PCR (Polymerase Chain Reaction) chip and kit for detecting expression quantity of functional genes related to microbial degradation of chlorinated hydrocarbon and application of PCR chip and kit

Abstract

The invention relates to the technical field of molecular biology and environmental biology, and discloses a PCR (Polymerase Chain Reaction) chip and a kit for detecting the expression quantity of functional genes related to microbial degradation of chlorinated hydrocarbon and application of the PCR chip and the kit. The PCR chip comprises a primer pair and a primer pair, the primer pair is used for specifically amplifying dehalogenation functional genes bvcA, cfrA, mbrA, pceA, pteA, tceA and vcrA respectively, the primer pair is used for specifically amplifying reference genes, and nucleotide sequences of the primer pair for specifically amplifying the dehalogenation functional genes are shown as SEQ ID NO. 1 to SEQ ID NO. 14. The PCR chip provided by the invention can specifically amplify seven functional genes related to the degradation of the chlorinated hydrocarbon microorganisms, is good in specificity, high in accuracy and good in repeatability, and can accurately detect the expression condition of the seven dehalogenation functional genes related to the degradation of the chlorinated hydrocarbon microorganisms through a qPCR technology, so that the existence condition of chlorinated hydrocarbon degrading bacteria can be reflected; the method has reliability in the aspects of chlorohydrocarbon polluted site biodegradability evaluation, degradation process research, repair effect verification and the like.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and environmental biotechnology, specifically to a PCR chip, kit, and application for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons. Background Technology

[0002] Chlorinated hydrocarbon solvents (CHS) are compounds in which the hydrogen atoms of a hydrocarbon molecule are replaced by chlorine. These include chlorinated alkanes, chlorinated alkenes, and chlorinated aromatics. As important organic solvents and intermediates, they are widely used in chemical, pharmaceutical, and pesticide industries. These compounds are hydrophobic, lipophilic, and slowly biodegradable, and persist in groundwater and sediments. Chlorinated hydrocarbons accumulate in the food chain, posing a long-term threat to humans and ecosystems. Chlorinated hydrocarbons and their degradation products contain acute toxins, carcinogens, mutagens, and endocrine disruptors, seriously endangering human health and ecological safety.

[0003] Chlorinated hydrocarbons are a class of pollutants commonly found in environmental media. Their entry into the environment mainly includes: production processes; entry as chemical intermediates during use; entry as impurities of downstream products; entry through sudden accidents or improper handling and disposal, such as waste incineration and its dumping grounds; entry through direct use, such as chlorobenzene and hexachlorobenzene entering the environment directly as pesticides; and the natural formation of low-chlorinated hydrocarbons through the degradation of high-chlorinated benzenes in nature. For example, hexachlorobenzene can be converted to pentachlorobenzene in the natural environment, and tetrachloroethylene and trichloroethylene can be degraded into dichloroethylene and vinyl chloride, etc.

[0004] For contaminated groundwater, it is particularly important to adopt appropriate remediation technologies. Commonly used remediation technologies include physical, chemical, and microbial remediation techniques.

[0005] Physical removal technologies include gas-phase extraction and adsorption, but chlorinated hydrocarbons cannot be degraded into environmentally harmless substances through physical removal alone; further effective methods are needed to degrade chlorinated hydrocarbons. Chemical remediation technologies mainly include chemical oxidation and reduction. This method requires the application of large amounts of strong oxidizing agents or substances with high reducing power, making it the preferred method for areas with complex pollution, but it carries the risk of secondary pollution.

[0006] Bioremediation technologies include phytoremediation and microbial remediation. Microbial remediation, which utilizes the growth and metabolic activities of microorganisms to degrade chlorinated hydrocarbons, is a widely used method characterized by its green and economical nature. Based on the nutrient requirements of the microorganisms, it is mainly divided into aerobic and anaerobic methods. Under aerobic conditions, chlorinated hydrocarbons can provide the necessary energy for microbial growth, or they can undergo co-metabolic reactions with various pollutants such as toluene, xylene, and phenol to produce carbon dioxide and water. Under anaerobic conditions, chlorinated hydrocarbons are gradually reduced and dechlorinated. Deep soil and groundwater are inherently favorable anaerobic environments; therefore, anaerobic microbial reduction is a promising bioremediation method.

