Primer group, kit and application thereof, and method for detecting biotoxicity of environmental sample

Abstract

The invention relates to the technical field of molecular biology and environment, and discloses a primer group, a kit and application thereof, and a method for detecting biotoxicity of an environmental sample, the primer group comprises a primer pair for specific amplification of an hsp70.3 gene and an mt2 gene, and a primer pair for specific amplification of a reference gene; the primer pair for specifically amplifying the hsp70.3 gene comprises 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; the primer pair for specifically amplifying the mt2 gene comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6. The primer group disclosed by the invention can be used for monitoring the content change of hsp70.3 and mt2 in a living body, so that the biotoxicity of a to-be-detected environmental sample can be analyzed, the early warning of heavy metal pollutants is realized, particularly the existence of the heavy metal pollutants in the to-be-detected environmental sample can be prompted, and the heavy metal detection can be further combined for analysis in the later period.

Description

Technical Field

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

[0002] Metallothionein (MT), chemically known as metallothioneine trimethylammonium, is a class of low-molecular-weight, cysteine-rich, non-enzymatic metal-binding proteins widely distributed in the biological world. Since its discovery in 1957, MT research has become a hot topic in basic scientific research due to its unique physicochemical properties and biological functions. MT exhibits widespread and high inducibility. Research results show that heavy metals, hormones, physiological factors, and pathological factors can all induce MT synthesis, suggesting the broad scope and importance of its functions in organisms. MT participates in heavy metal metabolism, possesses a strong metal-binding capacity, and is closely related to the in vivo metabolism of essential elements such as zinc and copper. For example, as an important zinc-binding protein, MT can regulate zinc participation in biological metabolic processes by modulating cellular zinc levels. The role of MT in heavy metal detoxification has been extensively studied; exposing animals or in vitro cultured cells to heavy metal conditions can enhance MT gene transcription and synthesis. Synthesized MT binds to free heavy metals, reducing their toxicity. In addition, metalloproteinases (MT) also have the function of combating ionizing radiation and scavenging free radicals. Due to the strong reducing agent effect of the sulfhydryl groups in glutathione when scavenging free radicals, the abundant cysteine ​​sulfhydryl groups in MT have attracted widespread attention. Some speculate that it may play an important role in combating free radical damage and maintaining free radical balance in the body. MT participates in DNA replication and transcription, as well as protein synthesis and degradation. Whether it is cellular metabolism or protein and energy metabolism, the participation of metalloenzymes or metalloproteins is indispensable. Most of these enzymes use metals as cofactors, especially zinc and copper, and these metals are closely related to metabolism.

[0003] Heat shock proteins (HSPs) are proteins synthesized in response to physical, chemical, and biological stimuli in the environment. Also known as heat shock proteins, they are a highly conserved class of proteins, widely found in prokaryotes and eukaryotes. The binding or dissociation of HSPs with polypeptides is closely related to their binding with ADP or ATP. The C-terminal "EEVD" sequence is a key site for the connection between HSPs and the proteins they regulate. HSPs enable organisms to develop resistance to heat stress when pre-exposed to sublethal temperatures. Numerous studies have shown that the accumulation of induced HSPs determines the heat tolerance of eukaryotic cells. HSPs possess antioxidant biological activity within cells, increasing the synthesis and release of endogenous antioxidants and providing strong resistance to stress. They can inhibit NADPH oxidase, a key enzyme in the production of oxygen free radicals, thereby reducing the production of oxygen free radicals through feedback mechanisms. HSPs can directly release and increase the levels of endogenous peroxidases such as superoxide dismutase (SOD), which catalyzes the dismutation of oxygen free radicals, thus eliminating them. HSPs participate in immune responses; high temperatures can enhance the phagocytic capacity of monocytes and macrophages, which are the host's first line of defense. During stress, these cells produce high concentrations of reactive oxygen species (ROS) and cytokines to induce HSP expression; conversely, HSPs can protect cells and tissues from inflammatory damage by inhibiting ROS and cytokines. HSPs can regulate apoptosis; many factors induce apoptosis, such as drugs, ROS, heavy metals, bacteria, and viruses. Studies have found that HSPs participate in multiple stages of the apoptosis pathway and play a crucial role. HSPs can also be used for environmental pollution monitoring; the production of HSPs during stress can be rapidly detected, making them a promising biomarker for environmental pollution monitoring. Numerous studies have shown that HSPs produced by organisms in contaminated soil and ocean can be used to study cell stress markers.

[0004] The use of oilfield additives in China is increasing year by year, and their composition is quite complex. A large number of chemical agents containing heavy metals are being used in the oil and gas extraction process. Meanwhile, it has been found that oily wastewater generated from petroleum extraction, processing, refining, and coking in the chemical industry is characterized by its wide range of sources, large volume, poor biodegradability, and high hazard. It mainly contains organic matter such as oils and soaps, 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. These metals are characterized by high stability, inherent toxicity, bioaccumulation, long decay periods, and difficulty in treatment. When heavy metals accumulate to a certain concentration in organisms, poisoning can occur.

