Primer group, kit and application thereof

Abstract

The invention relates to the field of molecular biology and environmental biology, and discloses a primer group, a kit and application thereof.The primer group comprises primer pairs used for specific amplification of a cyp1a1 gene and a primer pair used for specific amplification of an ahr2 gene respectively, and the primer pairs used for specific amplification of the cyp1a1 gene comprise an upstream primer with the nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with the nucleotide sequence shown as SEQ ID NO.2; the primer pair for specifically amplifying the ahr2 gene comprises an upstream primer with the nucleotide sequence shown as SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown as SEQ ID NO.4. The invention further discloses a kit for detecting the ahr2 gene. By utilizing the primer group provided by the invention, the toxic reaction of an organism to an environmental sample (petrochemical pollutants) can be represented, and the backtracking of benzene series in the petrochemical pollutants is realized; the primer group can be applied to component optimization of oilfield auxiliaries, improvement of a sewage treatment technology, evaluation of biotoxicity and ecotoxicity of benzene series in a petrochemical polluted site and provision of a basis for guiding remediation of the petrochemical polluted site.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and environmental biotechnology, specifically to a primer set, a reagent kit, and their applications. Background Technology

[0002] Benzene is a crucial raw material for the production of various chemicals and petrochemical products, including styrene, ethylbenzene, cumene, phenol, and cyclohexane. It is also used as an organic solvent and gasoline additive to increase octane rating. In 2012, the annual production of benzene, toluene, and xylene was 95 million tons, while annual consumption of benzene alone was approximately 40 million tons. Due to the toxicity of benzene compounds, they must be removed from industrial wastewater before it is discharged into receiving water bodies. Benzene compounds are frequently leaked, highly toxic, and have the potential to accumulate in humans through the food chain, placing them prominently on the priority pollutant lists of the U.S. Toxic Substances Administration and the Disease Registry. Human exposure to benzene can damage several organs, including the liver, kidneys, lungs, heart, and brain; cause DNA strand breaks and chromosome damage; and have severe effects on the blood, potentially leading to plastic anemia and acute myeloid leukemia.

[0003] With the increasing demand for petroleum in production and daily life, crude oil extraction continues, leading to severe environmental pollution problems both domestically and internationally. Petroleum pollution primarily arises during the extraction, refining, transportation, use, and disposal of petroleum. In the petrochemical industry, leaks from production facilities, underground storage tanks and pipelines, uncontrolled discharge of pollutants, and sudden environmental incidents can cause serious environmental pollution. Petroleum pollutants contaminate surface water, groundwater, soil, and air to varying degrees, making it a type of pollutant with a wide-ranging impact.

[0004] Currently, chemical methods for detecting benzene compounds are relatively well-established, enabling both qualitative and quantitative detection. However, these methods only provide the composition and concentration of benzene pollutants and cannot directly reflect their toxic effects on organisms. Current research on the toxicity of oilfield additives and produced water is primarily limited to ecotoxicity studies. Oilfield additive toxicity studies mainly utilize luminescent bacteria methods, while produced water toxicity studies focus on offshore oil exploration and development, with test organisms primarily including shrimp larvae, Artemia, Daphnia mongolica, and Goby. These biotoxicity testing methods can only obtain acute toxicity data for benzene pollutants. They lack explanations of specific toxicity effects, toxic endpoints, toxic pathways, and toxic mechanisms, and also lack health toxicity data. They cannot reveal teratogenic, carcinogenic, or mutagenic effects, hindering the optimization of additive components, improvement of pollution treatment technologies, and comprehensive assessment of the biotoxicity and ecotoxicity of benzene pollutants. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a primer set, a reagent kit and its application. This primer set can be used to directly reflect the toxic effects of benzene series compounds on organisms and realize the tracing of benzene series compounds in petrochemical pollutants.

[0006] To achieve the above objectives, the present invention provides a primer set comprising primer pairs for specifically amplifying the cyp1a1 gene and the ahr2 gene, respectively, and primer pairs for specifically amplifying an internal reference gene.

[0007] The primer pairs used for specific amplification of the cyp1a1 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2;

[0008] Primer pairs used for specific amplification of the ahr2 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4.

[0009] Preferably, the primer pair used for specifically amplifying the internal reference gene has the nucleotide sequences of the upstream and downstream primers as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0010] A second aspect of the present invention provides a kit comprising the primer set as described above.

[0011] Preferably, the kit further comprises reverse transcriptase, DNA polymerase, deoxyribonucleoside triphosphate, MgSO4, reference dye, and RNase-free water.

[0012] A third aspect of the present invention provides the application of the primer set as described above or the kit as described above in detecting the expression levels of the cyp1a1 gene and the ahr2 gene.

[0013] A fourth aspect of the present invention provides the application of the primer set or kit described above in the detection of benzene series compounds in environmental samples.

[0014] Preferably, the environmental sample is produced water from an oilfield, an oilfield additive, or an oilfield soil.

[0015] The fifth aspect of the present invention provides the role of the primer set or kit described above in detecting the biotoxicity of environmental samples.

[0016] Preferably, it is specifically used in detecting the biotoxicity caused by benzene compounds in environmental samples.

