is selected from oilfield produced water raw water, oilfield produced water three-phase separation effluent, oilfield produced water primary sedimentation effluent, oilfield produced water secondary sedimentation effluent, or oilfield produced water filtration effluent;

[0019] The oilfield auxiliaries are selected from water damage treatment agents, water lock damage treatment agents, gel-like plugging agents, composite chromium cross-linking agents for polymer flooding, high-temperature acidizing corrosion inhibitors, or organic retarded acids.

[0020] The present invention also provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish, including the following steps:

[0021] Set up an experimental group solution and a control group solution;

[0022] Place zebrafish embryos at the cell stage in the experimental group solution and the control group solution for hatching respectively, collect the surviving embryos and larvae, extract the total RNA of the surviving embryos and larvae, and then reverse transcribe the total RNA as a template to obtain cDNA;

[0023] Mix the cDNA and the PCR chip for RT-qPCR reaction to obtain CT values;

[0024] Calculate the relative expression fold of the target gene relative to the reference gene based on the CT value, and then analyze using the relative expression fold of the gene to evaluate the toxic effect of petrochemical pollutants on zebrafish.

[0025] In one embodiment, the experimental group solution is obtained by diluting the sample to be tested with standard dilution water, and the control group solution is standard dilution water;

[0026] The hatching conditions are a temperature of 20 °C and a time of 96 h;

[0027] The reaction conditions for reverse transcription are: incubation at 42 °C for 15 min and heating at 85 °C for 5 s;

[0028] The RT-qPCR reaction is to mix the cDNA, the PCR chip described in any one of claims 1-5, the qPCR reaction premix, and RNase-free water to perform the RT-qPCR reaction to obtain the CT value;

[0029] The qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye, and ribonucleoside triphosphate;

[0030] The RT-qPCR reaction program is: pre-denaturation at 94 °C for 30 s, then denaturation at 94 °C for 5 s, and then annealing and extension at 60 °C.

[0031] Compared with the prior art, the advantages and positive effects of the present invention are:

[0032] 1. The present invention provides a PCR chip, a kit, and its application for evaluating the toxic effect of zebrafish. The PCR chip is a composite ecotoxicity chip for the detoxification metabolism, neurodevelopmental toxicity, and endocrine disruption toxicity of zebrafish embryos related to petrochemical chemicals and pollutants. By co-loading a total of 59 genes in 8 pathways such as aryl hydrocarbon receptor, phase I metabolism, phase II metabolism, hypothalamic-pituitary-gonad-liver axis, hypothalamic-pituitary-thyroid axis, hypothalamic-pituitary-adrenal axis, growth-related, and neurotoxicity-related, and 2 reference genes, a large chip for comprehensive toxicity effect analysis is formed;

[0033] 2. The present invention provides a PCR chip, a kit, and its application for evaluating the toxic effect of zebrafish. The operation method of the PCR chip is simple, and a large amount of toxicity data can be obtained at one time. It can more comprehensively and detailedly study the comprehensive toxicity effect mechanism of petrochemical pollutants on zebrafish embryos, providing data support for a deeper study of the toxic mechanism of heavy metal pollutants. Description of the Drawings

[0034] Figure 1 is the PCRArray design;

[0035] Figure 2 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the produced water from the oilfield.

[0036] Figure 3 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the three-phase separated produced water from the oilfield.

[0037] Figure 4 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the primary sedimentation effluent of the produced water from the oilfield.

[0038] Figure 5 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the secondary sedimentation effluent of the produced water from the oilfield.

[0039] Figure 6 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the filtered effluent of the produced water from the oilfield.

[0040] Figure 7 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the existing aids in Shengli Oilfield (a certain water damage treatment agent / water block damage treatment agent).

[0041] Figure 8 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the existing aids in Shengli Oilfield (gel type / room temperature gel type plugging agent).

[0042] Figure 9 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the existing aids in Shengli Oilfield (crosslinking agent / composite chromium type crosslinking agent for polymer flooding).

[0043] Figure 10 It is a schematic diagram of the change results of the expression levels of genes related to the exposed chip of the existing aids in Shengli Oilfield (for acidizing / high temperature acidizing corrosion inhibitor).

[0044] Figure 11 It is the change of the expression levels of genes related to the exposed chip of the existing aids in Shengli Oilfield (organic retarded acid). Detailed implementation manners

[0045] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] An embodiment of the present invention provides a PCR chip for evaluating the toxic effects of zebrafish, which includes 59 pairs of primers designed for target genes related to detoxification metabolism, neurodevelopment, and endocrine disruption. Each pair of primers includes an upstream primer and a downstream primer, and the sequences of the 59 pairs of target gene primers are shown in SEQ ID NO.1 - SEQ ID NO.118 respectively.