[0005] Environmental samples from petrochemical contaminated sites (such as produced water, oilfield additives, and surrounding soil) contain heavy metals. Heavy metal pollution is characterized by its high hazard, persistence, and difficulty in remediation. It can eventually enter the human body through degradation and bioaccumulation in the food chain, and is difficult to eliminate from the body. Even low concentrations can disrupt normal physiological activities and seriously damage human health. Currently, chemical detection methods are relatively complete, enabling qualitative and quantitative detection of heavy metals. However, chemical methods can only obtain the composition and concentration of heavy metals in environmental samples and cannot directly reflect the toxic effects of heavy metals on organisms.

[0006] Quantitative real-time PCR (qPCR) is a method that uses fluorescent chemicals to measure the total amount of product after each polymerase chain reaction (PCR) cycle during DNA amplification. It is a method for quantitative analysis of specific DNA sequences in the sample using internal or external controls. Real-time PCR monitors the PCR process in real time using fluorescence signals. However, there are currently no reports on using qPCR technology to detect the biotoxicity of heavy metal pollutants in environmental samples from petrochemical contaminated sites. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems existing in the prior art and provide a primer set, a reagent kit, and their application, as well as a method for detecting the biotoxicity of environmental samples. This method is characterized by high sensitivity, speed, and high reproducibility, and can achieve early warning of heavy metal pollutants in environmental samples, especially indicating the presence of heavy metal pollutants.

[0008] To achieve the above objectives, the present invention provides a primer set comprising primer pairs for specifically amplifying the hsp70.3 gene and the mt2 gene, respectively, and primer pairs for specifically amplifying an internal reference gene;

[0009] The primer pair used for specific amplification of the hsp70.3 gene includes: 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;

[0010] Primer pairs used for specific amplification of the mt2 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6.

[0011] 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.1 and SEQ ID NO.2.

[0012] A second aspect of the present invention provides a kit comprising the primer set as described above.

[0013] Preferably, the kit further comprises a reverse transcription reaction mixture, a gDNA removal solution, a qPCR reaction mixture, a dye, and water.

[0014] Preferably, the reverse transcription reaction mixture contains reverse transcriptase, MgCl2, dNTPs, and Oligo(dT).

[0015] Preferably, the qPCR reaction mixture includes DNA polymerase, dNTPs, and MgCl2.

[0016] 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 hsp70.3 gene and the mt2 gene.

[0017] The fourth aspect of the present invention provides the application of the primer set as described above or the kit as described above in the detection of heavy metal pollutants in environmental samples.

[0018] The fifth aspect of the present invention provides the application of the primer set or kit described above in detecting the biotoxicity of environmental samples.

[0019] Preferably, the environmental sample is produced water from an oilfield, an oilfield additive, or an oilfield soil.

[0020] The sixth aspect of this invention provides a method for detecting biotoxicity of environmental samples based on RT-qPCR, the method comprising the following steps:

[0021] (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.

[0022] (2) The extracted total RNA was used as a template for reverse transcription to obtain cDNA. Then, using the cDNA as a template, the primer pairs in the primer set as described above were used as amplification primers for qPCR reaction.

[0023] (3) Calculate the relative expression fold of the target gene relative to the internal reference gene based on the CT value obtained from the qPCR reaction, and analyze the biotoxicity based on the relative expression fold.

[0024] Preferably, in step (1), the incubation conditions include: a temperature of 25-30°C and a time of 80-120 hours.

[0025] Preferably, in step (2), the reverse transcription conditions include: incubation at 40–60°C for 12–18 min, followed by heating at 82–88°C for 4–6 s.

[0026] The primer set provided by this invention contains primer pairs for specifically amplifying zebrafish genes hsp70.3 and mt2, respectively. This primer set enables the monitoring of changes in the content of hsp70.3 and mt2 in organisms. Since genes hsp70.3 and mt2 are highly sensitive to toxic exposure to heavy metal pollutants, they can provide early warning of the toxic effects of heavy metals in environmental samples. Therefore, this primer set can be applied to the detection of biotoxicity in environmental samples from petrochemical contaminated sites.

[0027] The method provided by this invention first incubates zebrafish embryos with environmental samples to be tested, obtaining surviving embryos and hatched larvae. Then, using primer pairs from the primer set provided by this invention as amplification primers, and based on RT-qPCR technology, the changes in the expression levels of genes hsp70.3 and mt2 in the organism after incubation treatment can be obtained. This allows for the analysis of the biotoxicity of the environmental samples and the early warning of heavy metal pollutants, especially indicating the presence of heavy metal pollutants in the environmental samples. Further analysis can be performed in conjunction with heavy metal detection. This method selects hsp70.3 and mt2, genes that are highly sensitive to heavy metal pollutants, as target genes, resulting in a small number of target genes, simple operation, and high sensitivity. It is suitable for high-throughput detection of environmental samples, is rapid, and highly reproducible. By using a two-step RT-qPCR method, a stable cDNA library that can be stored for a long time and used for multiple reactions can be established. By selecting zebrafish genes with high homology to human genes as target genes, the toxic effects of environmental samples on humans can be better reflected. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of some of the reagents used in the detection method provided by the present invention;

[0029] Figure 2 This is a graph showing the relative fold increase of target genes when detecting produced water from an oilfield in Example 1.