[0017] A sixth aspect of the present invention provides a method for detecting the biotoxicity of environmental samples, the method comprising the following steps:

[0018] (1) Zebrafish embryos at the 4-128 cell stage were placed in the sample solution to be tested or blank dilution solution for incubation. Surviving embryos and hatched larvae were collected, and total RNA was extracted from the embryos and larvae.

[0019] (2) Using the extracted total RNA as a template, and using the primer pairs in the primers described above as amplification primers, perform RT-qPCR reaction;

[0020] (3) Calculate the relative expression fold of the target gene relative to the internal reference gene based on the CT value obtained from the RT-qPCR reaction, and analyze the biotoxicity based on the relative expression fold. Preferably, in step (1), the incubation conditions include: a temperature of 25-30°C and a time of 80-120 h.

[0021] Preferably, in step (1), the test sample solution is obtained by diluting the test environment sample with a blank diluent.

[0022] Preferably, the conditions for the RT-qPCR reaction include:

[0023] Incubate at 45–55°C for 12–18 min, then heat at 93–96°C for 1–3 min; then proceed to the qPCR cycling phase for a total of 35–45 cycles: heat at 95°C for 15 s, then react at 60°C for 30 s.

[0024] The primer set provided in this invention is designed for the genes cyp1a1 and ahr2, which are highly sensitive to benzene compounds. Using this primer set, it is possible to characterize the toxic responses of organisms to environmental samples from contaminated sites (such as petrochemical pollutants), and to trace benzene compounds within petrochemical pollutants. Therefore, this primer set is suitable for the preliminary screening of complex environmental samples, can efficiently trace benzene compounds, and is suitable for large-scale application. Simultaneously, this primer set can be applied to the optimization of oilfield additives, the improvement of wastewater treatment technologies, the assessment of the biotoxicity and ecotoxicity of petrochemical pollutants, and to provide a basis for guiding the remediation of petrochemical contaminated sites.

[0025] The detection method provided by this invention utilizes the primer set or kit and RT-qPCR technology to rapidly obtain changes in the expression levels of genes cyp1a1 and ahr2 after exposure to toxicity from environmental samples. These changes in gene expression levels directly reflect the toxic effects of benzene compounds in the environmental samples on organisms, enabling retrospective analysis of benzene compounds in the samples. This detection method, employing a one-step RT-qPCR approach, reduces experimental errors and improves efficiency. By selecting target genes cyp1a1 and ahr2, which are particularly sensitive to benzene compounds, the number of target genes is reduced, resulting in high sensitivity, suitability for high-throughput amplification / screening, rapid operation, and high reproducibility. This method can retrospectively analyze benzene compounds in various environmental media. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the composition of the reagent kit provided by the present invention;

[0027] Figure 2 This refers to the changes in the expression levels of relevant genes during exposure to produced water from the oilfield in Example 1.

[0028] Figure 3 This is the melting curve of the primer amplification process of the relevant genes when the produced water from the oilfield is exposed in Example 1;

[0029] Figure 4 This refers to the changes in the expression levels of relevant genes during the exposure of produced water from the three-phase separation process in oilfields in Example 2.

[0030] Figure 5 This is the melting curve of the primer amplification process of related genes during the exposure of produced water from the three-phase separation process in oilfield in Example 2;

[0031] Figure 6 This refers to the changes in the expression levels of relevant genes when the produced water from the oilfield was exposed after filtration in Example 3.

[0032] Figure 7 This is the melting curve of the primer amplification process of the relevant genes when the produced water is exposed after filtration in Example 3.

[0033] Figure 8 This refers to the changes in the expression levels of relevant genes when the produced water samples were exposed before injection in Example 4.

[0034] Figure 9 This is the melting curve of the primer amplification process of the relevant genes when the water sample was exposed before water injection in the oilfield produced water in Example 4;

[0035] Figure 10 This refers to the changes in the expression levels of relevant genes when soil samples were exposed at a depth of 1m in Example 5.

[0036] Figure 11 This is the melting curve of the primer amplification process of the relevant gene when the soil was exposed at a depth of 1m in Example 5;

[0037] Figure 12 This refers to the changes in the expression levels of relevant genes when soil samples were exposed at a depth of 2m in Example 6.

[0038] Figure 13 This is the melting curve of the primer amplification process of the relevant gene when the soil was exposed at a sampling depth of 2m in Example 6;

[0039] Figure 14 This refers to the changes in the expression levels of relevant genes when soil samples were exposed at a depth of 3m in Example 7.

[0040] Figure 15 This is the melting curve of the primer amplification process of the relevant gene when the soil was exposed at a sampling depth of 3m in Example 7;

[0041] Figure 16 This refers to the changes in the expression levels of relevant genes when soil samples were exposed at a depth of 4m in Example 8.

[0042] Figure 17 This is the melting curve of the primer amplification process of the relevant gene when the soil was exposed at a sampling depth of 4m in Example 8;

[0043] Figure 18 This refers to the changes in the expression levels of relevant genes when soil samples were exposed at a depth of 5m in Example 9.