[0047] The 59 pairs of primers described in the above embodiments correspond to the target genes cyp1a1, cyp1b1, cyp1c1, cyp1c2, ahr2, ahrra, ahrrb, ugt1a1, ugt1b5, ugt5a1, ugt5a4, ugt5c2, ugt5c3, sult1st5, sult1st7, sult3st2, inhbaa, rpl8, igf1ra, igf1rb, igf2r, crh, ttr, ghra, igf1a, slc5a5, tg, dio1, dio2, trh, gnrhr2, gnrhr3, gnrhr4, fshr, lhb, lhcgr, vtg1, vtg3, esr1, esr2a, erβ, ar, star, 3β-hsd, 17β-hsd, cyp19a1b, cyp17a1, cyp19a1a, hmgcra, glsa, grin1b, gad1b, gad2, gabrg2, abat, th1, drd2a, drd2b, and slc6a3 related to detoxification metabolism, neurodevelopment, and endocrine disruption; among them, ahr2, ahrra, ahrrb correspond to the aryl hydrocarbon receptor-related genes, cyp1a1, cyp1b1, cyp1c1, cyp1c2 correspond to the phase I metabolism-related genes, ugt1a1, ugt1b5, ugt5a1, ugt5a4, ugt5c2, ugt5c3, sult1st5, sult1st7, sult3st2 correspond to the phase II metabolism-related genes, gnrhr2, gnrhr3, gnrhr4, fshr, lhb, lhcgr, vtg1, vtg3, esr1, esr2a, erβ, ar, star, 3β-hsd, 17β-hsd, cyp19a1a, cyp19a1b, cyp17a1, hmgcra correspond to the hypothalamic-pituitary-gonad-liver axis-related genes, ttr, slc5a5, tg, dio1, dio2, trh correspond to the hypothalamic-pituitary-thyroid axis-related genes, crh corresponds to the hypothalamic-pituitary-adrenal axis-related genes, nhbaa, rpl8, igf1ra, igf1rb, igf2r, ghra, igf1a correspond to the growth-related genes, glsa, grin1b, gad1b, gad2, gabrg2, abat, th1, drd2a, drd2b, slc6a3 correspond to the neurotoxicity-related genes, a total of 8 related pathways, and the genes and primer sequences are shown in Table 1.

[0048] Table 1 - Primer Sequences of Genes Loaded on PCRArray

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] Furthermore, the 59 genes related to the above 8 pathways were screened by the following methods:

[0056] (1) Screening genes related to the induction of zebrafish embryo toxicity effects through the GO database of the network bioinformatics database NCBI; obtaining some genes through literature retrieval of toxicity effect studies on multiple species such as humans, rats, mice, chickens, double-crested cormorants, Daphnia magna, and zebrafish; screening some genes from the multi-species PCRArray commercial chips produced by Qiagen; screening some differentially expressed genes through experimental gene expression profile analysis;

[0057] (2) Selecting a total of 59 genes and 2 internal reference genes related to 8 pathways such as aryl hydrocarbon receptor, phase I metabolism, phase II metabolism, hypothalamic-pituitary-gonad-liver axis, hypothalamic-pituitary-thyroid axis, hypothalamic-pituitary-adrenal axis, growth-related, and neurotoxicity-related from the gene bank and primer bank and loading them together to form a large chip for comprehensive toxicity effect analysis, which can be used for preliminary toxicity analysis.

[0058] Even further, the primer sequence design process of the above 59 genes is as follows:

[0059] Search for the target gene or protein and mRNA in the NCBI gene database, copy the coding region, use the Primer-BLAST module of the NCBI website or Primer Premier 5 software for primer design, and use Oligo software to verify and evaluate the designed primers. It is necessary to ensure that the absolute value of the 3'△G of the primer does not exceed 4.5 kcal / mol to prevent the formation of double-stranded structures at the mismatch sites; the number of binding base pairs does not exceed 3 to prevent the formation of dimers and hairpin structures between primers; the GC content of the primer is generally between 40% and 60%, and the GC content of the upstream and downstream primers does not differ much, and the Tm is generally between 59 and 68 °C. After determining the primers, perform a homologous sequence search through NCBI Blast and use the Ensembl database for cross-exon verification to finally determine the primers of the target gene. The obtained gene primer sequences are shown in Table 1.

[0060] In a specific embodiment, the PCR chip further includes two pairs of internal reference gene primers designed for gadph and β-actin. Each pair of the internal reference gene primers includes an upstream primer and a downstream primer, and their sequences are shown in SEQ ID NO.119 - SEQ ID NO.122.

[0061] In a specific embodiment, the PCR chip further includes a carrier, and the carrier is a 384-well plate.