[0030] Figure 3 This is a graph showing the relative fold increase of target genes during the three-phase separation of produced water from an oilfield, as shown in Example 2.

[0031] Figure 4 This is a graph showing the relative fold increase of the target gene when detecting water samples from a certain oilfield before water injection in Example 3;

[0032] Figure 5 This is a graph showing the relative fold increase of target genes when detecting an active adjuvant (room temperature gel plugging agent) in an oilfield in Example 4;

[0033] Figure 6 This is a graph showing the relative fold increase of the target gene when detecting an active adjuvant (pressure-reducing and injection-enhancing surfactant) in an oilfield in Example 5;

[0034] Figure 7 This is a graph showing the relative fold increase of target genes when soil samples from a residential area surrounding a petrochemical zone were tested in Example 6.

[0035] Figure 8 This is a graph showing the relative fold expression of target genes when soil samples from the green belt surrounding a petrochemical zone were tested in Example 7.

[0036] Figure 9 This is a graph showing the relative fold increase of the target gene when detecting the heavy metal zinc in Example 8;

[0037] Figure 10 This is a graph showing the relative fold increase of the target gene when detecting lead in Example 9;

[0038] Figure 11 This is a graph showing the relative fold increase of the target gene mt2 when detecting the heavy metal cadmium in Example 10;

[0039] Figure 12 This is a graph showing the relative fold increase of the target gene hsp70.3 when detecting the heavy metal cadmium in the test case;

[0040] Figure 13 This is a graph showing the relative fold increase of target genes when testing the biotoxicity of an environmental sample (wastewater from a textile industry) in a test case. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] Currently, chemical detection methods are relatively well-established, enabling qualitative and quantitative detection of heavy metals. However, these methods can only determine the composition and concentration of heavy metals in environmental samples, and cannot directly reflect the toxic effects of heavy metals on organisms. Therefore, to maximize the protection of susceptible populations from heavy metal hazards, this invention provides a primer set that can be used to monitor changes in the levels of HSP70.3 and MT2 in organisms. Based on these changes, the level of heavy metal exposure and the degree of heavy metal pollution in organisms can be predicted.

[0044] In this invention, the primer set includes primer pairs for specifically amplifying the hsp70.3 gene and the mt2 gene, respectively, and primer pairs for specifically amplifying the internal reference gene.

[0045] The primer pair used to detect the hsp70.3 gene includes: 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;

[0046] Primer pairs used for detecting the mt2 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6.

[0047] In this invention, zebrafish are used as a model organism to analyze the toxic effects of pollutants from petrochemical contaminated sites on zebrafish. Zebrafish have 87% genome homology with human genes and possess advantages such as ease of rearing, high reproductive capacity, and rapid development. Therefore, selecting zebrafish for research can better reflect the toxic effects of pollutants on humans, enabling early detection and early prevention.

[0048] In this invention, the design process of specific primers includes the following steps:

[0049] Step S10: Screen for pathways and genes related to the toxic effects of heavy metal pollutants on zebrafish and embryos, and verify the expression and endpoint information of the gene to be designed. After identifying the target gene, search for the relevant gene name in the NCBI gene database, copy the coding region of the target gene, and design primers for the target gene using Primer Premier5 software. The following principles should be followed when designing primers: Melting temperature (Tm): The Tm value of the primer should be 55–60℃, and the difference in Tm between the two primers in the primer pair should be 2–3℃; Length: To maximize binding properties, the primer length should be 18–30 nucleotides; Secondary structure: Avoid any inverted repeat sequences in the primers, as inverted repeat sequences can form stable hairpin structures, preventing effective primer binding; Avoid genomic DNA: In real-time fluorescent RT-PCR experiments, false positives can occur due to the amplification of genomic DNA. Therefore, primers should be designed on the flanking parts of long introns or multiple short introns, or across exon-exon junctions. GC content: The primer GC content should be around 50%; Sequence: The last five bases at the 3' end of each primer should contain one G or C. A single G or C at the 3' end can reduce non-specific amplification during PCR. However, too many Gs or Cs at the 3' end may cause the primer to bind non-specifically to the target site, ultimately leading to erroneous extension. The 3' end of the primer pair should not contain any complementary sequences that could lead to primer dimer formation.

[0050] Step S20: The designed primers were validated and evaluated using Oligo software. Evaluation indicators included a Tm generally around 60℃; primer GC content between 40% and 60%, with minimal difference in GC content between upstream and downstream primers; and an absolute value of ΔG at the 3' end not exceeding 4.5 kcal / mol. The primer pair with the highest score was selected, and homologous sequence searches were performed in NCBI Blast to ensure primer specificity. Finally, cross-exon validation was performed using the Ensembl database. Gene primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the PAGE method. Transcriptome sequencing identified the key detoxification metabolism genes most sensitive to heavy metal pollutant toxicity exposure: hsp70.3 and mt2.