[0007] Current research on microbial degradation and remediation technologies goes beyond macroscopic issues such as reaction conditions, removal efficiency, and intermediate products. It needs to delve into the microscopic realm, exploring the genes and regulatory mechanisms by which microorganisms control organic matter degradation. Further analysis of the dynamic changes in the number and population structure of microorganisms within the reaction system is crucial to understanding the relationship between organic matter degradation and microorganisms. This will allow for more effective analysis of the feasibility of microbial bioremediation, monitoring of the remediation process, adjustment of remediation protocols, and comparison of remediation effectiveness. This requires the assistance of techniques such as molecular biology and genetic engineering.

[0008] Currently, commonly used techniques for analyzing microbial community structure mainly include 16S rRNA sequencing, metagenomic sequencing, and quantitative real-time PCR (qPCR). 16S rRNA is a highly conserved gene segment in bacteria and archaea, exhibiting a degree of variability. Sequencing 16S rRNA genes can identify different genera and species within a microbial community and assess their relative abundance. Metagenomic sequencing analyzes the total DNA of the entire microbial community in environmental samples, elucidating the relationship between microbial function and the environment, and identifying and studying specific functional genes and metabolic pathways. It not only provides information on the diversity and abundance of the microbial community but also allows for the clustering and analysis of functional genes. qPCR technology utilizes changes in fluorescence signals to detect changes in the amount of amplification product in each cycle of the PCR amplification reaction. Qualitative analysis is performed on the corresponding gene in the test template by analyzing the Ct value relationship between the internal reference gene and the target gene.

[0009] Compared to qPCR, which has a clearly defined target gene, sequencing technology may face the challenge of not detecting results due to low abundance of bacterial genus / species in the community. Furthermore, 16S rRNA sequencing primarily targets community species and is insufficient for functional gene studies, while metagenomic sequencing mainly targets community DNA and cannot detect the expression of functional genes. Total RNA sequencing can compensate for this deficiency, but it still faces the challenge of sequencing sensitivity.

[0010] Currently, the main focus of qPCR technology is on using it to study the microbial community structure of contaminated sites (i.e., designing primers for target bacterial species that can degrade the pollutant, and then detecting the number of the target bacterial species) to assess the possibility of microbial degradation of pollutants in contaminated sites. However, the detection methods for microbial community structure are cumbersome and difficult to detect all bacterial species that can degrade the pollutant, inevitably resulting in missed detections. Furthermore, the detected bacterial species may have issues with whether the corresponding reduction and dehalogenation genes are expressed, leading to poor specificity. Summary of the Invention

[0011] The purpose of this invention is to overcome the problems existing in the prior art and provide a PCR chip, kit, and application for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons. This PCR chip can detect common functional genes related to reductive dehalogenation and has high sensitivity and reliability in detecting the biodegradation of chlorinated hydrocarbon pollution.

[0012] To achieve the above objectives, the present invention provides a PCR chip for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons. The PCR chip includes primer pairs for specifically amplifying the dehalogenation functional genes bvcA, cfrA, mbrA, pceA, pteA, tceA, and vcrA, and primer pairs for specifically amplifying the internal reference gene.

[0013] The nucleotide sequences of the upstream and downstream primers of the primer pair used for specific amplification of the bvcA gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0014] The primer pair used for specific amplification of the cfrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0015] The primer pair used for specific amplification of the mbrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0016] The primer pair used for specific amplification of the pceA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0017] The primer pair used for specific amplification of the pteA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0018] The primer pair used for specific amplification of the tceA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0019] The primer pair used for specific amplification of the vcrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0020] Preferably, the primer pair used for specific amplification of the internal reference gene has the sequences of the upstream and downstream primers as shown in SEQ ID NO.15 and SEQ ID NO.16.

[0021] Preferably, the PCR chip further includes a chip plate, wherein each primer pair exists individually in the detection well of the chip plate.

[0022] A second aspect of the present invention provides a kit comprising a PCR chip as described above.

[0023] The third aspect of the present invention provides the application of the PCR chip or the kit described above in detecting the expression levels of seven dehalogenation functional genes related to the microbial degradation of chlorinated hydrocarbons.