[0062] The present invention also provides a PCR chip kit, which is characterized by including the PCR chip according to any of the above embodiments, a qPCR reaction premix, and RNase-free water;

[0063] Wherein, the qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye, and deoxyribonucleotide triphosphate.

[0064] The present invention also provides an application of the PCR chip or the PCR chip kit in evaluating the toxic effects of petrochemical pollutants on zebrafish, and the toxic effects include zebrafish detoxification metabolism, neurodevelopment, and endocrine disruption.

[0065] In a specific embodiment, the petrochemical pollutants are selected from oilfield produced water or oilfield additives;

[0066] Wherein, the oilfield produced water is selected from oilfield produced water influent, oilfield produced water three-phase separation effluent, oilfield produced water primary sedimentation effluent, oilfield produced water secondary sedimentation effluent, or oilfield produced water filtration effluent;

[0067] The oilfield additives are selected from water damage treatment agents, water lock damage treatment agents, gel-like plugging agents, composite chromium crosslinking agents for polymer flooding, high-temperature acidizing corrosion inhibitors, or organic retarded acids.

[0068] The present invention further provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish, including the following steps:

[0069] S1. Set up an experimental group solution and a control group solution;

[0070] S2. Place zebrafish embryos at the cell stage in the experimental group solution and the control group solution for hatching respectively, collect the surviving embryos and larvae, extract the total RNA of the surviving embryos and larvae, and then reverse transcribe the total RNA as a template to obtain cDNA;

[0071] S3. Mix the cDNA and the PCR chip for RT-qPCR reaction to obtain CT values;

[0072] S4. Calculate the relative expression fold of the target gene relative to the reference gene based on the CT value, and then analyze using the relative expression fold of the gene to evaluate the toxic effect of petrochemical pollutants on zebrafish.

[0073] In a specific embodiment, the experimental group solution is obtained by diluting the sample to be tested with standard dilution water, and the control group solution is standard dilution water;

[0074] The hatching conditions are a temperature of 20 °C and a time of 96 h;

[0075] The reaction conditions for reverse transcription are: incubation at 42 °C for 15 min and heating at 85 °C for 5 s;

[0076] The RT-qPCR reaction is to mix the cDNA, the PCR chip according to any one of claims 1-5, the qPCR reaction premix, and RNase-free water for the RT-qPCR reaction to obtain the CT value;

[0077] The qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye, and ribonucleoside triphosphate;

[0078] The RT-qPCR reaction program is: pre-denaturation at 94 °C for 30 s, then denaturation at 94 °C for 5 s, and then annealing and extension at 60 °C.

[0079] To introduce more clearly and in detail a PCR chip, kit, and its application for evaluating the toxic effect of petrochemical pollutants on zebrafish provided by the embodiments of the present invention, the following will be described in conjunction with specific embodiments.

[0080] Example 1

[0081] This example provides a method for evaluating the toxic effect of petrochemical pollutants on zebrafish using a PCR chip, specifically as follows:

[0082] (1) Select a collection site in Shengli Oilfield, use a clean sampling bottle to collect the incoming water from a collection site in Shengli Oilfield, store the sample at 4 °C, and transport it to the laboratory for freezing at -20 °C within 24 h;

[0083] (2) Before detection, equilibrate the incoming water sample at 26 °C and aerate it for 30 min to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L, and dilute it immediately before use;

[0084] (3)Collect zebrafish embryos at the 4 - 128 cell stage, and conduct zebrafish embryo toxicity experiments according to "HJ 1069-2019 Determination of Acute Toxicity of Water Quality - Zebrafish Egg Method", "OECD 236 Fish Embryo Acute Toxicity (FET) Test" and "GB / T 21807-2008 Short-term Toxicity Test of Chemicals at the Fish Embryo and Yolk Sac Larval Stages"; place 1 embryo and 2 ml of the test dilution or blank dilution in each well of a 24-well plate. Collect the surviving embryos and incubate them at 20 °C. After 96 h of incubation, collect the surviving embryos and hatched larvae;

[0085] (4)Use the centrifugal column method to extract the total RNA of zebrafish embryos and larvae using the Shanghai Feijie Total RNA Rapid Extraction Kit. Operate the whole process in a low-temperature environment. To ensure that the extracted RNA is not contaminated or degraded, use RNase-free pipette tips and EP tubes. Aspirate all the exposure liquid in the culture well plate, rinse the embryos twice with pre-cooled distilled water, mash the embryos with a tissue grinder, then add 500 μL of RA2 lysis buffer to each tube. After thoroughly pipetting and mixing, transfer the sample to the inner tube and let it stand for 1 min. Then place the inner tube (already inserted into the outer tube) into a high-speed low-temperature centrifuge and centrifuge at 4 °C and 13,000 g for 1 min. Take out the inner tube, aspirate the liquid in the outer tube and then put the inner tube back. Add 500 μL of wash buffer and centrifuge for 1 min. Then repeat this washing step once. Take out the inner tube, aspirate the liquid in the outer tube and then put the inner tube back. Without adding wash buffer, centrifuge for 1 min. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube. Add 25 μL of elution buffer to the center of the inner tube membrane, let it stand for 1 min, and then centrifuge for 1 min to obtain the total RNA. Measure the concentration and quality of the RNA with a full-wavelength microplate reader. When the ratio of OD 260 / OD 280 is between 1.9 - 2.1, it indicates that the extracted RNA has good quality and can be used for subsequent reverse transcription experiments;

