PCR chip, application thereof and method for evaluating toxic effect of heavy metal pollutants on zebra fish

Abstract

The invention relates to the technical field of molecular biology and environmental biology, and discloses a PCR chip, application thereof and a method for evaluating the toxic effect of petroleum pollutants on zebra fish. The PCR chip comprises 22 pairs of target gene primers, each pair of target gene primers comprises an upstream primer and a downstream primer, and the sequences of the 22 pairs of target gene primers are as shown in SEQ ID NO. 1 to SEQ ID NO. 44. According to the invention, 22 specific related genes are specifically selected according to a research result, and a PCR chip for researching the toxic effect of the heavy metal pollutants on the zebra fish is integrated, so that chip customization based on the heavy metal pollutants is realized; the operation method of the PCR chip is mature and simple, a large amount of toxicity data can be obtained at a time, the toxic effect mechanism of the heavy metal pollutants on the zebra fish embryos can be researched more comprehensively and in detail, and data support is provided for deeper research on the toxic mechanism of the heavy metal pollutants.

Description

Technical Field

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

[0002] At present, the amount of domestic oilfield additives used is increasing year by year, and the types and compositions are quite complex. A large number of chemical agents containing heavy metals are used in the process of oil and gas extraction. At the same time, it is found that the oily wastewater produced by the extraction, processing, refining and coking of petroleum in the chemical industry has the characteristics of wide sources, large water volume, difficult biodegradation and high hazards. It mainly contains organic matter such as oil and surfactants, sulfides, phenols, acetone, aromatics and some heavy metals. Studies have found that heavy metals in petroleum wastewater include copper, cadmium, lead, zinc, nickel and arsenic, which have the characteristics of high stability, inherent toxicity, bioaccumulation, long decay period and difficulty in treatment. When heavy metals accumulate to a certain concentration in the body, acute and chronic poisoning will occur.

[0003] Since heavy metal petroleum pollutants are highly toxic to organisms, this study chose zebrafish, a vertebrate widely used worldwide, as a model organism to study the toxic effects of heavy metal petroleum pollutants on zebrafish, as the zebrafish genome has 87% homology with human genes and has the advantages of being easy to raise, having strong fertility and rapid development.

[0004] Scholars studied the toxic effects of heavy metal cadmium on fish and found that cadmium, as an environmental endocrine disruptor, can cause changes in the morphological structure of fish gonads, resulting in a decrease in the number of sperm in male fish, and can also interfere with reproductive behavior. The effect of cadmium on the reproduction of higher vertebrates is mainly through the synthesis of related hormones on the reproductive axis. The hypothalamus-pituitary-gonad axis of fish is sensitive to cadmium exposure. Heavy metal arsenic can cause pericardial edema, abnormal heart rate and abnormal head development in zebrafish embryos. When zebrafish embryos are exposed to heavy metal arsenic, the survival rate, hatching rate, heart rate, body length and motor ability of zebrafish are reduced, and deformities increase. Low concentrations of arsenic can interfere with the transcription levels of metallothionein (MT) and heat shock protein HSP70 mRNA in early-life zebrafish, and cause oxidative stress and damage in early-life zebrafish, thereby reducing the survival rate of zebrafish embryos. Arsenic mainly induces oxidative stress and affects the normal physiological activities of cells, causing toxic effects including cytotoxicity, neurotoxicity and developmental toxicity. Environmental concentrations of heavy metal copper can cause ocular dysplasia in zebrafish embryos, i.e., obvious microphthalmia, by inducing oxidative stress. Copper can also affect the abnormal expression of genes related to neurodevelopment, causing abnormal neurodevelopment in zebrafish embryos. Abnormal neurodevelopment can inhibit embryonic development, and copper ions can pass through the chorion and accumulate continuously in the embryo, causing strong teratogenic effects on the embryo, such as embryonic tail bending, hatching failure, and embryonic death. Copper ions can inhibit the reproduction of aquatic animals by delaying sexual maturity, reducing fertilization rate, and reducing egg production. The effects of copper ions on the early development of zebrafish embryos are mainly manifested in inhibiting hatching, inhibiting growth and development, and accelerating heart rate. Heavy metal lead is a neurotoxin that can cause neurodegenerative diseases, cell signal transduction disorders, and neurotransmission disorders caused by brain damage, brain morphological changes, and cognitive impairment. Chronic lead exposure is toxic to the central nervous system of fish and can lead to behavioral and cognitive dysfunction. Calcium is a key ion for the release and regulation of neurotransmitters, and lead can simulate calcium ions to compete with receptors. Therefore, lead exposure can cause changes in calcium homeostasis, thereby affecting various physiological functions such as neurotransmission. Studies have shown that lead-induced neurotoxicity occurs by inhibiting the activity of cholinesterase. The reduction of acetylcholinesterase will lead to the accumulation of acetylcholine, which will lead to overstimulation and continuous nerve excitement. Severe neurotoxicity can lead to death. It has been reported that the turning speed and swimming time of zebrafish exposed to lead decrease with the increase of lead concentration, and the startle response decreases. Exposure to heavy metals nickel and zinc in the environment also causes damage to the antioxidant system and nervous system of environmental organisms.