[0051] In this invention, two key target genes for detoxification and metabolism, hsp70.3 and mt2, with high sensitivity were selected and the operation is simple. Two pairs of specific primers were designed for these two genes. The primer set containing these two pairs of specific primers can be used for early warning of heavy metal pollutants and is suitable for preliminary screening of samples in complex environments. It can be further combined with heavy metal detection for analysis in the later stage.

[0052] In one specific embodiment, the primer pair used for specifically amplifying the internal reference gene has upstream and downstream primer nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0053] In a preferred embodiment, the primer sequences, product lengths, and annealing temperatures of the two target genes and one internal reference gene (β-actin) in the primer set are shown in Table 1 below.

[0054] Table 1

[0055]

[0056] The present invention also proposes a kit comprising the primer set as described above.

[0057] In a preferred embodiment, the kit further comprises a reverse transcription reaction mixture, a gDNA removal solution, a qPCR reaction mixture, dye, and water. The design of these components enables the kit to be used for RT-qPCR (real-time quantitative polymerase chain reaction). It is understood that each component in the kit is stored separately, and each primer pair in the primer set is stored separately.

[0058] In the kit described in this invention, the water is RNase-free water.

[0059] In the kit described in this invention, preferably, the reverse transcription reaction mixture contains reverse transcriptase, MgCl2, dNTPs, and Oligo(dT).

[0060] In the kit described in this invention, preferably, the qPCR reaction mixture includes DNA polymerase, dNTPs, and MgCl2.

[0061] Among them, dNTP (deoxyribonucleoside triphosphate) refers to the collective term for four types of deoxyribonucleoside triphosphates (dATP, dGTP, dTTP and dCTP).

[0062] This invention also proposes the application of the primer set or kit described above in detecting the expression levels of the hsp70.3 and mt2 genes. By observing changes in the expression levels of the hsp70.3 and mt2 genes, early warning of pollutants can be provided, especially indicating the possible presence of heavy metal pollutants. Further analysis can be conducted in conjunction with heavy metal detection in the later stages.

[0063] The present invention also proposes the application of the primer set or kit described above in the detection of heavy metal pollutants in environmental samples.

[0064] The present invention also proposes the application of the primer set or kit described above in the detection of biotoxicity in environmental samples.

[0065] In the application described in this invention, the specific type of environmental sample is not limited, and it can originate from petrochemical contaminated sites. In a specific embodiment, the environmental sample is oilfield produced water, oilfield additives, or oilfield soil. Oilfield soil refers to soil surrounding a petrochemical area, such as soil from a residential area or greenbelt surrounding a petrochemical area.

[0066] This invention also proposes a method for detecting biotoxicity of environmental samples based on RT-qPCR, the method comprising the following steps:

[0067] (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.

[0068] (2) The extracted total RNA was used as a template for reverse transcription to obtain cDNA. Then, using the cDNA as a template, the primer pairs in the primer set as described above were used as amplification primers for qPCR reaction.

[0069] (3) Calculate the relative expression fold of the target gene relative to the internal reference gene based on the CT value obtained from the qPCR reaction, and analyze the biotoxicity based on the relative expression fold.

[0070] This invention employs a relatively quantitative detection method to analyze the expression levels of hsp70.3 and mt2 genes in zebrafish, which have high homology with humans after exposure to heavy metal pollution. By analyzing changes in gene expression levels, early warning of heavy metal pollutants in environmental samples can be provided, especially indicating the possible presence of heavy metal pollutants. Further analysis can be conducted in conjunction with heavy metal detection in subsequent stages.

[0071] In step (1), zebrafish embryos are subjected to toxicity exposure. Understandably, zebrafish embryos viable at the 4-128 cell stage are selected for the toxicity exposure experiment.

[0072] In this invention, the specific steps of step (1) can be carried out with reference to "HJ 1069-2019 Determination of Acute Toxicity of Water by Zebrafish Egg Method", "OECD 236 Fish Embryo Acute Toxicity (FET) Test" and "GB / T 21807-2008 Short-term Toxicity Test of Chemicals in Fish Embryos and Yolk Sac Larvae", such as the selection of zebrafish embryos and the preparation of the sample solution to be tested.

[0073] In this invention, step (1) involves preparing the sample solution to be tested by diluting the environmental sample to be tested with a blank diluent.

[0074] 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.

[0075] In the method described in this invention, step (2) is a two-step RT-qPCR, that is, reverse transcription and qPCR are performed in two steps. By choosing the two-step RT-qPCR, a stable cDNA library can be established for multiple qPCR reactions, realizing the long-term preservation of experimental samples; at the same time, the two-step method can optimize the reaction buffer and reaction conditions of a single reaction process, making the results more sensitive and accurate.

[0076] In this invention, step (2) is performed using the kit described in this invention.