[0086] (5)Using the extracted total RNA as a template, perform reverse transcription according to the operating steps of the TransGen Biotech AT341-01 Reverse Transcription Kit. In a 20 μL reaction system, there is 1 μg of total RNA template, 4 μL of 5× All-in-one SuperMix for qPCR, 1 μL of gDNA Remover, and RNase-free water. Reaction conditions: Incubate at 42 °C for 15 min, heat at 85 °C for 5 s. The 20 μL of cDNA obtained by reverse transcription is diluted to 100 μL with RNase-free water, and then can be used for RT-qPCR experiments;

[0087] (6) RT-qPCR experiments were carried out using the QPCRArray plate containing 59 target genes related to detoxification metabolism, neurodevelopment and endocrine disruption independently developed by the present invention. The well positions of the 384-well plate were set in 16 rows, with 24 wells in each row. A 20 μL reaction system was placed in each well. The specific gene arrangement order is as Figure 1 shown. In the 20 μL reaction system, there were 2 μL of reverse transcription cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of qPCR reaction premix and RNase-free water. The reaction conditions were: pre-denaturation at 94 °C for 30 s, then denaturation at 94 °C for 5 s, and then annealing and extension at 60 °C. The CT value was obtained. The relative expression fold of the target gene relative to the internal reference gene was calculated from the CT value. Then, using the relative expression fold of the gene, the changes in the expression levels of the target genes related to detoxification metabolism, neurodevelopment and endocrine disruption were interpreted.

[0088] Result analysis: As Figure 2 shown, the neurotoxicity-related results of zebrafish embryos treated with raw water exposure showed that the expression levels of most genes related to neurodevelopment decreased, indicating that raw water exposure inhibited neurodevelopment. At the same time, raw water exposure also interfered with the expression of genes related to the endocrine hypothalamic-pituitary-gonad-liver axis, hypothalamic-pituitary-thyroid axis and hypothalamic-pituitary-adrenal axis. Except for the local up-regulation of genes such as vtg3, 3β-hsd, dio2 and igf2r, raw water exposure inhibited the expression of most genes related to endocrine disruption. The genes related to the detoxification metabolism pathway were highly expressed, especially the genes related to phase I metabolism and phase II metabolism, indicating that when zebrafish embryos were stimulated by exogenous organic pollutants, the detoxification metabolism pathway could be activated to protect the body.

[0089] Example 2

[0090] This example provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR chip, specifically:

[0091] The operation process was the same as that in Example 1, except for step (1): Select a collection site in Shengli Oilfield, and use a clean sampling bottle to collect the three-phase separated effluent from a collection site in Shengli Oilfield. The sample was stored at 4 °C and transported to the laboratory for freezing storage at -20 °C within 24 h.

[0092] Result analysis: As Figure 3As shown in the figure, the test results of the composite toxicity PCRArray chip show that exposing zebrafish embryos to the effluent after three-phase separation can lead to a significant upregulation of the genes related to the detoxification and metabolism pathway, while downregulating the expression of genes in the neurotoxicity pathway and the endocrine disruption pathway. However, the interference effect on the toxicity pathway is higher than that of the influent. The function of the three-phase separator is to separate oil, gas, and water. If only considering the physical process, the toxicity of the effluent should decrease due to the reduction in the content of oil, gas, etc. However, for the test results of the composite toxicity PCR Array chip, the effluent after three-phase separation shows a higher toxicity than the influent. It is suspected that the high toxicity may be related to the oilfield additives such as demulsifiers added in the three-phase separation stage to improve the separation efficiency.

[0093] Example 3

[0094] This example provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR chip, specifically as follows:

[0095] The operation process is the same as that in Example 1, with the only difference being in step (1): Select a collection site in the Shengli Oilfield and collect the effluent from the first-stage sedimentation at a certain collection site in the Shengli Oilfield using a clean sampling bottle. The sample is stored at 4°C and transported to the laboratory for freezing storage at -20°C within 24 hours.