[0005] The current PCR chip technology can study the expression of genes related to signal pathways or biological functions from the whole genome scale and has been widely developed and applied. PCR array chip technology can measure the expression differences of multiple genes on a set of chips and is a high-throughput toxicity testing technology. PCR chip combines the detection characteristics of SYBR Green real-time fluorescence quantitative PCR, and has the advantages of good repeatability, high sensitivity and strong specificity. The biggest advantage is that it can quantitatively detect the expression of multiple genes at the same time. As a high-quality experimental platform with high cost performance and convenient use, PCR chip provides researchers with a new method to focus on the expression of specific genes. PCRArray 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 the toxic endpoint, the toxic pathways activated by pollutants are interpreted and the toxicity mechanism is revealed. However, there is no relevant report on how PCR chips can intuitively and comprehensively evaluate the toxic effects of heavy metal pollutant exposure on zebrafish. Summary of the invention

[0006] The purpose of the present invention is to overcome the problem that the existing PCR chip cannot intuitively and comprehensively evaluate the toxic effects of heavy metal pollutants on zebrafish, and to provide a PCR chip and its application as well as a method for evaluating the toxic effects of heavy metal pollutants on zebrafish. The PCR chip can comprehensively and thoroughly study the toxic effects of heavy metal pollutants on zebrafish embryos.

[0007] In order to achieve the above objectives, the present invention provides a PCR chip, which includes 22 pairs of target gene primers, each pair of target gene primers includes an upstream primer and a downstream primer, and the sequences of the 22 pairs of target gene primers are shown in SEQ ID NO.1 to SEQ ID NO.44.

[0008] Preferably, each pair of target gene primers is in solution form.

[0009] Preferably, the concentration of each pair of target gene primers is 8-12 μM.

[0010] Preferably, the PCR chip further comprises a pair of internal reference gene primers, wherein the internal reference gene primers comprise an upstream primer and a downstream primer, and the sequences thereof are shown in SEQ ID NO.45 to SEQ ID NO.46.

[0011] Preferably, the internal reference gene primer is in the form of a solution.

[0012] Preferably, the concentration of each pair of internal reference gene primers is 8-12 μM.

[0013] Preferably, the PCR chip further comprises a vector.

[0014] Preferably, the carrier is a 96-well plate.

[0015] A second aspect of the present invention provides a PCR chip kit, characterized in that the PCR chip kit comprises the PCR chip described above, a qPCR reaction premix containing a dye, and RNase-free water.

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

[0017] A third aspect of the present invention provides an application of the PCR chip or the PCR chip kit described above in evaluating the toxic effects of heavy metal pollutants on zebrafish.

[0018] Preferably, the heavy metal pollutants are selected from one or more of oilfield produced water, oilfield additives, heavy metal salts and heavy metal oxides.

[0019] Preferably, the oilfield produced water is oilfield produced water and / or oilfield produced water obtained by three-phase separation.

[0020] Preferably, the oilfield additive is selected from gel plugging agents and / or pressure-reducing and injection-increasing surfactants.

[0021] Preferably, the heavy metal elements contained in the heavy metal pollutants are selected from one or more of copper, cadmium, lead, zinc, nickel and arsenic.

[0022] A fourth aspect of the present invention provides a method for evaluating the toxic effects of heavy metal pollutants on zebrafish, the method comprising the following steps:

[0023] (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;

[0024] (2) mixing the cDNA and the PCR chip according to any one of claims 1 to 5 to perform RT-qPCR reaction to obtain a CT value;

[0025] (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.

[0026] Preferably, in step (1), the incubation conditions include: a temperature of 20 to 30° C. and a time of 90 to 100 hours.

[0027] Preferably, in step (1), 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.

[0028] 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.

[0029] Preferably, step (2) specifically comprises: mixing the cDNA, the PCR chip according to any one of claims 1 to 5, a qPCR reaction premix containing a dye and RNase-free water to perform RT-qPCR reaction to obtain a CT value.

[0030] Preferably, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBR Green I fluorescent dye and ribonucleotide triphosphate.

[0031] Preferably, in step (2), the conditions of the RT-qPCR reaction include: pre-denaturation at 94°C for 30 seconds, denaturation at 94°C for 5 seconds, and then annealing and extension at 60°C for 15 seconds.