[0024] The fourth aspect of the present invention provides the application of the PCR chip or the kit described above in the detection of chlorinated hydrocarbon degrading bacteria.

[0025] The fifth aspect of the present invention provides the application of the PCR chip or the kit described above in assessing the microbial degradation capacity of chlorinated hydrocarbon contaminated sites.

[0026] The sixth aspect of the present invention provides the application of the PCR chip or the kit described above in assessing the feasibility of microbial remediation of chlorinated hydrocarbon contaminated sites.

[0027] The seventh aspect of this invention provides a method for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons, the method comprising the following steps:

[0028] A1. Collect the bacterial community in the sample to be tested and extract the total RNA of the bacterial community;

[0029] A2. Using the total RNA as a template, reverse transcription was performed to obtain cDNA;

[0030] A3. Using the cDNA as a template, qPCR reaction is performed using the PCR chip described above to obtain the Ct values ​​of the dehalogenated functional gene and the internal reference gene.

[0031] A4. Calculate the relative expression fold of the dehalogenation functional gene relative to the internal reference gene based on the Ct value.

[0032] An eighth aspect of the present invention provides a method for assessing the microbial degradation capacity of a site contaminated with chlorinated hydrocarbons, the method comprising the following steps:

[0033] (1) Collect water samples from the contaminated site and collect the microbial community in the water samples or treated water samples;

[0034] (2) Extract total RNA from the bacterial community and use the total RNA as a template for reverse transcription to obtain cDNA;

[0035] (3) Using the cDNA as a template, qPCR reaction was performed using the PCR chip described above to obtain the Ct values ​​of the dehalogenated functional gene and the internal reference gene.

[0036] (4) Calculate the relative expression fold of the dehalogenation functional gene relative to the internal reference gene based on the Ct value, and evaluate the degradation ability of microorganisms in the contaminated site for chlorinated hydrocarbons by the relative expression fold.

[0037] Preferably, the reverse transcription process includes: mixing the total RNA, reverse transcription reaction mixture, gDNA removal agent and RNase-free water to form a reverse transcription reaction system, performing a reverse transcription reaction to obtain cDNA.

[0038] Preferably, the conditions for the reverse transcription reaction include: incubation at 40–45°C for 12–18 min, and heating at 82–88°C for 4–6 s.

[0039] Preferably, the conditions for the qPCR reaction include: heating at 90–95°C for 5–10 seconds, followed by reaction at 55–65°C for 20–60 seconds, for 40–60 cycles.

[0040] The PCR chip provided by this invention can specifically amplify seven functional genes related to the microbial degradation of chlorinated hydrocarbons (including pceA, cfrA, pteA, tceA, and mbrA genes related to the dehalogenation degradation of high-halogenated compounds, and vcrA and bvcA genes related to the dehalogenation degradation of low-halogenated compounds). It exhibits high specificity, accuracy, and reproducibility. Using qPCR technology, this PCR chip can accurately detect the expression of these seven dehalogenation functional genes related to the microbial degradation of chlorinated hydrocarbons, thereby reflecting the presence of chlorinated hydrocarbon-degrading bacteria. It is reliable for assessing the biodegradation capacity of chlorinated hydrocarbon-contaminated sites, studying the degradation process, and verifying remediation effectiveness. Therefore, using this PCR chip, the expression of functional genes related to the microbial degradation of chlorinated hydrocarbons can be accurately detected, the feasibility of bioremediation of chlorinated hydrocarbon pollution can be explored, the bioremediation process of chlorinated hydrocarbon pollution can be evaluated, and further guidance can be provided for improving bioremediation methods for chlorinated hydrocarbon pollution.

[0041] The detection method provided by this invention utilizes the PCR chip, which features high sensitivity through the selection of target genes, is suitable for high-throughput amplification / screening, and is rapid and highly reproducible. This method can detect functional genes related to chlorinated hydrocarbon degradation in various environmental media and cultures. Attached Figure Description

[0042] Figure 1 This shows the arrangement of primer pairs used for specific amplification of various genes on the PCR chip plate.