[0044] Figure 19 This is the melting curve of the primer amplification process of the relevant gene when the soil was exposed at a depth of 5m in Example 9;

[0045] Figure 20 This refers to the changes in the expression levels of related genes during benzene exposure in Example 10;

[0046] Figure 21 This is the melting curve of the primer amplification process of the relevant gene during benzene exposure in Example 10. Detailed Implementation

[0047] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0048] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0049] This invention provides a primer set comprising primer pairs for specifically amplifying the cyp1a1 gene and the ahr2 gene, respectively, and primer pairs for specifically amplifying an internal reference gene.

[0050] The primer pairs used for specific amplification of the cyp1a1 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2;

[0051] Primer pairs used for specific amplification of the ahr2 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4.

[0052] Because petroleum pollutants are highly toxic to organisms, and zebrafish, whose genome shares 87% homology with human genes and possesses advantages such as ease of rearing, high reproductive capacity, and rapid development, were chosen as the model organism in this invention. By selecting zebrafish embryos, which share high genetic homology with humans, this approach can better reflect the biotoxicity of environmental samples to humans. To better understand the health and ecological impacts of exposure to benzene series pollutants, reduce costs and time, and improve testing efficiency, this invention focuses on the carcinogenic mechanisms of benzene series compounds in petroleum, selecting the most critical and sensitive genes cyp1a1 and ahr2 to characterize the organism's toxic response to petrochemical pollutants.

[0053] In this invention, the design process of specific primer sequences includes the following steps:

[0054] First, the pathways and genes related to the toxic effects of aromatic petroleum pollutants on zebrafish and embryos were screened, and the expression and endpoint information of the designed genes were verified. After identifying the target gene, the relevant gene name was searched in the NCBI gene database, the coding region of the target gene was copied, primers for the target gene were designed using Primer Premier 5 software, and the designed primers were validated and evaluated using Oligo software to avoid problems such as hairpin structures and primer dimers. After confirming that the primers were of good quality, homologous sequence search was performed in the NCBI Blast module to ensure the specificity of the primers. At the same time, the designed primers were validated across exons using the Ensembl database, and the annealing temperature of the primers was ensured to be around 60℃. Finally, the target gene primers that met the above requirements were screened. Through preliminary experiments, ahr2 and cyp1a1, which are relatively sensitive to the toxicity of aromatic petroleum pollutants, were screened.

[0055] In a specific implementation, the primer pair used for specifically amplifying the internal reference gene has upstream and downstream nucleotide sequences as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0056] In this invention, the primer sequences, product lengths, annealing temperatures, and sequence numbers for the two target genes and one internal reference gene (gapdh) are shown in Table 1 below.

[0057] Table 1

[0058]

[0059] The present invention also proposes a kit comprising the primer set as described above.

[0060] In one specific implementation, 0.8 μL of upstream and downstream primers for the cyp1a1, ahr2, and gapdh genes are added to an RT-qPCR reaction tube, respectively. The well opening is sealed with a sealing film and a hole is poked with a needle to facilitate water vapor evaporation. The tube is then placed in a metal bath and heat-treated at 60°C for 10 min. Because the total amount of primer solution is small, it is completely dried in 10 min. After removal, the tube opening is sealed with a new sealing film and placed at 4°C for use when needed for detection.

[0061] Please refer to the following: Figure 1 In a preferred embodiment, the kit further comprises an enzyme mixture and a reaction mixture (containing polymerase cofactor Mg). 2+ The kit contains deoxyribonucleoside triphosphates (DRPs), dyes, and RNase-free water. The enzyme mixture includes reverse transcriptase and DNA polymerase (with the addition of Platinum Taq antibody to ensure the DNA polymerase participates in the qPCR reaction at high temperature). Based on the design of these components, this kit can be used for one-step RT-qPCR to detect benzene contamination in the sample. It is understood that each component in the kit is stored separately, and each primer pair in the primer set is stored separately.

[0062] In a specific implementation, the kit contains a primer set, reverse transcriptase, DNA polymerase, deoxyribonucleoside triphosphate, MgSO4, a reference dye, and RNase-free water.

[0063] In a preferred embodiment, the components and contents of the kit are shown in Table 2 below.

[0064] Table 2

[0065]

[0066] The present invention also provides the application of the primer set as described above or the kit as described above in detecting the expression levels of the cyp1a1 gene and the ahr2 gene.

[0067] This invention also proposes the application of the primer set or kit described above in the detection of benzene series compounds in environmental samples. Since the primer set and kit include primer pairs for detecting the cyp1a1 and ahr2 genes, which are most sensitive to benzene series compounds, the primer set and kit can be used to detect the presence of benzene series compounds in environmental samples.

[0068] The present invention does not limit the specific source of the environmental samples, which can originate from sites contaminated with conventional benzene series compounds. In a specific embodiment, the environmental samples are petrochemical pollutants, such as produced water from oil fields, oilfield additives, or oilfield soil.

[0069] This invention also proposes the role of the primer set or kit described above in detecting the biotoxicity of environmental samples. More specifically, it proposes the role in detecting the biotoxicity caused by benzene compounds in environmental samples.

[0070] Using the primer set and kit, RT-qPCR reaction can be performed to obtain the relative fold increase of the target gene after exposure to the toxicity of the sample. The relative fold increase can reflect the biotoxicity of the environmental sample. Since the target gene is a gene sensitive to benzene series compounds, the presence and biotoxicity of benzene series compounds in the environmental sample can be inferred by the biotoxicity of the environmental sample.