[0032] The model organism selected by the present invention is zebrafish embryo, which has the advantages of transparent embryo and high homology of genes with humans. The mechanism of petroleum pollutants proposed in the prior art, especially heavy metals in petroleum pollutants, on zebrafish pathogenicity is not clear, and it is impossible to know which gene is affected to cause zebrafish pathogenicity. Through a lot of investigation and research, the present invention found that when the environmental organism zebrafish is exposed to heavy metals such as copper, cadmium, lead, zinc, nickel and arsenic, it is more sensitive to 7 toxic pathways, namely neurotoxicity-related, metallothionein, antioxidant, heat shock protein, hypothalamus-pituitary-thyroid axis, growth-related and hypothalamus-pituitary-gonad axis; the combined toxic effects of heavy metal petroleum pollutants such as oilfield produced water and oilfield adjuvants on zebrafish embryos were also studied. The present invention has targetedly selected a total of 22 specific related genes through research results, integrated a PCR chip for studying the toxic effects of heavy metal pollutants on zebrafish, and realized chip customization based on heavy metal pollutants; the PCR chip operation method is mature and simple, and a large amount of toxicity data can be obtained at one time, which can be used to more comprehensively and thoroughly study the toxic effect mechanism of heavy metal pollutants on zebrafish embryos, and provide data support for deeper research on the toxic mechanism of heavy metal pollutants.

[0033] Compared with the existing technology, the present invention is more targeted, has simpler data processing, can intuitively evaluate the toxic effects of heavy metal pollutant exposure on organisms, and can trace back pollutants based on molecular pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is a diagram of the reaction system setup in a PCR experiment;

[0035] Figure 2 The changes in gene expression levels of the chip-related genes exposed to oilfield produced water in Example 1;

[0036] Figure 3 The changes in gene expression levels of the three-phase separation of produced water from oilfields in Example 2 exposed to water chip;

[0037] Figure 4 The changes in gene expression levels of the active additives (normal temperature gel plugging agents) exposed to the chip in Shengli Oilfield in Example 3;

[0038] Figure 5 It is the changes in gene expression levels of the active adjuvant (pressure-reducing and injection-increasing surfactant) exposed to the chip in Example 4 Shengli Oilfield;

[0039] Figure 6 It is the change in gene expression related to the heavy metal petroleum pollutant cadmium exposure chip in Example 5;

[0040] Figure 7 The changes in gene expression levels related to the heavy metal petroleum pollutant arsenic exposure chip in Example 6;

[0041] Figure 8 It is the change in gene expression related to the copper exposure chip of heavy metal petroleum pollutants in Example 7;

[0042] Fig. 9 The changes in gene expression levels of the heavy metal petroleum pollutant lead exposure chip related to Example 8;

[0043] Fig.10 The changes in gene expression levels of the nickel exposure chip related to heavy metal petroleum pollutants in Example 9;

[0044] Fig.11 This is the change in gene expression levels related to the zinc exposure chip of heavy metal petroleum pollutants in Example 10. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] On one hand, the present invention provides a PCR chip, which includes 22 pairs of target gene primers, each pair of target gene primers includes an upstream primer and a downstream primer, and the sequences of the 22 pairs of target gene primers are shown in SEQ ID NO.1 to SEQ ID NO.44.

[0048] The 22 pairs of target gene primers on the PCR chip of the present invention correspond to zebrafish neurotoxicity-related, metallothionein, antioxidant, heat shock protein, hypothalamus-pituitary-thyroid axis, growth-related and hypothalamus-pituitary-gonad axis-related genes SOD1, CAT, hsp70.3, Ghrh, Gh1, igf1, igf2a, igf2b, trα, trβ, gnrh3, gnrhr1, MT2, tph1b, tph2, tph2, slc6a4a, Mao, Gfap, ACHE, MBP, Shha and Manf ; Among them, tph1b, tph2, slc6a4a, Mao, Gfap, ACHE, MBP, Shha and Manf correspond to neurotoxicity-related genes, MT2 corresponds to metallothionein-related genes, SOD1 and CAT correspond to antioxidant-related genes, hsp70.3 corresponds to heat shock protein-related genes, trα and trβ correspond to hypothalamus-pituitary-thyroid axis-related genes, Ghrh, Gh1, igf1, igf2a and igf2b correspond to growth-related genes, and gnrh3 and gnrhr1 correspond to hypothalamus-pituitary-gonad axis-related genes. The gene and primer sequences are shown in Table 1 below.

[0049] Preferably, the PCR chip further comprises a pair of internal reference gene primers, the internal reference gene primers comprising an upstream primer and a downstream primer, and the sequences thereof are shown in SEQ ID NO. 45 to SEQ ID NO. 46. The gene and primer sequences are shown in Table 1 below.