[0096] Result analysis: As Figure 4 shown, the test results of the composite toxicity PCRArray chip show that the toxicity of the effluent from the first-stage sedimentation is relatively low, and the interference on the expression of genes related to the detoxification and metabolism pathways, neurotoxicity, and endocrine disruption pathways is relatively small, indicating that sedimentation treatment can significantly reduce the biological toxicity of the produced water from the oilfield.

[0097] Example 4

[0098] This example provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR chip, specifically as follows:

[0099] The operation process is the same as that in Example 1, with the only difference being in step (1): Select a collection site in the Shengli Oilfield and collect the effluent from the second-stage sedimentation at a certain collection site in the Shengli Oilfield using a clean sampling bottle. The sample is stored at 4°C and transported to the laboratory for freezing storage at -20°C within 24 hours.

[0100] Result analysis: As Figure 5 shown, the test results of the composite toxicity PCRArray chip show that the toxicity of the effluent from the second-stage sedimentation is relatively low, and the interference on the expression of genes related to the detoxification and metabolism pathways, neurotoxicity, and endocrine disruption pathways is relatively small, indicating that sedimentation treatment can significantly reduce the biological toxicity of the produced water from the oilfield.

[0101] Example 5

[0102] This embodiment provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR chip, specifically as follows:

[0103] The operation process is the same as that in Embodiment 1, except for step (1): Select a collection site in Shengli Oilfield, and use a clean sampling bottle to collect the filtered effluent from a collection site in Shengli Oilfield. The sample is stored at 4°C and transported to the laboratory within 24 hours for freezing storage at -20°C.

[0104] Result analysis: As Figure 6 shown, the test results of the combined toxicity PCRArray chip show that the toxicity of the filtered effluent is relatively high, and it has a greater interference effect on the expression of genes related to the detoxification metabolism pathway, neurotoxicity, and endocrine disruption pathway. In particular, the high expression of genes related to phase I metabolism and phase II metabolism suggests that there may be organic components in the pollutants, and it is speculated that it may be related to additives such as backwashing filter media cleaning agents added during filtration.

[0105] Embodiment 6

[0106] This embodiment provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR chip, specifically as follows:

[0107] (1) Select the active additives in Shengli Oilfield (a certain water damage treatment agent / water lock damage treatment agent) as the test substance. The sample is stored at room temperature and transported to the laboratory within 24 hours for storage in the dark at room temperature;

[0108] (2) Dilute the oilfield additives before detection; Collect zebrafish embryos at the 4-128 cell stage, and conduct zebrafish embryo toxicity experiments according to "HJ 1069-2019 Determination of Acute Toxicity of Water Quality - Zebrafish Egg Method", "OECD 236 Fish Embryo Acute Toxicity (FET) Test" and "GB / T 21807-2008 Short-Term Toxicity Test for Chemicals at the Fish Embryo and Yolk Sac Larval Stages". Each well of the 24-well plate contains 1 embryo and 2 ml of the test dilution or blank dilution. Collect the surviving embryos and incubate them at 20°C. After 96 hours of incubation, collect the surviving embryos and hatched larvae;

[0109] (3) The total RNA of zebrafish embryos and larvae was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit by the centrifugal column method. The operation was carried out under low-temperature conditions throughout the process. To ensure that the extracted RNA was not contaminated or degraded, RNase-free pipette tips and EP tubes were used. The exposed liquid in the culture well plate was aspirated completely, and the embryos were rinsed twice with pre-cooled distilled water. The embryos were mashed with a tissue grinder, and then 500 μL of RA2 lysis buffer was added to each tube. After thoroughly pipetting and mixing, the samples were transferred to the inner tube and left to stand for 1 min. Then, the inner tube (already inserted into the outer tube) was placed in a high-speed low-temperature centrifuge and centrifuged at 4 °C and 13,000 g for 1 min. The inner tube was taken out, the liquid in the outer tube was aspirated and discarded, and then the inner tube was put back. 500 μL of wash buffer was added and centrifuged for 1 min. Then this washing step was repeated once. The inner tube was taken out, the liquid in the outer tube was aspirated and discarded, and then the inner tube was put back without adding wash buffer and centrifuged for 1 min. Finally, the inner tube was transferred to a new 1.5 mL RNase-free centrifuge tube, 25 μL of elution buffer was added to the center of the inner tube membrane, and after standing for 1 min, it was centrifuged for 1 min to obtain the total RNA. The concentration and quality of the RNA were measured with a full-wavelength microplate reader. When the ratio of OD 260 / OD 280 was between 1.9 and 2.1, it indicated that the extracted RNA had good quality and could be used for subsequent reverse transcription experiments;

[0110] (4) Using the extracted total RNA as a template, reverse transcription was carried out according to the operating steps of the TransGen Biotech AT341-01 Reverse Transcription Kit. In a 20 μL reaction system, it contained 1 μg of total RNA template, 4 μL of 5× All-in-one SuperMix for qPCR, 1 μL of gDNA Remover, and RNase-free water. The reaction conditions were incubation at 42 °C for 15 min and heating at 85 °C for 5 s. The 20 μL of cDNA obtained by reverse transcription was diluted to 100 μL with RNase-free water and could then be used for RT-qPCR experiments.