[0071] This invention also proposes a method for detecting the biotoxicity of benzene compounds in environmental samples, the method comprising the following steps:

[0072] (1) Zebrafish embryos at the 4-128 cell stage were placed in the sample solution to be tested or blank dilution solution for incubation. Surviving embryos and hatched larvae were collected, and total RNA was extracted from the embryos and larvae.

[0073] (2) Using the extracted total RNA as a template, and using the primer pairs in the primer set as described above as amplification primers, perform RT-qPCR reaction;

[0074] (3) Calculate the relative expression fold of the target gene relative to the internal reference gene based on the CT value obtained from the RT-qPCR reaction, and analyze the biotoxicity caused by benzene series compounds in the environmental samples based on the relative expression fold.

[0075] In this invention, a one-step RT-qPCR reaction is used, in which reverse transcription and PCR amplification are performed simultaneously. Since both steps are completed in a single tube, this method has fewer experimental errors, and fewer pipetting steps reduce the risk of contamination. This method is suitable for high-throughput amplification / screening, is rapid, and highly reproducible.

[0076] In step (1), a toxicity exposure experiment is conducted on zebrafish embryos. Specifically, the zebrafish embryo toxicity experiment can be carried out in accordance with "HJ 1069-2019 Determination of Acute Toxicity of Water by Zebrafish Egg Method" and "OECD 236 Fish Embryo Acute Toxicity (FET) Test".

[0077] In a specific implementation, the incubation conditions in step (1) include: a temperature of 25 to 30°C and a time of 80 to 120 hours.

[0078] In a specific embodiment, in step (1), the sample solution to be tested is obtained by diluting the environmental sample to be tested using a blank diluent. In one specific embodiment, the environmental sample is a petrochemical pollutant, such as produced water from an oilfield, oilfield additives, or oilfield soil.

[0079] In a specific implementation, step (2) is performed using the kit described above.

[0080] Furthermore, in step (2), the reaction system for the RT-qPCR reaction includes: total RNA, amplification primers (upstream and downstream primers), reverse transcriptase, DNA polymerase, deoxyribonucleoside triphosphate, MgSO4, reference dye, and RNase-free water.

[0081] In this invention, the amplification of each gene is performed in different reaction tubes. Specifically, primer pairs for the specific amplification of genes cyp1a1, ahr2, and gapdh are added to RT-qPCR reaction tubes, respectively. Then, total RNA, reverse transcriptase, DNA polymerase, deoxyribonucleoside triphosphate, MgSO4, reference dye, and RNase-free water are added to each reaction tube to form reaction systems for detecting genes cyp1a1, ahr2, and gapdh, respectively.

[0082] Preferably, the conditions for the RT-qPCR reaction include:

[0083] Incubate at 45–55°C for 12–18 min, then heat at 93–96°C for 1–3 min; then proceed to the qPCR cycling phase for a total of 35–45 cycles: heat at 95°C for 15 s, then react at 60°C for 30 s.

[0084] In the method described in this invention, in step (3), the CT value refers to the cyclic threshold of the sample.

[0085] In the method described in this invention, in step (3), the CT value is obtained by RT-qPCR reaction, and then the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh are calculated based on the CT value. The relative expression folds are calculated as follows: ΔCt = Target gene Ct - Internal reference gene Ct, ΔΔCt = ΔCt of the test sample - ΔCt of the blank sample, and the expression ratio = 2. -ΔΔCt .

[0086] In the method described in this invention, in step (3), when the relative expression fold of the target gene relative to the internal reference gene is greater than 2, it indicates that exposure to the environmental sample to be tested will interfere with gene expression in the organism. The larger the relative expression fold, the greater the biotoxicity of the environmental sample to be tested, and the greater the possibility of benzene series pollution in the environmental sample to be tested.

[0087] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0088] Example 1

[0089] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (oilfield produced water) using the primer set or kit described in this invention.

[0090] Environmental samples to be tested: Select a sampling site in an oilfield and collect incoming water from that site using a clean sampling bottle (toluene 372 mg / L, phenol 410 mg / L, 2-methylphenol 182 mg / L, 3-methylphenol 175 mg / L, 2,5-dimethylphenol 165 mg / L, 3,4-dimethylphenol 147 mg / L, naphthalene 207 mg / L); the samples are stored at 4℃ and transported to the laboratory for freezing at -20℃ within 24 hours; before testing, the incoming water samples are equilibrated at 26℃ and aerated for 30 minutes to ensure that the initial dissolved oxygen concentration is not lower than 4 mg / L, thus obtaining the water sample to be tested; dilute immediately before use.

[0091] Blank dilution: CaCl2 294 mg / L, MgSO4 123.25 mg / L, NaHCO3 63 mg / L, KCl 5.5 mg / L, balance water;

[0092] Test sample solution: Take 40 mL of water sample and dilute it to 1000 mL with blank diluent.