[0050] Table 1

[0051]

[0052]

[0053] In the present invention, the target gene primers can be in various forms when used in the RT-qPCR experiment. Preferably, each pair of target gene primers is in the form of a solution. More preferably, the concentration of each pair of target gene primers is 8 to 12 μM.

[0054] In a specific embodiment, the internal reference gene primers and the target gene primers are used in the same manner and under the same conditions for RT-qPCR reaction. Preferably, the internal reference gene primers are in the form of a solution. More preferably, the concentration of each pair of internal reference gene primers is 8 to 12 μM.

[0055] According to the present invention, specifically, the PCR chip further comprises a carrier; preferably, the carrier is a 96-well plate.

[0056] When conducting the experiment, different target gene primers and internal reference gene primers are placed in different wells of the carrier to obtain the PCR chip; standard dilution water is placed in one well as a blank control. At the same time, according to the needs of the RT-qPCR experiment, a qPCR reaction premix containing a dye and RNase-free water need to be added to each well.

[0057] A second aspect of the present invention provides a PCR chip kit, which comprises the PCR chip described above, a qPCR reaction premix containing a dye, and RNase-free water.

[0058] In a preferred embodiment, the dye-containing qPCR reaction premix contains Taq hot start enzyme, SYBRGreenⅠ fluorescent dye and oxyribonucleotide triphosphate.

[0059] A third aspect of the present invention provides an application of the PCR chip or the PCR chip kit described above in evaluating the toxic effects of heavy metal pollutants on zebrafish.

[0060] In the present invention, the source of the heavy metal pollutants is not limited. Specifically, the heavy metal pollutants are selected from one or more of oilfield produced water, oilfield additives, heavy metal salts and heavy metal oxides. Herein, the oilfield produced water and oilfield additives both contain heavy metal elements.

[0061] In a specific embodiment, the oilfield produced water is oilfield produced water and / or oilfield produced water obtained by three-phase separation.

[0062] In a specific embodiment, the oilfield additive is selected from gel plugging agents and / or pressure-reducing and injection-increasing surfactants.

[0063] In the present invention, the heavy metal elements contained in the heavy metal pollutants are selected from one or more of copper, cadmium, lead, zinc, nickel and arsenic.

[0064] A fourth aspect of the present invention provides a method for evaluating the toxic effects of heavy metal pollutants on zebrafish, the method comprising the following steps:

[0065] (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;

[0066] (2) mixing the cDNA with the PCR chip described above to perform RT-qPCR reaction to obtain a CT value;

[0067] (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.

[0068] In the present invention, in step (1), the experimental group solution is obtained by diluting the sample to be tested with standard dilution water; the control group solution is standard dilution water. Specifically, the sample to be tested is the heavy metal pollutant mentioned above.

[0069] 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".

[0070] 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.

[0071] In a specific implementation manner, the sample to be tested is stored at 4°C after collection, transported to the laboratory within 24 hours and frozen at -20°C, and needs to be balanced and aerated at 26°C for 30 minutes before testing to ensure that the initial dissolved oxygen concentration is not less than 4 mg / L; it is diluted before use.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Further, step (2) specifically comprises: mixing the cDNA, the PCR chip described above, the qPCR reaction premix containing the dye and RNase-free water to perform RT-qPCR reaction to obtain the CT value. Preferably, the RT-qPCR reaction system contains 10 volume % of the cDNA, 2 volume % of the forward primer, 2 volume % of the reverse primer, 50 volume % of the qPCR reaction premix containing the dye and the remainder of RNase-free water.

[0076] 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.

[0077] In the method described in the present invention, in step (2), the conditions of the RT-qPCR reaction include: pre-denaturation at 94°C for 30 seconds, denaturation at 94°C for 5 seconds, and then annealing and extension at 60°C for 15 seconds.

[0078] Compared with the prior art, preferably, the present invention targets 7 toxic pathways that are more sensitive when zebrafish are exposed to heavy metals such as copper, cadmium, lead, zinc, nickel and arsenic, namely neurotoxicity-related, metallothionein, antioxidant, heat shock protein, hypothalamus-pituitary-thyroid axis, growth-related and hypothalamus-pituitary-gonad axis, and selects 22 related genes in a targeted manner, integrating a PCR chip for studying the toxic effects of heavy metal petroleum pollutants on zebrafish. It is more targeted, simple to process data, can intuitively evaluate the toxic effects of heavy metal exposure in petrochemical wastewater on organisms, and can trace back pollutants based on molecular pathways. A large amount of toxicity data can be obtained at one time, providing data support for a deeper study of the toxic mechanism of heavy metals in petrochemical wastewater. The data can be displayed in a friendly manner to non-biological professionals while improving the utilization of the data.