[0111] (5) RT-qPCR experiments were carried out using the QPCRArray plate containing 59 target genes related to detoxification metabolism, neurodevelopment, and endocrine disruption independently developed by the present invention. The well positions of the 384-well plate were arranged in 16 rows, with 24 wells in each row. 20 μL of reaction system was placed in each well. The specific gene arrangement order was as Figure 1As shown, in a 20-μL reaction system, there are 2 μL of reverse-transcribed cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of qPCR reaction premix, and RNase-free water. The reaction conditions are as follows: pre-denaturation at 94°C for 30 s, then denaturation at 94°C for 5 s, and then annealing and extension at 60°C. The CT value is obtained. The relative expression fold of the target gene relative to the internal reference gene is calculated using the CT value. Then, the relative expression fold of the gene is used to interpret the target genes related to detoxification metabolism, neurodevelopment, and endocrine disruption.

[0112] Result analysis: As Figure 7 shown, the exposure to a certain water damage treatment agent / water lock damage treatment agent can significantly induce the expression of phase I metabolism, phase II metabolism, and aryl hydrocarbon receptor in zebrafish embryos. Moreover, except for upregulating the neurotoxic gene abat, it inhibits the expression of genes related to neurodevelopment and endocrine. This is consistent with the result of the high-concentration group of the water damage treatment agent / water lock damage treatment agent inhibiting embryo hatching in the previous experimental results, verifying the reliability of the microarray results.

[0113] Example 7

[0114] This example provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR microarray, specifically as follows:

[0115] The operation process is the same as that in Example 6, with the only difference being in step (1): Select the currently used additives in Shengli Oilfield (gel-type / room-temperature gel plugging agents) as the test substances. The samples are stored at room temperature and transported to the laboratory for storage at room temperature in the dark within 24 h.

[0116] Result analysis: As Figure 8 shown, through the preliminary scanning of the composite microarray, it is found that the gel-type / room-temperature gel plugging agents can induce the activation of the detoxification metabolism pathway, inhibit the neurodevelopment pathway and the endocrine disruption pathway. Moreover, in the previous experimental results, it was found that the gel-type / room-temperature gel plugging agents significantly inhibited the hatching of zebrafish embryos. According to the ingredient description provided by the additive manufacturer, the gel-type / room-temperature gel plugging agents contain trivalent chromium, and it is reported in the literature that chromium can inhibit the hatching of zebrafish embryos. It is speculated that the inhibition of embryo hatching may be due to the toxic effect of chromium, which is also consistent with the test results of this microarray.

[0117] Example 8

[0118] This example provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR microarray, specifically as follows:

[0119] The operation process is the same as that in Example 6, with the only difference being in step (1): Select the currently used additives in Shengli Oilfield (crosslinking agent / composite chromium-based crosslinking agent for polymer flooding) as the test substances. The samples are stored at room temperature and transported to the laboratory for storage at room temperature in the dark within 24 h.

[0120] Result analysis: As Figure 9 shown, through the preliminary scanning of the composite large chip, it is found that the composite chromium-based crosslinking agent for crosslinking agent / polymer flooding can induce the activation of the detoxification metabolic pathway, inhibit the neurodevelopment pathway and the endocrine disruption pathway, and in the previous experimental results, it is found that the composite chromium-based crosslinking agent for crosslinking agent / polymer flooding significantly inhibits the hatching of zebrafish embryos. According to the ingredient description provided by the additive manufacturer, the composite chromium-based crosslinking agent for crosslinking agent / polymer flooding contains trivalent chromium, and it is reported in the literature that chromium inhibits the hatching of zebrafish embryos. It is speculated that the inhibition of embryo hatching may be the toxic effect of chromium, which is also consistent with the test results of this chip.

[0121] Example 9

[0122] This example provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR chip, specifically as follows:

[0123] The operation process is the same as that of Example 6, except for step (1): Select the currently used additives in Shengli Oilfield (acidification / high-temperature acid corrosion inhibitor) as the test substances. The samples are stored at room temperature and transported to the laboratory for storage at room temperature in the dark within 24 hours.