[0093] (1) Zebrafish embryo toxicity exposure and RNA extraction

[0094] The next day, after the light exposure begins, open the spawning box cover, remove the inner tank, discard the water in the outer tank, and then put it back into the inner tank. Carefully add blank diluent along the inner tank wall, ensuring the water level is about two body widths of the broodstock from the bottom of the inner tank to avoid harming the broodstock. Remove the partition to allow the male and female broodstock to mate and spawn. After 30 minutes, check the spawning status of the broodstock in each tank. Collect the corresponding fish eggs from each spawning tank, rinse them with blank diluent, and transfer them to crystallization dishes. After standing in a constant temperature incubator or constant temperature room for 45 minutes, observe the crystallization dishes with a black background and a side light. In each crystallization dish, screen out unfertilized, damaged, deformed, and dead fish eggs to avoid harming them. Collect the surviving zebrafish embryos at the 4-128 cell stage.

[0095] The zebrafish embryos collected at the 4-128 cell stage were placed in 24-well plates, and the test sample solution or blank diluent was added. One embryo and 2 mL of test sample solution or blank diluent were added to each well of the 24-well plate. The 24-well plates were incubated at 28°C. After incubation for 96 hours, the surviving embryos and hatched larvae were collected.

[0096] Total RNA was extracted from zebrafish embryos and larvae using the Shanghai Feijie Total RNA Rapid Extraction Kit via centrifugation column method. This experiment was conducted at low temperature, using RNase-free pipette tips and EP tubes to ensure no contamination or degradation of the extracted RNA. After aspirating the exposure solution from 1.5 mL of RNase-free EP tubes, the embryos were rinsed twice with pre-cooled distilled water. The embryos were then homogenized using a 1 mL RNase-free pipette tip. 500 μL of RA2 lysis buffer was added to each tube, and the mixture was thoroughly mixed by pipetting. The sample was then transferred to an inner tube and allowed to stand for 1 minute. The inner tube (already inserted into the outer tube) was then centrifuged at 4°C and 13000 g for 1 minute. The inner tube was removed, the liquid in the outer tube was aspirated, and the inner tube was returned to the inner tube. 500 μL of washing buffer was added, and the mixture was centrifuged for 1 minute. This washing step was repeated once. The inner tube was then removed, the liquid in the outer tube was aspirated, and the inner tube was returned to the inner tube without adding washing buffer, and the mixture was centrifuged for 1 minute. 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 stand for 1 min, and then centrifuge for 1 min to obtain total RNA. Measure the concentration and quality of RNA using a full-wavelength microplate reader. When the OD... 260 / OD 280 When the ratio 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 experiments.

[0097] (2) One-step RT-qPCR reaction

[0098] Using the extracted total RNA as a template, RT-qPCR was performed using the kit shown in Table 2. Specifically, the reaction system is shown in Table 3 below; the reaction conditions are: incubation at 50℃ for 15 minutes (cDNA synthesis temperature range 42-60℃), followed by heating at 95℃ for 2 minutes; then, the qPCR cycle was performed for a total of 40 times: heating at 95℃ for 15 seconds, followed by reaction at 60℃ for 30 seconds.

[0099] Table 3

[0100] reagents 20μL reaction system Total RNA Calculate the volume of 200 ng RNA based on concentration. enzyme mixture 1μL reaction mixture 10μL <![CDATA[ROX TM Reference dye (25 μM) 0.4 / 0.04μL RNase-free water Add to 20μL upstream primer 0.4μL Downstream primer 0.4μL

[0101] The primer pair sequences used to amplify the genes cyp1a1, ahr2 and gapdh are shown in Table 1.

[0102] ROX TM The amount of reference dye added was determined based on the qPCR instrument system, in which Applied Biosystems... TM 7000,7300,7700,7900HT,7900HT Fast,StepOne TM and StepOnePlus TM The system requires the addition of 0.4 μL of dye; Applied Biosystems TM 7500, ViiA TM 7, and QuantStudio TM 3,5,6Flex,7Flex,and 12k Flexsystems; Agilent TM Mx3000P TM Mx3005P TM and Mx4000 TM The system needs to be supplemented with 0.04 μL of dye.

[0103] (3) Data Analysis

[0104] Using the cyp1a1 gene as an internal reference, the target gene was standardized, and CT value analysis was performed using StepOne software. -ΔΔCT The relative fold increase in gene expression is calculated as follows: ΔCT = CT of target gene - CT of internal reference gene, ΔΔCT = ΔCT of test sample - ΔCT of blank sample, and the expression ratio = 2. -ΔΔCT Here, blank sample refers to blank diluent.

[0105] Test results are as follows Figure 2-3 As shown.

[0106] Depend on Figure 2It can be seen that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh are 23.61 and 4.62, respectively. This indicates that exposure to environmental samples in this embodiment interferes with gene expression in organisms, suggesting the presence of benzene series compounds in the environmental samples (petrochemical pollutants). This is consistent with the previous analysis results of the incoming water composition, which proves the accuracy and feasibility of the kit described in this invention.

[0107] Depend on Figure 3 As can be seen, the melting curves of cyp1a1, ahr2, and gapdh are all single-peaked, indicating that the primers have good specificity and that only a single product exists in the amplification, which proves the reliability of the detection method provided by this invention.

[0108] Example 2

[0109] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (three-phase separated effluent from oilfield produced water) using the primer set or kit described in this invention.

[0110] The method described in Example 1 was implemented, except that the environmental sample to be tested was: a sampling site in an oil field was selected, and a clean sampling bottle was used to collect the three-phase separated water (toluene 301 mg / L, 2,6-dimethylphenol 123 mg / L, 2,4-dimethylphenol 118 mg / L, phenol 319 mg / L) from the sampling site in the oil field.