[0079] The present invention designs toxic pathway-related gene primers based on heavy metal petrochemical characteristic pollutants, forming an independent gene primer library, which can realize the traceability of pollutants based on molecular pathways, and can also study the molecular effect differences of petrochemical pollutants with similar components such as heavy metals. This gene chip technology will play a huge role in environmental safety and human health research.

[0080] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0081] The following examples carry out zebrafish embryo toxicity experiments according to "HJ 1069-2019 Determination of acute toxicity of water quality - zebrafish egg method", "OECD 236 Fish Embryo Acute Toxicity (FET) Test" and "GB / T 21807-2008 Short-term toxicity test of chemicals for fish embryos and yolk sac larvae".

[0082] In the following examples, the base sequences in the target gene primers and the internal reference gene primers were independently developed and designed and synthesized by Shanghai Shenggong Biotechnology Co., Ltd.; the qPCR reaction premix containing the dye was a commercial kit purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0083] Example 1

[0084] This example is used to illustrate the process of using the zebrafish PCR chip described in the present invention to evaluate the toxic effects of oilfield produced water (containing Ba 50.88 mg / L, Fe 8.076 mg / L, As 0.961 mg / L, Cu 0.725 mg / L) from a certain collection site of Shengli Oilfield on zebrafish.

[0085] 1) Incubation process

[0086] The produced water sample (sample 1 to be tested) from a collection station of Shengli Oilfield was collected and stored at 4°C, transported to the laboratory and frozen at -20°C within 24 hours, balanced and aerated at 26°C for 30 minutes before testing to ensure that the initial dissolved oxygen concentration was not less than 4 mg / L, and diluted before use;

[0087] Experimental group solution: dilute 10 mL of sample 1 with 90 mL of standard dilution water;

[0088] Control solution: standard dilution water;

[0089] The incubation process includes:

[0090] 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.

[0091] 2) Extraction of total RNA

[0092] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0093] 3) Reverse transcription

[0094] 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 5× 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.

[0095] 4) RT-qPCR reaction

[0096] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (components are Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in the figure. Figure 2 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0097] from Figure 2 It can be seen that the difference in the expression of neurotoxicity-related genes in zebrafish embryos exposed to oilfield produced water shows that the expression of most neural development-related genes is reduced, indicating that oilfield produced water exposure inhibits neural development. The high expression of metallothionein mt2 and heat shock protein hsp70.3 indicates that oilfield produced water contains metals. After entering the embryo, heavy metals activate antioxidant genes (sod1, cat) in the body and inhibit the expression of genes related to the hypothalamic pituitary thyroid axis, growth-related and hypothalamic pituitary gonad axis.

[0098] Example 2

[0099] This example is used to illustrate the process of using the zebrafish PCR chip described in the present invention to evaluate the toxic effects of produced water before reinjection at a certain collection site in Shengli Oilfield (containing metal elements such as iron, arsenic, tin, barium, selenium, boron and lithium, wherein the arsenic content is greater than 0.05 mg / L, and is defined as Class V water according to the national "Quality standard for ground water GB / T 14848-93").

[0100] 1) Incubation process

[0101] A water sample (sample 2 to be tested) was collected from a collection station in Shengli Oilfield before reinjection and stored at 4°C. It was transported to the laboratory and frozen at -20°C within 24 hours. It was balanced and aerated at 26°C for 30 minutes before testing to ensure that the initial dissolved oxygen concentration was not less than 4 mg / L. It was diluted before use.

[0102] Experimental solution: dilute 10 mL of sample 2 with 90 mL of standard dilution water;

[0103] Control solution: standard dilution water;

[0104] The incubation process includes:

[0105] 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.

[0106] 2) Extraction of total RNA

[0107] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low temperature environment. To ensure that the extracted RNA was not contaminated or dissolved, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0108] 3) Reverse transcription

[0109] 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 5× 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.

[0110] 4) RT-qPCR reaction

[0111] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 3 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0112] from Figure 3 It can be seen that the differential expression of neurotoxicity-related genes in zebrafish embryos exposed to oilfield produced water before reinjection shows that although oilfield produced water exposure before reinjection still inhibits neural development, the degree of damage is lower than that of oilfield produced water. The high expression of metallothionein mt2 and heat shock protein hsp70.3 indicates that the water before oilfield produced water reinjection still contains metals. After entering the embryo, heavy metals activate the antioxidant pathway in the body and inhibit the expression of genes related to the hypothalamic pituitary thyroid axis, growth-related and hypothalamic pituitary gonad axis, but the specific interference degree is less than that of the oilfield produced water sample. The experimental results show that the toxicity of the incoming water is reduced after the sewage treatment process, but it still retains some toxicity.