[0124] Result analysis: As Figure 10 shown, considering that the gonads of zebrafish embryos and larvae are not yet mature; therefore, a composite chip combining some detoxification metabolic pathways, endocrine disruption, and neurodevelopment pathway genes is selected to conduct research on the petrochemical pollutant pathway with the greatest toxicity. Exposure of zebrafish embryos to the acidification / high-temperature acid corrosion inhibitor was found to have significant expression differences in genes related to the aryl hydrocarbon receptor, such as ahr2, ahrra, and ahrrb, genes related to phase I metabolism, such as cyp1a1, cyp1b1, cyp1c1, and cyp1c2, and genes related to phase II metabolism, such as ugt1a1, ugt1b1, ugt5a1, ugt5a4, ugt5c2, ugt5c3, sult1st5, sult1st7, and sult3st2, all of which were significantly upregulated. At the same time, the expression of genes related to neurodevelopment and endocrine disruption was also inhibited, indicating that the organic substances in the acidification / high-temperature acid corrosion inhibitor can activate the expression of the aryl hydrocarbon receptor and genes related to detoxification metabolism, and it was also found that the acidification / high-temperature acid corrosion inhibitor can inhibit the expression of genes related to neurodevelopment and endocrine disruption, which is consistent with the previous experimental result that the acidification / high-temperature acid corrosion inhibitor inhibits the hatching of zebrafish embryos.

[0125] Example 10

[0126] This example provides a method for evaluating the toxic effects of petrochemical pollutants on zebrafish using a PCR chip, specifically as follows:

[0127] The operation process is the same as that of Example 6, except for step (1): Select the currently used assistant agent (organic retarded acid) in Shengli Oilfield as the test substance. The sample is stored at room temperature and transported to the laboratory for storage at room temperature in the dark within 24 hours.

[0128] Result analysis: As Figure 11 shown, after the organic retarded acid enters the zebrafish embryos, it causes the high expression of genes related to the detoxification metabolism pathway, especially the genes related to phase I metabolism and phase II metabolism, indicating that when the zebrafish embryos are stimulated by exogenous organic pollutants, they can activate the detoxification metabolism pathway to protect the body. At the same time, the expressions of genes in the neurotoxicity pathway and the endocrine disruption pathway are mostly down-regulated. The combined toxicity of the organic retarded acid is stronger than that of water damage treatment agents / water block damage treatment agents, gel-type / room temperature gel plugging agents, cross-linking agents / composite chromium-type cross-linking agents for polymer flooding, acidification / high temperature acid corrosion inhibitors. The components of oilfield assistant agents are complex, with different properties, acids and bases. The ecological toxicity is related to heavy metal components, surfactants, bactericides, oily solvents and acidic substances, etc., and is also related to the synergistic toxicity caused by the combined action of these substances.

Claims

1. A PCR chip for evaluating the toxic effects of zebrafish, characterized in that, 59 pairs of primers were designed for target genes related to detoxification metabolism, neurodevelopment and endocrine disruption. Each pair of primers includes an upstream primer and a downstream primer, and the sequences of the 59 pairs of target gene primers are shown in SEQ ID NO.1 - SEQ ID NO.118 respectively.

2. The PCR chip according to claim 1, wherein The target genes related to detoxification metabolism, neurodevelopment and endocrine disruption include 59 genes including cyp1a1, cyp1b1, cyp1c1, cyp1c2, ahr2, ahrra, ahrrb, ugt1a1, ugt1b5, ugt5a1, ugt5a4, ugt5c2, ugt5c3, sult1st5, sult1st7, sult3st2, inhbaa, rpl8, igf1ra, igf1rb, igf2r, crh, ttr, ghra, igf1a, slc5a5, tg, dio1, dio2, trh, gnrhr2, gnrhr3, gnrhr4, fshr, lhb, lhcgr, vtg1, vtg3, esr1, esr2a, erβ, ar, star, 3β - hsd, 17β - hsd, cyp19a1b, cyp17a1, cyp19a1a, hmgcra, glsa, grin1b, gad1b, gad2, gabrg2, abat, th1, drd2a, drd2b and slc6a3.