[0077] In a specific implementation, step (2) includes:

[0078] A1. Using the extracted total RNA as a template, reverse transcription was performed in reverse transcription reaction buffer to obtain cDNA;

[0079] A2. Using the cDNA as a template, perform a qPCR reaction in qPCR reaction buffer.

[0080] Please refer to the following: Figure 1 In a specific implementation, in step A1, the reverse transcription (i.e., reverse transcription) reaction buffer contains a reverse transcription reaction mixture, a gDNA removal solution, and RNase-free water.

[0081] In a specific implementation, step A2 involves a qPCR reaction buffer containing a qPCR reaction mixture, a reference dye, and primers. The primers are either β-actin upstream / downstream primers, HSP70 upstream / downstream primers, or MT2 upstream / downstream primers.

[0082] In a preferred embodiment, the reverse transcription conditions in step (2) include: incubation at 40–60°C for 12–18 min, followed by heating at 82–88°C for 4–6 s.

[0083] In a preferred embodiment, the conditions for the qPCR reaction in step (2) include: heating at 94°C for 30 seconds, and then entering the cycling stage for 40 to 45 cycles, wherein each cycle consists of 5 seconds at 94°C and 30 seconds at 58 to 62°C.

[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, step (3), the process of calculating the relative expression fold of the target gene relative to the internal reference gene based on the CT value includes: ΔCt = target gene Ct - reference gene Ct, ΔΔCt = ΔCt of the test sample - ΔCt of the blank sample, and 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 the environmental sample exposure interferes with gene expression in the organism. The larger the relative expression fold, the greater the biotoxicity of the environmental sample and the greater the possibility of heavy metal pollutants in the environmental sample.

[0087] In the method described in this invention, zebrafish genes hsp70.3 and mt2 are selected as environmental monitoring markers. Based on real-time quantitative PCR technology, the relative expression fold (i.e., expression level change) of target genes hsp70.3 and mt2 relative to the internal reference gene β-actin can be calculated. The change in the expression level of target genes can reflect the toxic effects of heavy metal pollutant exposure on the key metabolic genes hsp70.3 and mt2, thereby enabling the analysis of biotoxicity caused by heavy metal pollutants in environmental samples.

[0088] The method provided by this invention has a small number of target genes, is simple to operate, and has high sensitivity. It can perform high-throughput analysis of the biotoxicity of environmental samples, is rapid and highly reproducible, and has broad application prospects. It can be applied to the optimization of oilfield additives, the improvement of wastewater treatment technology, the assessment of the biotoxicity and ecotoxicity of pollutants, and to provide a basis for guiding the remediation of heavy metal contaminated sites. Therefore, this invention can provide technical support for the risk monitoring of heavy metal ions and can also provide new methods and ideas for research in other related fields.

[0089] 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.

[0090] In the following examples, the components and contents of the kit used to perform two-step RT-qPCR are shown in Table 2.

[0091] Table 2

[0092]

[0093] In Table 2, the nucleotide sequences of the primers (internal reference gene β-actin, target genes hsp70.3 and mt2) are shown in SEQ ID NO.1 to SEQ ID NO.6.

[0094] Example 1

[0095] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (produced water from an oilfield) based on the primer set or kit described in this invention.

[0096] Environmental samples to be tested: Select a sampling site in an oil field and use a clean sampling bottle to collect water samples from the sampling site (Fe 1.583 mg / L, As 0.2 mg / L, Ba 31.93 mg / L).

[0097] The collected incoming water samples were stored at 4℃ and transported to the laboratory for freezing at -20℃ within 24 hours. Before testing, the incoming water samples were equilibrated at 26℃ and aerated for 30 minutes to ensure that the initial dissolved oxygen concentration was not lower than 4 mg / L, thus obtaining the water sample to be tested. The sample was diluted immediately before use.

[0098] Test sample solution: Take 40 mL of water sample and dilute it to 1000 mL with blank diluent;

[0099] Blank dilution (i.e. blank sample): CaCl2 294 mg / L, MgSO4 123.25 mg / L, NaHCO3 63 mg / L, KCl 5.5 mg / L, balance water;

[0100] (1) Toxicity exposure and RNA extraction in zebrafish embryos

[0101] The following day, after the daylighting begins, open the spawning box cover, remove the inner tank, discard the water from the outer tank, and then place 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, avoiding 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 eggs from each spawning tank, rinse them with blank diluent, and transfer them to crystallization dishes. After standing in a constant temperature incubator or room for 45 minutes, observe the crystallization dishes with a black background and a side light. In each crystallization dish, remove unfertilized, damaged, deformed, and dead eggs to avoid harming them.

[0102] The selected zebrafish embryos were placed in 24-well plates, with one zebrafish embryo and 2 mL of the test sample solution or blank diluent added to each well. The plates were incubated at 28°C for 96 hours, and the surviving embryos and hatched larvae were collected.

[0103] Total RNA was extracted from surviving embryos and larvae collected after hatching 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 or larvae were rinsed twice with pre-cooled distilled water. The embryos or larvae 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 tube 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 tube 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.