[0113] Example 3

[0114] This example is used to illustrate the process of using the zebrafish PCR chip described in the present invention to evaluate the toxic effects of the current additives in Shengli Oilfield (room temperature gel plugging agent, purchased from Huabin Chemical, containing 0.5% polyacrylamide; 0.1% chromium chloride; 0.25% cross-linking agent; 98.7% water) on zebrafish.

[0115] 1) Incubation process

[0116] The room temperature gel plugging agent sample (sample 3 to be tested) was stored at room temperature 25°C, transported to the laboratory within 24 hours and stored at room temperature and away from light, and diluted before testing;

[0117] Experimental group solution: dilute 3 mL of sample 3 with 97 mL of standard dilution water;

[0118] Control solution: standard dilution water;

[0119] The incubation process includes:

[0120] 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.

[0121] 2) Extraction of total RNA

[0122] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0123] 3) Reverse transcription

[0124] 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 5× 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.

[0125] 4) RT-qPCR reaction

[0126] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 4 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0127] from Figure 4 It can be seen that the results of the exposure experiment of room-temperature gel plugging agents on zebrafish embryos show that the high expression of metallothionein mt2 and heat shock protein hsp70.3 indicates that the room-temperature gel plugging agents contain metals. After entering the embryos, heavy metals activate the antioxidant genes sod1 and cat in the body, and the genes related to the detoxification metabolic pathway are significantly highly expressed. According to the ingredient description provided by the manufacturer of room-temperature gel plugging agents, the room-temperature gel plugging agents in this embodiment contain trivalent chromium, which means that room-temperature gel plugging agents will inhibit the expression of genes related to the hypothalamic pituitary thyroid axis, growth-related and hypothalamic pituitary gonadal axis.

[0128] Example 4

[0129] This example is used to illustrate the process of using the zebrafish PCR chip described in the present invention to evaluate the toxic effects of the current adjuvants in Shengli Oilfield (pressure-reducing and injection-increasing surfactants, purchased from Dongying Lifeng, containing 20% ​​alkylphenol polyoxyethylene ether; 3% fluorocarbon surfactant; 5% sodium alkylphenol polyoxyethylene ether sulfonate; 0.2% chromium chloride; 71.8% water) on zebrafish.

[0130] 1) Incubation process

[0131] The pressure-reducing and injection-increasing surfactant sample (sample 4 to be tested) was stored at room temperature of 25°C, transported to the laboratory within 24 hours and stored at room temperature and away from light, and diluted before testing;

[0132] Experimental group solution: dilute 3 mL of sample 4 with 97 mL of standard dilution water;

[0133] Control solution: standard dilution water;

[0134] The incubation process includes:

[0135] 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.

[0136] 2) Extraction of total RNA

[0137] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0138] 3) Reverse transcription

[0139] 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 5× 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.

[0140] 4) RT-qPCR reaction

[0141] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 5 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0142] from Figure 5 It can be seen that the results of the exposure experiment of zebrafish embryos to antihypertensive surfactants show that the high expression of metallothionein mt2 and heat shock protein hsp70.3 indicates that the antihypertensive surfactants contain metals. After entering the embryos, heavy metals activate the antioxidant genes sod1 and cat in the body, and the genes related to the detoxification metabolic pathway are significantly highly expressed. According to the ingredient description provided by the antihypertensive surfactant manufacturer, the antihypertensive surfactant in this embodiment contains trivalent chromium, which means that the antihypertensive surfactant will inhibit the expression of hypothalamic pituitary thyroid axis, growth-related and hypothalamic pituitary gonadal axis-related genes.

[0143] Example 5

[0144] This example is used to illustrate the process of evaluating the toxic effect of cadmium chloride on zebrafish using the zebrafish PCR chip described in the present invention.

[0145] 1) Incubation process

[0146] Store cadmium chloride (sample 5) in a reagent storage filter reagent cabinet and dilute before testing;

[0147] Experimental group solution: 218.77 μg of the sample 5 to be tested was diluted with 10 mL of standard dilution water;

[0148] Control solution: standard dilution water;

[0149] The incubation process includes:

[0150] 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.

[0151] 2) Extraction of total RNA

[0152] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0153] 3) Reverse transcription

[0154] 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 5× 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.

[0155] 4) RT-qPCR reaction

[0156] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1 As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 6 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0157] from Figure 6 As can be seen in the results of the heavy metal cadmium exposure experiment on zebrafish embryos, cadmium exposure causes high expression of metallothionein mt2 and heat shock protein hsp70.3 in zebrafish embryos. After entering the embryo, heavy metal cadmium activates the antioxidant and detoxification metabolic pathways in the body, and cadmium inhibits the expression of genes related to the hypothalamus-pituitary-thyroid axis, growth-related and hypothalamus-pituitary-gonad axis.

[0158] Example 6

[0159] This example is used to illustrate the process of evaluating the toxic effect of arsenic trioxide on zebrafish using the zebrafish PCR chip described in the present invention.