3. The PCR chip according to claim 2, wherein, In the PCR chip, the usage concentration of the 59 pairs of primers shown in SEQ ID NO.1 - SEQ ID NO.118 is 10 μM; The product of the target gene cyp1a1 related to detoxification metabolism, neurodevelopment, and endocrine disruption is 224 bp in size.The product size of cyp1b1 is 159bp, the product size of cyp1c1 is 160bp, the product size of cyp1c2 is 338bp, the product size of ahr2 is 454bp, the product size of ahrra is 187bp, the product size of ahrrb is 339bp, the product size of ugt1a1 is 153bp, the product size of ugt1b5 is 358bp, the product size of ugt5a1 is 379bp, the product size of ugt5a4 is 259bp, the product size of ugt5c2 is 153bp, the product size of ugt5c3 is 266bp, the product size of sult1st5 is 246bp, the product size of sult1st7 is 336bp, the product size of sult3st2 is 131bp, the product size of inhbaa is 127bp, the product size of rpl8 is 195bp, the product size of igf1ra is 365bp, the product size of igf1rb is 207bp, the product size of igf2r is 466bp, the product size of crh is 150bp, the product size of ttr is 166bp, the product size of ghra is 299bp, the product size of igf1a is 300bp, the product size of slc5a5 is 443bp, the product size of tg is 436bp, the product size of dio1 is 142bp, the product size of dio2 is 414bp, the product size of trh is 371bp, the product size of gnrhr2 is 308bp, the product size of gnrhr3 is 372bp, the product size of gnrhr4 is 229bp, the product size of fshr is 373bp, the product size of lhb is 108bp, the product size of lhcgr is 398bp, the product size of vtg1 is 101bp, the product size of vtg3 is 286bp, the product size of esr1 is 452bp, the product size of esr2a is 265bp, the product size of erβ is 353bp, the product size of ar is 370bp, the product size of star is 148bp, the product size of 3β-hsd is 150bp, the product size of 17β-hsd is 138bp, the product size of cyp19a1b is 477bp, the product size of cyp17a1 is 212bp, the product size of cyp19a1a is 117bp, the product size of hmgcra is 406bp, the product size of glsa is 181bp, the product size of grin1b is 242bp, the product size of gad1b is 402bp, the product size of gad2 is 137bp, the product size of gabrg2 is 117bp, the product size of abat is 114bp, the product size of th1 is 390bp, the product size of drd2a is 145bp, the product size of drd2b is 130bp and the product size of slc6a3 is 195bp., 4. The PCR chip according to claim 1, wherein, It also includes 2 pairs of internal reference gene primers designed for gadph and β - actin. Each pair of the internal reference gene primers includes an upstream primer and a downstream primer, and their sequences are shown in SEQ ID NO.119 - SEQ ID NO.

122.

5. The PCR chip according to claim 1, wherein It also includes a carrier, and the carrier is a 384 - well plate.

6. A PCR chip kit, characterized in that, It includes the PCR chip, qPCR reaction premix and RNase - free water described in any one of claims 1 - 5; Among them, the qPCR reaction premix contains Taq hot - start enzyme, SYBR Green Ⅰ fluorescent dye and deoxyribonucleotide triphosphate.

7. Use of the PCR chip according to any one of claims 1-5 or the PCR chip kit according to claim 6 in evaluating the toxic effects of petrochemical pollutants on zebrafish, characterized in that, The toxic effects include zebrafish detoxification metabolism, neurodevelopment and endocrine disruption.

8. The application according to claim 7, characterized in that The petrochemical pollutants are selected from oil - field produced water or oil - field additives; Among them, the oil - field produced water is selected from the incoming water of oil - field produced water, the three - phase separated water of oil - field produced water, the first - stage sedimentation water of oil - field produced water, the second - stage sedimentation water of oil - field produced water or the filtered water of oil - field produced water; The oil - field additives are selected from water damage treatment agents, water lock damage treatment agents, gel - type plugging agents, composite chromium - type cross - linking agents for polymer flooding, high - temperature acidizing corrosion inhibitors or organic retarded acids.

9. A method for evaluating the toxic effects of petrochemical pollutants on zebrafish, characterized in that, It includes the following steps: Set up experimental group solutions and control group solutions; The zebrafish embryos at the cell stage were respectively placed in the experimental group solution and the control group solution for hatching. The surviving embryos and larvae were collected, and the total RNA of the surviving embryos and larvae was extracted. Then, cDNA was reverse transcribed using the total RNA as a template; The cDNA was mixed with the PCR chip according to any one of claims 1-5 to perform an RT-qPCR reaction to obtain CT values; The relative expression fold of the target gene relative to the internal reference gene was calculated based on the CT values, and then the relative expression fold of the gene was used for analysis to evaluate the toxic effects of petrochemical pollutants on zebrafish.

10. The method for evaluating the toxic effect of petrochemical pollutants on zebrafish according to claim 9, characterized in that, The experimental group solution was obtained by diluting the test sample with standard dilution water, and the control group solution was standard dilution water; The hatching conditions were a temperature of 20°C and a time of 96 h; The reaction conditions for the reverse transcription were: incubation at 42°C for 15 min and heating at 85°C for 5 s; The RT-qPCR reaction was performed by mixing the cDNA, the PCR chip according to any one of claims 1-5, the qPCR reaction premix, and RNase-free water to obtain CT values; The qPCR reaction premix contained Taq hot start enzyme, SYBR Green I fluorescent dye, and deoxyribonucleotide triphosphate; The RT-qPCR reaction program was: pre-denaturation at 94°C for 30 s, then denaturation at 94°C for 5 s, and then annealing and extension at 60°C.

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