[0111] The test results of this embodiment are as follows: Figure 4-5 As shown.

[0112] Depend on Figure 4 It can be seen that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 20.17 and 3.23, respectively, indicating that environmental sample exposure in this embodiment interferes with gene expression in the organism, suggesting the presence of benzene compounds in the environmental samples. This is consistent with the previous component analysis results of the three-phase separated water. Furthermore, after the three-phase separation water treatment step, the naphthalene content was zero, indicating the effectiveness of the water treatment process at this site. This is also reflected in the reduced CT value, which proves the sensitivity, accuracy, and feasibility of the kit described in this invention.

[0113] Figure 5 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0114] Example 3

[0115] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (water filtered from oilfield produced water) using the primer set or kit described in this invention.

[0116] The method described in Example 1 was implemented, except that the environmental sample to be tested was: a sampling station in an oil field was selected, and a clean sampling bottle was used to collect the filtered water from the sampling station in the oil field (toluene 256 mg / L, phenol 223 mg / L, 2-methylphenol 101 mg / L, p-methylphenol 86 mg / L, 2,6-dimethylphenol 72 mg / L, 2,4-dimethylphenol 75 mg / L, 3,4-dimethylphenol 65 mg / L).

[0117] The test results of this embodiment are as follows: Figure 6-7 As shown.

[0118] Figure 6 The results showed that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 16.35 and 2.61, respectively, indicating that environmental sample exposure in this embodiment interferes with gene expression in organisms and suggests the presence of benzene series compounds in the environmental samples. This is consistent with the component analysis results of the effluent after filtration in the early stage, which proves the accuracy and feasibility of the kit described in this invention.

[0119] Figure 7 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0120] Example 4

[0121] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (oilfield produced water samples before injection) using the primer set or kit described in this invention.

[0122] The method described in Example 1 was implemented, except that the environmental samples to be tested were: a water sample was collected from a certain oilfield sampling site before water injection using a clean sampling bottle (toluene 152 mg / L, phenol 123 mg / L, 2-methylphenol 52 mg / L, 2,6-dimethylphenol 32 mg / L, 3,5-dimethylphenol 29 mg / L, 3,4-dimethylphenol 15 mg / L).

[0123] The test results of this embodiment are as follows: Figure 8-9 As shown.

[0124] Figure 8 The results showed that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 10.19 and 1.82, respectively, indicating that environmental sample exposure in this embodiment interferes with gene expression in the organism, suggesting the presence of benzene compounds in the environmental sample, and the content of benzene compounds was reduced compared with that before treatment, which is consistent with the previous component analysis results, and can prove the accuracy and feasibility of the kit described in this invention.

[0125] Figure 9 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers had good specificity.

[0126] Example 5

[0127] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (soil sampling depth 1m) using the primer set or kit described in this invention.

[0128] The method described in Example 1 was implemented, except that the environmental samples to be tested were: a contaminated site of an abandoned oil refinery in the Loess Plateau of Northwest China; sampling was conducted using PVC pipes disinfected with alcohol, with a sampling depth of 1m; the collected soil and rock samples were immediately placed in sterile sampling bags, preserved with dry ice during field and transportation, and frozen at -20℃ in the laboratory; simultaneously, the concentration of petroleum hydrocarbons in the sediment samples was tested and analyzed, and the test results were: chlorobenzene 10.15mg / kg, benzene 2.32mg / kg, pyrene 1.74mg / kg. kg; 250 g of solid sample was extracted with 10 mL of analytical grade dichloromethane and 250 mL of analytical grade methanol respectively; the combined solvents were filtered through a GB-140 glass fiber membrane filter (90 mm in diameter, pore size = 0.4 μm); the extract was concentrated by rotary evaporation, then transferred to a glass vial and dried under nitrogen; the organic extract after nitrogen blowing was weighed and dissolved at a ratio of 0.01 mg of organic extract to 3 μL of dimethyl sulfoxide to prepare a stock solution; the blank diluent consisted of: CaCl2 294 mg / L, MgSO4 123.25 mg / L, NaHCO3 63 mg / L, and KCl 5.5 mg / L; the sample solution to be tested was prepared by diluting the stock solution 1000 times with the blank diluent.

[0129] The test results of this embodiment are as follows: Figure 10-11 As shown.

[0130] Figure 10 The results showed that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 6.82 and 3.12, respectively, significantly upregulated. This indicates that environmental sample exposure in this embodiment interferes with gene expression in vivo, and suggests that the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) is mainly mediated by the AhR-activated P450 monooxygenase cyp1a1. In other words, the metabolic activation of PAHs is related to the expression of cyp1a1 and ahr genes, which is consistent with previous physicochemical test results.

[0131] Figure 11 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0132] Example 6

[0133] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (soil sampling depth 2m) using the primer set or kit described in this invention.