[0160] 1) Incubation process

[0161] Arsenic trioxide (sample 6) was stored in a filter reagent cabinet in the reagent library and diluted before testing;

[0162] Experimental group solution: dilute 197.84 μg of the sample 6 to be tested with 10 mL of standard dilution water;

[0163] Control solution: standard dilution water;

[0164] The incubation process includes:

[0165] 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.

[0166] 2) Extraction of total RNA

[0167] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0168] 3) Reverse transcription

[0169] 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 5× 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.

[0170] 4) RT-qPCR reaction

[0171] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 7 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0172] from Figure 7 It can be seen that the results of the heavy metal arsenic exposure experiment on zebrafish embryos showed that arsenic would interfere with the mRNA transcription levels of metallothionein (MT) and heat shock protein HSP70 in early-life zebrafish, and cause oxidative stress and damage in early-life zebrafish, thereby reducing the survival rate of zebrafish embryos. Arsenic mainly induces oxidative stress, affects the normal physiological activities of cells, and causes toxic effects including cytotoxicity, neurotoxicity, and developmental toxicity.

[0173] Example 7

[0174] This example is used to illustrate the process of evaluating the toxic effect of copper sulfate on zebrafish using the zebrafish PCR chip described in the present invention.

[0175] 1) Incubation process

[0176] Store copper sulfate (sample 7) in a reagent storage filter reagent cabinet and dilute before testing;

[0177] Experimental group solution: 159.61 μg of the sample 7 to be tested was diluted with 10 mL of standard dilution water;

[0178] Control solution: standard dilution water;

[0179] The incubation process includes:

[0180] 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.

[0181] 2) Extraction of total RNA

[0182] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0183] 3) Reverse transcription

[0184] 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 5× 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.

[0185] 4) RT-qPCR reaction

[0186] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Figure 8 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0187] from Figure 8 It can be seen that the results of the heavy metal copper exposure experiment on zebrafish embryos showed that heavy metal copper can induce oxidative stress, and copper can also cause a decrease in the expression of nerve-related genes.

[0188] Example 8

[0189] This example is used to illustrate the process of evaluating the toxic effect of lead sulfate on zebrafish using the zebrafish PCR chip described in the present invention.

[0190] 1) Incubation process

[0191] Lead sulfate (test sample 8) was stored in a filter reagent cabinet in the reagent store and diluted before testing;

[0192] Experimental group solution: 303.26 μg of the sample 8 to be tested was diluted with 10 mL of standard dilution water;

[0193] Control solution: standard dilution water;

[0194] The incubation process includes:

[0195] 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.

[0196] 2) Extraction of total RNA

[0197] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0198] 3) Reverse transcription

[0199] 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 5× 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.

[0200] 4) RT-qPCR reaction

[0201] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Fig. 9 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0202] from Fig. 9 It can be seen that the results of the heavy metal lead exposure experiment on zebrafish embryos showed that heavy metal lead would cause the expression of neural development-related genes tph1b, tph2, slc6a4a, mao, gfap, ache, mbp, shha and manf to decrease.

[0203] Example 9

[0204] This example is used to illustrate the process of evaluating the toxic effect of nickel oxide on zebrafish using the zebrafish PCR chip described in the present invention.

[0205] 1) Incubation process

[0206] Store nickel oxide (sample 9) in a reagent storage filter reagent cabinet and dilute before testing;

[0207] Experimental group solution: dilute 146.71 μg of the sample 9 to be tested with 10 mL of standard dilution water;

[0208] Control solution: standard dilution water;

[0209] The incubation process includes:

[0210] 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.

[0211] 2) Extraction of total RNA

[0212] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0213] 3) Reverse transcription

[0214] 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 5× 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.

[0215] 4) RT-qPCR reaction

[0216] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Fig.10 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0217] from Fig.10 It can be seen that the results of the heavy metal nickel exposure experiment on zebrafish embryos showed that the expression of neural development-related genes Tph1b, Tph2, Slc6a4a, Mao, Gfap, Ache, Mbp, Shha and Manf decreased, while inducing high expression of metallothionein MT2 and heat shock protein Hsp70.3. After nickel enters the embryo, it activates the antioxidant genes in the body, namely SOD and CAT. This shows that exposure to heavy metal nickel can cause damage to the antioxidant system and nervous system of environmental organisms.

[0218] Example 10

[0219] This example is used to illustrate the process of evaluating the toxic effect of zinc sulfate on zebrafish using the zebrafish PCR chip of the present invention.

[0220] 1) Incubation process

[0221] Zinc sulfate (sample to be tested 10) was stored in a filter reagent cabinet in the reagent store and diluted before testing;

[0222] Experimental group solution: dilute 161.45 μg of the sample 10 to be tested with 10 mL of standard dilution water;

[0223] Control solution: standard dilution water;

[0224] The incubation process includes:

[0225] 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.