[0043] Figure 2 This refers to changes in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples collected from petrochemical plants.

[0044] Figure 3 This is the melting curve diagram from Example 1;

[0045] Figure 4 The changes in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples collected from petrochemical plants and enriched and cultured for 1 day were observed.

[0046] Figure 5 The changes in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples collected from petrochemical plants and enriched for 5 days;

[0047] Figure 6 The changes in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples collected from petrochemical plants and enriched for 10 days;

[0048] Figure 7 The change in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples cultured for 1 day after stimulation with 50 mg / L dichloroethane;

[0049] Figure 8 The change in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples cultured for 5 days after stimulation with 50 mg / L dichloroethane;

[0050] Figure 9 The change in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples enriched and cultured for 10 days with 50 mg / L dichloroethane;

[0051] Figure 10 The change in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples enriched and cultured for 1 day with 50 mg / L tetrachloroethane;

[0052] Figure 11 The change in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples cultured for 5 days after stimulation with 50 mg / L tetrachloroethane;

[0053] Figure 12 The change in the expression levels of functional genes related to the degradation of chlorinated hydrocarbons in water samples enriched and cultured for 10 days with 50 mg / L tetrachloroethane. Detailed Implementation

[0054] 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.

[0055] 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.

[0056] This invention provides a PCR chip for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons. The PCR chip includes primer pairs for specifically amplifying the dehalogenation functional genes bvcA, cfrA, mbrA, pceA, pteA, tceA, and vcrA, and primer pairs for specifically amplifying the internal reference gene.

[0057] The nucleotide sequences of the upstream and downstream primers of the primer pair used for specific amplification of the bvcA gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0058] The primer pair used for specific amplification of the cfrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0059] The primer pair used for specific amplification of the mbrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0060] The primer pair used for specific amplification of the pceA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0061] The primer pair used for specific amplification of the pteA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0062] The primer pair used for specific amplification of the tceA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0063] The primer pair used for specific amplification of the vcrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0064] The PCR chip provided by this invention can detect seven functional genes related to the bioreduction and dehalogenation of chlorinated hydrocarbons, namely pceA, cfrA, pteA, tceA and mbrA genes related to the dehalogenation degradation of high-halogenated hydrocarbons, and vcrA and bvcA genes related to the dehalogenation of low-halogenated hydrocarbons, thereby enabling the assessment of the biodegradation potential and stage of chlorinated hydrocarbons in contaminated sites.

[0065] The PCR chip described in this invention delves into the microscopic realm of exploring genes and regulatory mechanisms that control the degradation of chlorinated hydrocarbons by microorganisms. It can accurately detect the expression of functional genes related to the degradation of chlorinated hydrocarbons by microorganisms, explore the feasibility of bioremediation of chlorinated hydrocarbon pollution, evaluate the bioremediation process of chlorinated hydrocarbon pollution, and further assist in guiding the improvement of bioremediation methods for chlorinated hydrocarbon pollution.

[0066] In a specific implementation, the primer pair used for specifically amplifying the internal reference gene (16S rRNA) has the sequences of its upstream and downstream primers as shown in SEQ ID NO.15 and SEQ ID NO.16.

[0067] In this invention, the design process of primer pairs for specifically amplifying seven functional genes related to the bioreduction and dehalogenation of chlorinated hydrocarbons includes the following steps:

[0068] First, after reviewing relevant domestic and international literature on the biodegradation of chlorinated hydrocarbons and testing actual polluted water samples, dehalogenation functional genes related to the stepwise dehalogenation degradation process of different chlorinated hydrocarbons were screened, and the expression status 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, and the coding region of the target gene was copied. Since different species of microorganisms may have isoenzyme genes, all genes with the same name were screened out. Sequence comparison was performed using DNAMAN, and fragments with high base sequence similarity were selected. Corresponding primers were designed using Primer Premier 5 software, and the designed primers were verified and evaluated using Oligo software to avoid problems such as hairpin structures and primer dimers. After confirming that the primers had no quality issues, homologous sequence search was performed in the NCBI Blast module to ensure the specificity of the primers, and the annealing temperature of the primers was ensured to be around 60℃. Finally, primers that met the above requirements for the target gene were selected. Primer pairs with sharp single peaks in melting curves were screened by qPCR preliminary experiments, and primers with single bands in PCR products were screened by gel electrophoresis experiments, thus obtaining primer pairs that specifically amplify 7 functional genes related to bioreduction dehalogenation.