[0134] The method described in Example 5 was implemented, except that the sample to be tested was: a contaminated site of an abandoned oil refinery in the Loess Plateau of Northwest China was selected; sampling was carried out using PVC pipes disinfected with alcohol, with a sampling depth of 2m; the collected soil and rock samples were immediately placed in sterile sampling bags, preserved with dry ice in the field and during transportation, and frozen at -20℃ in the laboratory; at the same time, the concentration of petroleum hydrocarbons in the sediment samples was tested and analyzed, and the test results were 5.52 mg / kg of drossene, 1.2 mg / kg of benzene, and 0.9 mg / kg of pyrene. 250g of solid sample was extracted with 10 mL of analytical grade dichloromethane and 250 mL of analytical grade methanol respectively; the combined solvent was filtered through a GB-140 glass fiber membrane filter (90 mm in diameter, pore size = 0.4 μm); the extract was concentrated by rotary evaporation, and then transferred to a glass vial and dried under nitrogen; the organic extract after nitrogen blowing was weighed and dissolved at a ratio of 0.01 mg of organic extract to 3 μL of dimethyl sulfoxide to prepare a stock solution.

[0135] The test results of this embodiment are as follows: Figure 12-13 As shown.

[0136] Figure 12 The results showed that the relative fold increases of the target genes cyp1a1 and ahr2 compared to the internal reference gene gapdh were 4.17 and 3.39, respectively. The expression level of gene cyp1a1 significantly increased upon exposure to environmental samples, indicating that benzene compounds may have entered the embryo and induced the embryo to initiate detoxification and metabolic mechanisms. This also activated ahr ligands to participate in the metabolic clearance and detoxification of organic matter, suggesting that the environmental samples contained aromatic hydrocarbons as organic pollutants, which is consistent with previous physicochemical test results.

[0137] Figure 13 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0138] Example 7

[0139] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (soil sampling depth 3m) using the primer set or kit described in this invention.

[0140] The method described in Example 5 was implemented, except that the sample to be tested was: a contaminated site of an abandoned oil refinery in the Loess Plateau of Northwest China; sampling was conducted using PVC pipes disinfected with alcohol, with a sampling depth of 3m; the collected soil and rock samples were immediately placed in sterile sampling bags, preserved with dry ice during field and transportation, and frozen at -20℃ in the laboratory; simultaneously, the concentration of petroleum hydrocarbons in the sediment samples was tested and analyzed, and the results were: drylbenzene 5.6mg / kg, benzene 1.28mg / kg, and pyrene 0.96mg / kg. 250g of solid sample was extracted with 10mL of analytical grade dichloromethane and 250mL of analytical grade methanol respectively; the combined solvents were filtered through a GB-140 glass fiber membrane filter (90mm diameter, 0.4μm pore size). The extract was concentrated using rotary evaporation, then transferred to a glass vial and dried under nitrogen. The organic extract after nitrogen evaporation was weighed and dissolved at a ratio of 0.01mg of organic extract to 3μL of dimethyl sulfoxide to prepare a stock solution.

[0141] The test results of this embodiment are as follows: Figure 14-15 As shown.

[0142] Figure 14 The results showed that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 4.21 and 2.12, respectively. Previous physicochemical tests revealed that the composition and content of samples at sampling depths of 2m and 3m were similar. Therefore, at the molecular level, the upregulation folds of cyp1a1 and ahr2 expression were similar, indicating the presence of aromatic organic pollutants and verifying the stability of the kit described in this invention.

[0143] Figure 15 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0144] Example 8

[0145] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (soil sampling depth 4m) using the primer set or kit described in this invention.

[0146] The method described in Example 5 was followed, except that the environmental samples to be tested were from a contaminated site of an abandoned oil refinery in the Loess Plateau of Northwest China. Sampling was conducted using PVC pipes disinfected with alcohol at a depth of 4 m. The collected soil and rock samples were immediately placed in sterile sampling bags and stored on dry ice during field and transportation, and then frozen at -20°C in the laboratory. Simultaneously, the concentration of petroleum hydrocarbons in the sediment samples was analyzed, yielding 5.6 mg / kg chlorobenzyl, 1.28 mg / kg benzene, and 0.96 mg / kg pyrene. 250 g of solid samples were extracted with 10 mL of analytical grade dichloromethane and 250 mL of analytical grade methanol, respectively. The combined solvents were filtered through a GB-140 glass fiber membrane filter (90 mm diameter, 0.4 μm pore size). The extract was concentrated using rotary evaporation, then transferred to a glass vial and dried under nitrogen. The nitrogen-dried organic extract was weighed and dissolved in 3 μL of dimethyl sulfoxide at a ratio of 0.01 mg of organic extract per 3 μL to prepare a stock solution.

[0147] The test results of this embodiment are as follows: Figure 16-17 As shown.

[0148] Figure 16 The results showed that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 2.51 and 1.6, respectively. The expression levels of the two target genes were upregulated, but still decreased compared to the sample collection depths of 1m, 2m and 3m, suggesting that the content of aromatic organic compounds in the sample decreased with increasing sampling depth.

[0149] Figure 17 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0150] Example 9

[0151] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (soil sampling depth 5m) using the primer set or kit described in this invention.