[0104] (2) Two-step RT-qPCR

[0105] Two-step RT-qPCR was performed using the kits described in Table 2, and according to... Figure 1 The reagents shown were used for reverse transcription and qPCR reactions, respectively.

[0106] S1, reverse transcription

[0107] Using the extracted total RNA as a template, a reverse transcription reaction was performed to obtain cDNA. The reverse transcription reaction system consisted of 200 ng of total RNA, 100 μL of reverse transcription reaction mixture, 100 μL of gDNA removal solution, and 2 mL of RNase-free water. The reverse transcription reaction conditions were: incubation at 42°C for 15 minutes (cDNA synthesis temperature range is 42-60°C), followed by heating at 85°C for 5 seconds.

[0108] S2, qPCR reaction

[0109] The cDNA synthesized in step S1 was used as a template for qPCR reaction;

[0110] The qPCR reaction system includes: 2 μL of cDNA, 100 μL of qPCR reaction mixture, 100 μL of reference dye, 0.4 μL of upstream primer and 0.4 μL of downstream primer; specific amplification reaction systems for hsp70.3 gene, mt2 gene and internal reference gene are prepared in different qPCR reaction tubes respectively.

[0111] The qPCR reaction conditions are: first heat at 94℃ for 30s, then enter the cycling phase for 40-45 cycles, with each cycle consisting of 5s at 94℃ and 30s at 60℃.

[0112] (3) Data Analysis

[0113] Using the β-actin 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 = Target gene Ct - Internal reference gene Ct, ΔΔCt = ΔCt of the test sample - ΔCt of the blank sample, and the expression ratio = 2. -ΔΔCt .

[0114] Test results are as follows Figure 2 As shown, the results indicated that the relative expression folds of the target genes hsp70.3 and mt2 relative to the internal reference gene β-actin were 12.15 and 5.71, respectively, suggesting the possible presence of heavy metal pollutants in the environmental samples. This is consistent with the composition analysis results of the incoming water (Fe 1.583 mg / L, As 0.2 mg / L, Ba 31.93 mg / L), indicating that this method has an early warning effect on heavy metal pollutants.

[0115] Example 2

[0116] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (three-phase separated water from produced water in an oilfield) based on the primer set or kit described in this invention.

[0117] The method described in Example 1 was implemented, except that the environmental sample to be tested was: a three-phase separated water (Fe 1.647 mg / L, As 0.43 mg / L, Ba 36.65 mg / L) was collected from a certain oil field sampling site using a clean sampling bottle.

[0118] The results of this embodiment are as follows: Figure 3As shown, the results indicate that the relative expression folds of the target genes hsp70.3 and mt2 relative to the internal reference gene β-actin were 13.62 and 6.23, respectively. These folds were slightly higher than those in the incoming water sample, consistent with the slight increase in heavy metal content in the three-phase separated effluent (Fe 1.647 mg / L, As 0.43 mg / L, Ba 36.65 mg / L) observed in the physicochemical tests. It should be noted that the three-phase separator separates oil, gas, and water. Considering only the physical process, the effluent should show a decrease in heavy metal content due to the reduction in oil and gas content. However, the heavy metal content increased here, which may be related to oilfield additives such as demulsifiers added during the three-phase separation stage to improve separation efficiency.

[0119] Example 3

[0120] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (water samples from an oilfield produced water before injection) based on the primer set or kit described in this invention.

[0121] The method described in Example 1 was implemented, except that the environmental sample to be tested was: a water sample (Fe 1.156 mg / L, As 0.935 mg / L, Ba 29.09 mg / L) was collected from a certain collection site in an oil field before water injection using a clean sampling bottle.

[0122] The results of this embodiment are as follows: Figure 4 As shown, the results indicate that the relative expression folds of the target genes hsp70.3 and mt2 relative to the internal reference gene β-actin were 10.42 and 4.11, respectively, suggesting the presence of heavy metals in the pollutants. Furthermore, the pollutant content was reduced compared to before treatment, which is consistent with the results of physicochemical analysis and demonstrates the early warning function of this method.

[0123] Example 4

[0124] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (currently used adjuvants in an oilfield) based on the primer set or kit described in this invention.

[0125] The method described in Example 1 was implemented, except that the environmental sample to be tested was: an active additive in an oilfield (a room-temperature gel plugging agent, purchased from Huabin Chemical, containing the following components by mass fraction: 0.5% polyacrylamide; 0.1% chromium chloride; 0.25% crosslinking agent; and 99.15% water).

[0126] The results of this embodiment are as follows: Figure 5As shown, the results indicated that the relative expression folds of the target genes hsp70.3 and mt2 relative to the internal reference gene β-actin were 19.15 and 10.75, respectively. The upregulation of target gene expression suggests that this adjuvant interferes with the body's digestive metabolism and indicates that the adjuvant contains metals. This is consistent with the trivalent chromium contained in the adjuvant manufacturer's ingredient list (0.5% polyacrylamide; 0.1% chromium chloride; 0.25% crosslinking agent; 99.15% water), which proves the early warning function of this method.

[0127] Example 5

[0128] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (currently used adjuvants in an oilfield) based on the primer set or kit described in this invention.