[0226] 2) Extraction of total RNA

[0227] The total RNA of zebrafish embryos and larvae in each well of a 24-well plate was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit using the centrifugal column method. The whole process was operated in a low-temperature environment. To ensure that the extracted RNA was not contaminated or degraded, an RNase-free pipette tip and EP tube were used to aspirate the exposure solution in the culture well plate, and the embryos were rinsed twice with pre-cooled enzyme-free water. The embryos were crushed with a tissue grinder, and then 500 μL of RA2 lysis solution was added to each tube. After being fully blown and mixed, the sample was transferred to the inner cannula and allowed to stand for 1 minute. The inner cannula (which had been inserted into the outer cannula) was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 13000g for 1 minute. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, 500 μL of washing solution was added, and centrifuged for 1 minute. This washing step was repeated once. The inner cannula was removed, the liquid in the outer cannula was aspirated and put back into the inner cannula, and no washing solution was added, and centrifuged for 1 minute. Finally, transfer the inner tube to a new 1.5 mL RNase-free centrifuge tube, add 25 μL of elution solution to the center of the inner tube membrane, let it stand for 1 minute, and centrifuge for 1 minute to obtain total RNA. Use a full-wavelength microplate reader to measure the concentration and quality of RNA. When the OD260 / OD280 ratio is between 1.9-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent reverse transcription experiments.

[0228] 3) Reverse transcription

[0229] 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 5× 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.

[0230] 4) RT-qPCR reaction

[0231] RT-qPCR experiments were performed in a 96-well plate using 22 pairs of target gene primers and 1 pair of reference gene primers. The primer sequences and annealing temperatures are shown in Table 1. The 96-well plate has 8 rows of wells, 12 wells per row, and 20 μL of reaction system is placed in each well. The specific gene arrangement order is as follows: Figure 1As shown. In each 20μL reaction system, there are 200ng of reverse transcribed cDNA, 0.4μL of forward primer (10μM), 0.4μL of reverse primer (10μM), 10μL of qPCR reaction premix containing dye (Taq hot start enzyme, SYBR GreenⅠ fluorescent dye and ribonucleotide triphosphate) and RNase-free water. The reaction conditions are: pre-denaturation at 94℃ for 30s, denaturation at 94℃ for 5s, and then annealing and extension at 60℃ for 15s. The CT value obtained by the reaction was recorded, and the relative expression multiple of the target gene relative to the internal reference gene was calculated based on the CT value. The results are shown in Fig.11 As shown, the relative expression multiples of genes were then used to analyze and interpret the expression effects and toxic effects of heavy metal petroleum pollutant exposure on zebrafish metabolism-related genes.

[0232] from Fig.11 It can be seen that the results of the heavy metal zinc exposure experiment on zebrafish embryos showed that the expression of neural development-related genes tph1b, tph2, slc6a4a, mao, gfap, ache, mbp, shha and manf decreased, while inducing high expression of metallothionein mt2 and heat shock protein hsp70.3. Zinc entered the embryo and activated the antioxidant genes in the body, namely sod1 and cat. This shows that heavy metal zinc exposure can cause damage to the antioxidant system and nervous system of environmental organisms.

[0233] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A PCR chip, characterized in that: The PCR chip comprises 22 pairs of target gene primers, each pair of target gene primers comprises an upstream primer and a downstream primer, and the sequences of the 22 pairs of target gene primers are shown as SEQ ID NO.1 to SEQ ID NO.

44.

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

3. The PCR chip according to claim 1 or 2, characterized in that: The PCR chip also includes a pair of internal reference gene primers, which include an upstream primer and a downstream primer, and their sequences are shown in SEQ ID NO.45 to SEQ ID NO.

46.

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

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

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

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

8. The use according to claim 7, characterized in that: The heavy metal pollutants are selected from one or more of oilfield produced water, oilfield additives, heavy metal salts and heavy metal oxides; Preferably, the oilfield produced water is oilfield produced water and / or three-phase separated oilfield produced water; Preferably, the oilfield additive is selected from gel plugging agents and / or pressure-reducing and injection-increasing surfactants.

9. The use according to claim 7 or 8, characterized in that: The heavy metal elements contained in the heavy metal pollutants are selected from one or more of copper, cadmium, lead, zinc, nickel and arsenic.

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

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

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

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

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

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

16. The method according to any one of claims 10 to 15, characterized in that: In step (2), the conditions of the RT-qPCR reaction include: pre-denaturation at 94°C for 30 seconds, denaturation at 94°C for 5 seconds, and then annealing and extension at 60°C for 15 seconds.

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