[0069] In a specific implementation, the primer sequences, product lengths, annealing temperatures, and sequence numbers of the 7 bioreduction dehalogenation-related functional genes and 1 internal reference gene (16S rRNA) involved in the PCR chip are detailed in Table 1 below.

[0070] Table 1

[0071]

[0072] In a specific implementation, 0.8 μL of the upstream and downstream primers of the dehalogenase gene and the internal reference gene are added to the qPCR reaction tubes, respectively. The well openings can be temporarily sealed with a heat-sensitive sealing film and stored at 4°C for later use (only suitable for short-term storage, such as within 7 days).

[0073] In a specific implementation, the PCR chip further includes a chip plate, with each primer pair existing individually in a detection well of the chip plate. More specifically, the chip plate can be a 96-well plate.

[0074] Please refer to the following: Figure 1 In one specific implementation, the PCR chip can simultaneously perform qPCR experiments with three biological replicates at each of four test sites. Through the design of the PCR chip, it can acquire the expression status of chlorinated hydrocarbon degradation functional genes at multiple sampling points or sampling times in a single experiment, thereby reflecting the chlorinated hydrocarbon degradation status.

[0075] The PCR chip provided by this invention is simple to operate and features seven highly sensitive target genes, making it suitable for preliminary screening and degradation studies of samples from complex environments, and suitable for widespread application. This PCR chip can be used for feasibility assessment of in-situ enhanced bioremediation of chlorinated hydrocarbon-contaminated sites, tracking of chlorinated hydrocarbon biodegradation processes in contaminated sites, evaluation of the effectiveness of chlorinated hydrocarbon bioremediation, and improvement of chlorinated hydrocarbon bioremediation technologies, providing data and theoretical support for chlorinated hydrocarbon bioremediation.

[0076] The present invention also proposes a kit comprising the PCR chip described above.

[0077] In a specific embodiment, the kit may further include reagents for performing qPCR reactions. This invention does not limit the specific components and ratios of the reagents used for qPCR reactions; they can be commonly used qPCR reagents in the art.

[0078] In other embodiments, the kit may also contain reagents for performing reverse transcription.

[0079] The present invention also proposes the application of the PCR chip or the kit described above in detecting the expression levels of seven dehalogenation functional genes related to the microbial degradation of chlorinated hydrocarbons.

[0080] This invention also proposes the application of the PCR chip or kit described above in the detection of chlorinated hydrocarbon-degrading bacteria. Chlorinated hydrocarbon-degrading bacteria refer to microorganisms capable of degrading chlorinated hydrocarbons. By using the aforementioned PCR chip or kit, the expression levels of functional genes related to chlorinated hydrocarbon biodegradation in the sample can be detected, thereby enabling the assessment of the presence of chlorinated hydrocarbon-degrading bacteria.

[0081] The present invention also proposes the application of the PCR chip or the kit described above in assessing the microbial degradation capacity of chlorinated hydrocarbon contaminated sites. By using the PCR chip or kit described above, the expression levels of functional genes related to the biodegradation of chlorinated hydrocarbons in the test sample can be detected, thereby enabling the analysis of the microbial degradation capacity of chlorinated hydrocarbon contaminated sites.

[0082] The present invention also proposes the application of the PCR chip or the kit described above in assessing the feasibility of microbial remediation of chlorinated hydrocarbon contaminated sites. By using the PCR chip or kit described above, the expression of chlorinated hydrocarbon biodegradation functional genes can be detected, thereby assessing the feasibility of microbial remediation of chlorinated hydrocarbon contaminated sites.

[0083] This invention also proposes a method for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons, the method comprising the following steps:

[0084] A1. Collect the bacterial community in the sample to be tested and extract the total RNA of the bacterial community;

[0085] A2. Using the total RNA as a template, reverse transcription was performed to obtain cDNA;

[0086] A3. Using the cDNA as a template, qPCR reaction is performed using the PCR chip described above to obtain the Ct values ​​of the dehalogenated functional gene and the internal reference gene.