[0129] The method described in Example 1 was implemented, except that the environmental sample to be tested was: Shengli Oilfield's existing additive (pressure reducing and injection enhancing surfactant, purchased from Dongying Lifeng, containing the following components by mass fraction: alkylphenol polyoxyethylene ether 20%; fluorocarbon surfactant 3%; sodium alkylphenol polyoxyethylene ether sulfonate 5%; chromium chloride 0.2%; water 71.8%).

[0130] The results of this embodiment are as follows: Figure 6 As shown, the results indicate that the relative expression folds of the target genes hsp70.3 and mt2 relative to the internal reference gene β-actin were 21.73 and 12.02, respectively, both showing high expression levels. This suggests that a certain antihypertensive and injection-enhancing surfactant interferes with the body's detoxification metabolism and indicates that the adjuvant contains metals. According to the ingredient list provided by the adjuvant manufacturer (alkylphenol polyoxyethylene ether 20%; fluorocarbon surfactant 3%; sodium alkylphenol polyoxyethylene ether sulfonate 5%; chromium chloride 0.2%; water 71.8%), the adjuvant in this example contains trivalent chromium, which is consistent with the test results obtained by this method.

[0131] Example 6

[0132] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (soil from a residential area surrounding a petrochemical zone) based on the primer set or kit described in this invention.

[0133] The method described in Example 1 was implemented, except that the environmental samples to be tested were as follows: soil samples were collected from residential areas surrounding a petrochemical zone. During soil sample collection, at 3 to 5 points within a 100m radius of the sampling point, the topsoil was first scraped off with a clean bamboo spoon, and then a 20cm deep soil column was collected. Impurities were removed, and the sample was placed in a clean soil collection bag, resulting in approximately 1kg of mixed soil sample. After the collected soil sample was naturally air-dried, it was ground and reduced in size. The heavy metal content in the soil sample was determined to be Cd 0.187mg / kg, Pb 21.6mg / kg, and Zn 48.3mg / kg. The sample solution to be tested (i.e., the prepared soil extract) was prepared by adding 100ml of blank diluent to 100g of soil, stirring, and filtering with filter paper.

[0134] The results of this embodiment are as follows: Figure 7 As shown, the relative expression levels of metallothionein MT2 and heat shock protein HSP70.3 were 5.54 and 2.16, respectively. The upregulated expression of both target genes indicates the presence of heavy metals in the sample, consistent with the physicochemical analysis results (Cd 0.187 mg / kg, Pb 21.6 mg / kg, Zn 48.3 mg / kg), further validating the early warning function of this method. It also suggests that the surrounding residential areas may be affected by the petrochemical industry.

[0135] Example 7

[0136] This embodiment illustrates a method for detecting the biotoxicity of environmental samples (soil from a greenbelt surrounding a petrochemical zone) based on the primer set or kit described in this invention.

[0137] The method described in Example 1 was implemented, except that the environmental sample to be tested was: soil samples were collected from the green belt surrounding a petrochemical zone. During soil sample collection, at 3 to 5 points within a 100m radius of the sampling point, the surface soil was first scraped off with a clean bamboo spoon, and then a 20cm deep soil column was collected. The debris was removed and the sample was placed in a clean soil collection bag, resulting in a mixed soil sample of approximately 1kg. After the collected soil sample was naturally air-dried, it was ground and reduced in size. The heavy metal content in the soil sample was determined to be Cd 0.152mg / kg, Pb 15.9mg / kg, and Zn 39.75mg / kg. The sample solution to be tested (i.e., the prepared soil extract) was obtained by mixing 100g of soil with 100ml of blank diluent and filtering with filter paper.

[0138] The results of this embodiment are as follows: Figure 8As shown, the relative expression levels of metallothionein MT2 and heat shock protein HSP70.3 were 3.12 and 1.78, respectively, which were lower than those in soil samples from residential areas. This is consistent with the results of physicochemical analysis (Cd 0.152 mg / kg, Pb 15.9 mg / kg, Zn 39.75 mg / kg) showing a decrease in heavy metal content.

[0139] Example 8

[0140] This embodiment illustrates a method for detecting the biotoxicity of heavy metal zinc based on the primer set or kit described in this invention.

[0141] The method described in Example 1 was implemented, except that the sample to be tested was zinc sulfate; the sample solution to be tested was prepared by diluting 150 μg of zinc sulfate with 10 ml of blank diluent.

[0142] The results of this embodiment (i.e., the experiment in which zinc sulfate, a standard chemical, was selected as the test substance and zebrafish embryos were exposed) are as follows: Figure 9 As shown, the results indicated that the relative expression levels of metallothionein MT2 and heat shock protein HSP70.3 were 21.86 and 17.01, respectively, indicating that the entry of heavy metal zinc into zebrafish embryos induces high expression of the key genes MT2 and HSP70.3, verifying the sensitivity of this method.

[0143] Example 9

[0144] This embodiment illustrates a method for detecting the biotoxicity of heavy metal lead based on the primer set or kit described in this invention.