[0152] The method described in Example 5 was implemented, except that the environmental samples to be tested were from a contaminated site of an abandoned oil refinery in the Loess Plateau of Northwest China. Sampling was conducted using PVC pipes disinfected with alcohol at a depth of 5 m. The collected soil and rock samples were immediately placed in sterile sampling bags and stored on dry ice during field and transportation, and then frozen at -20°C in the laboratory. Simultaneously, the concentration of petroleum hydrocarbons in the sediment samples was analyzed, yielding 3.15 mg / kg chlorobenzyl, 0.72 mg / kg benzene, and 0.36 mg / kg pyrene. 250 g of solid samples were extracted using 10 mL of analytical grade dichloromethane and 250 mL of analytical grade methanol, respectively. The combined solvents were filtered through a GB-140 glass fiber membrane filter (90 mm diameter, 0.4 μm pore size). The extract was concentrated using rotary evaporation, then transferred to a glass vial and dried under nitrogen. The nitrogen-dried organic extract was weighed and dissolved in 3 μL of dimethyl sulfoxide at a ratio of 0.01 mg of organic extract per 3 μL to prepare a stock solution.

[0153] The test results of this embodiment are as follows: Figure 18-19 As shown.

[0154] Figure 18 The results showed that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 1.32 and 1.15, respectively, indicating that the sample exposure at a sampling depth of 5m had little interference with the expression of cyp1a1 and ahr2 genes in zebrafish embryos. This suggests that at this sampling depth, benzene compounds in the sample have a weak toxic effect on zebrafish embryos.

[0155] Figure 19 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0156] Example 10

[0157] This embodiment illustrates a method for detecting the biotoxicity of benzene using the primer set or kit described in this invention.

[0158] The method described in Example 1 was implemented, except that the sample to be tested was: benzene, a typical aromatic petroleum pollutant, was selected as the test substance, and the sample was stored in a filter-type reagent cabinet in the reagent library; blank diluent: CaCl2 294mg / L, MgSO4 123.25mg / L, NaHCO3 63mg / L, KCl 5.5mg / L; the sample solution to be tested was: 5mg of benzene was diluted with 10mL of blank diluent.

[0159] The test results of this embodiment are as follows: Figure 20-21 As shown.

[0160] Figure 20 The results showed that the relative expression folds of the target genes cyp1a1 and ahr2 relative to the internal reference gene gapdh were 30 and 27, respectively, which were significantly upregulated. This is consistent with the mechanism of action of benzene in the literature survey, that is, after benzene enters the human body, it forms epoxybenzene under the action of the cytochrome P450 family, which breaks the stable aromatic structure and can induce the toxic effect pathway of the aromatic hydrocarbon receptor (ahr) in the body's cells, thereby affecting cell function and leading to tumor development.

[0161] Figure 21 The results showed that the melting curves of cyp1a1, ahr2, and gapdh were all single-peaked, indicating that the primers provided by this invention have good specificity.

[0162] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combining the various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A primer set, characterized by, The primer set includes primer pairs for specifically amplifying the cyp1a1 and ahr2 genes, respectively, and primer pairs for specifically amplifying the internal reference gene. The primer pairs used for specific amplification of the cyp1a1 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; Primer pairs used for specific amplification of the ahr2 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

4.

2. The primer set according to claim 1, characterized in that, The primer pair used for specific amplification of the internal reference gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.5 and SEQ ID NO.

6.

3. A kit characterized in that, The kit contains the primer set as described in claim 1 or claim 2.

4. The kit of claim 3, wherein The kit also contains reverse transcriptase, DNA polymerase, deoxyribonucleoside triphosphate, MgSO4, reference dye, and RNase-free water.

5. The use of the primer set according to claim 1 or 2 or the kit according to any one of claims 3-4 in detecting the expression levels of the cyp1a1 gene and the ahr2 gene.

6. The use of the primer set according to claim 1 or 2 or the kit according to any one of claims 3-4 in the detection of benzene series compounds in environmental samples.

7. Use according to claim 6, characterized in that, The environmental samples are produced water, oilfield additives, or oilfield soil.

8. The role of the primer set according to claim 1 or 2 or the kit according to any one of claims 3-4 in detecting the biotoxicity of environmental samples.

9. Use according to claim 7, characterized in that, Specifically, it plays a role in detecting the biotoxicity of benzene compounds in environmental samples.

10. A method of detecting the biotoxicity of an environmental sample, characterized in that, The method includes the following steps: (1) Zebrafish embryos at the 4-128 cell stage were placed in the sample solution to be tested or blank dilution solution for incubation. Surviving embryos and hatched larvae were collected, and total RNA was extracted from the embryos and larvae. (2) Using the extracted total RNA as a template, and using the primer pairs in the primers of claim 1 or 2 as amplification primers, perform RT-qPCR reaction; (3) Calculate the relative expression fold of the target gene relative to the internal reference gene based on the CT value obtained from the RT-qPCR reaction, and analyze the biotoxicity based on the relative expression fold.

11. The method of claim 10, wherein, In step (1), the incubation conditions include: a temperature of 25-30°C and a time of 80-120 hours.

12. The method of claim 10, wherein, In step (1), the test sample solution is obtained by diluting the test environment sample with blank diluent.

13. The method of claim 10, wherein, The conditions for the RT-qPCR reaction include: Incubate at 45–55°C for 12–18 min, then heat at 93–96°C for 1–3 min; then proceed to the qPCR cycling phase for a total of 35–45 cycles: heat at 95°C for 15 s, then react at 60°C for 30 s.

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