[0145] The method described in Example 8 was implemented, except that the sample to be tested was lead sulfate, and the sample solution was prepared by diluting 350 μg of lead sulfate with 10 ml of blank diluent.

[0146] The results of this embodiment are as follows: Figure 10 As shown, the results indicated that the relative expression levels of metallothionein MT2 and heat shock protein HSP70.3 were 19.01 and 15.72, respectively, suggesting that lead in zebrafish embryos induces high expression of key genes involved in metabolism, thus verifying the sensitivity of this method.

[0147] Test case

[0148] This test case is used to evaluate the sensitivity and specificity of the detection method provided by the present invention.

[0149] 1. Sensitivity

[0150] Taking the detection of the biotoxicity of the heavy metal cadmium as an example, the sensitivity of the primer set, reagent kit and detection method described in this invention is explained.

[0151] Detection method: The method described in Example 8 was followed, except that the sample to be tested was cadmium chloride. CdCl2·2.5H2O was dissolved in blank diluent to obtain exposure solutions with Cd concentrations of 0 μg / L, 10 μg / L, 100 μg / L, 1000 μg / L, and 2000 μg / L, respectively.

[0152] The results of this embodiment are as follows: Figure 11 and Figure 12 As shown, the relative expression levels of metallothionein mt2 and heat shock protein hsp70.3 were 2.6 and 2.1 at a Cd concentration of 10 μg / L, respectively; 5.8 and 3.9 at a Cd concentration of 100 μg / L; 13.6 and 9.43 at a Cd concentration of 1000 μg / L; and 25.16 and 18.24 at a Cd concentration of 2000 μg / L. This suggests that cadmium, after entering zebrafish embryos, induces high expression of key genes involved in decomposition and metabolism, and its toxicity is higher than that of lead and zinc. Existing research results show that the content of metallothionein in the hepatopancreas of oysters is significantly increased at a Cd concentration of 100 μg / L, while in this method, the relative expression level of metallothionein mt2 was already increased at a Cd concentration of 10 μg / L, indicating that the primer set and detection method provided in this invention have good sensitivity.

[0153] 2. Specificity

[0154] Taking the detection of biotoxicity in an environmental sample (wastewater from a textile industry) as an example, the specificity of the primer set, reagent kit, and detection method described in this invention is illustrated.

[0155] Detection method: The method described in Example 1 was followed, except that the environmental sample to be tested was cotton textile wastewater, mainly composed of cotton lint and sizing agents. Sample solution to be tested: 100 mL of water sample was diluted to 1000 mL with blank diluent and filtered through filter paper.

[0156] The results of this embodiment are as follows: Figure 13 As shown, the relative expression folds of the target genes hsp70.3 and mt2 relative to the internal reference gene β-actin were 1.21 and 0.92, respectively, with no significant changes, indicating that the primer set and detection method provided by this invention have good specificity.

[0157] 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 combinations of 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 in that, The primer set includes primer pairs for specifically amplifying the hsp70.3 gene and the mt2 gene, respectively, and primer pairs for specifically amplifying the internal reference gene; The primer pair used for specific amplification of the hsp70.3 gene includes: 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; Primer pairs used for specific amplification of the mt2 gene include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

6.

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.1 and SEQ ID NO.

2.

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

4. The reagent kit according to claim 3, characterized in that, The kit also includes a reverse transcription reaction mixture, gDNA removal solution, qPCR reaction mixture, dye, and water.

5. The reagent kit according to claim 4, characterized in that, The reverse transcription reaction mixture contains reverse transcriptase, MgCl2, dNTPs, and Oligo(dT).

6. The reagent kit according to claim 4, characterized in that, The qPCR reaction mixture includes DNA polymerase, dNTPs, and MgCl2.

7. The use of the primer set according to claim 1 or 2 or the kit according to any one of claims 3-6 in detecting the expression levels of the hsp70.3 gene and the mt2 gene.

8. The use of the primer set according to claim 1 or 2 or the kit according to any one of claims 3-6 in the detection of heavy metal pollutants in environmental samples.

9. The use of the primer set according to claim 1 or 2 or the kit according to any one of claims 3-6 in detecting the biotoxicity of environmental samples.

10. The application according to claim 9, characterized in that, The environmental samples are produced water, oilfield additives, or oilfield soil.

11. A method for detecting biotoxicity of environmental samples based on RT-qPCR, 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, reverse transcription is performed to obtain cDNA. Then, using the cDNA as a template, qPCR is performed using the primer pairs in the primer set described in claim 1 or 2 as amplification primers. (3) Calculate the relative expression fold of the target gene relative to the internal reference gene based on the CT value obtained from the qPCR reaction, and analyze the biotoxicity based on the relative expression fold.

12. The method according to claim 11, characterized in that, In step (1), the incubation conditions include: a temperature of 25-30°C and a time of 80-120 hours.

13. The method according to claim 11, characterized in that, In step (2), the reverse transcription conditions include: first incubation at 40-60℃ for 12-18 min, and then heating at 82-88℃ for 4-6 s.

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