[0087] A4. Calculate the relative expression fold of the dehalogenation functional gene relative to the internal reference gene based on the Ct value.

[0088] By detecting the relative expression fold of dehalogenation functional genes in the test sample relative to the internal reference gene, we can indicate the presence of chlorinated hydrocarbon degrading bacteria in the test sample, explore the feasibility of bioremediation of chlorinated hydrocarbon pollution, evaluate the bioremediation process of chlorinated hydrocarbon pollution, and further assist in guiding the improvement of chlorinated hydrocarbon pollution bioremediation methods.

[0089] In the method described in this invention, in step A1, the sample to be tested can be an environmental sample or its culture from a contaminated site. Specifically, it can be a water sample from the contaminated site, or a culture of the water sample from the contaminated site. The water sample culture refers to an enrichment culture at approximately 30°C for 1-10 days by artificially adding appropriate nutrients and stress pollutants (such as organic components like beef extract, peptone, and yeast powder, inorganic salt ions, electron donors like acetic acid and sodium acetate, and chlorinated hydrocarbon components like 1,2-dichloroethane) to increase the microbial content.

[0090] In a preferred embodiment, step A2 includes the following steps: mixing the total RNA, reverse transcription reaction mixture, gDNA remover, and RNase-free water to form a reverse transcription reaction system, performing a reverse transcription reaction, and obtaining cDNA.

[0091] In one specific embodiment, in step A2, the reaction materials used for the reverse transcription reaction are provided by TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit.

[0092] In a preferred embodiment, the conditions for the reverse transcription reaction in step A2 include: incubation at 40–45°C for 12–18 min, and heating at 82–88°C for 4–6 s.

[0093] In a preferred embodiment, the conditions for the qPCR reaction in step A3 include: heating at 90–95°C for 5–10 seconds, followed by reaction at 55–65°C for 20–60 seconds, and repeating 40–60 times.

[0094] In this invention, primer pairs for specific gene amplification are individually present in each well of the PCR chip, so that specific amplification of each gene is performed in different wells. In a specific embodiment, step A3 includes: adding cDNA, qPCR reaction mixture, passive reference dye, and nuclease-free water to each well of the PCR chip containing primer pairs to form a reaction system, and then performing a qPCR reaction.

[0095] In one specific implementation, in step A3, the reaction materials used in the qPCR reaction are provided by the PerfectStart Green qPCR SuperMix kit from TransGen.

[0096] In a specific implementation, 2 is adopted. -ΔΔCT The relative fold increase of the target gene can be calculated using the following method:

[0097] ΔCt=Ct 目的基因 -Ct 内参基因

[0098] ΔΔCt=ΔCt 待测样品 -ΔCt 参比(对照)

[0099] Relative expression ratio = 2 -ΔΔCt

[0100] Therefore, in this embodiment, the method further includes: using the above method to detect the Ct values ​​of the target gene and the internal reference gene of the reference sample (control sample).

[0101] This invention also proposes a method for assessing the biodegradability of sites contaminated with chlorinated hydrocarbons, the method comprising the following steps:

[0102] (1) Collect water samples from the contaminated site and collect the microbial community in the water samples or treated water samples;

[0103] (2) Extract total RNA from the bacterial community and use the total RNA as a template for reverse transcription to obtain cDNA;

[0104] (3) Using the cDNA as a template, qPCR reaction was performed using the PCR chip described above to obtain the Ct values ​​of the dehalogenated functional gene and the internal reference gene.

[0105] (4) Calculate the relative expression fold of the dehalogenation functional gene relative to the internal reference gene based on the Ct value, and evaluate the degradation ability of microorganisms in the contaminated site for chlorinated hydrocarbons by the relative expression fold.

[0106] By detecting the relative expression fold of dehalogenation functional genes in water samples relative to internal reference genes, it is possible to indicate the presence of chlorinated hydrocarbon degrading bacteria in the water samples, assess the possibility and stage of chlorinated hydrocarbon biodegradation in water bodies, and evaluate the feasibility of in-situ enhanced bioremediation of contaminated sites.

[0107] In a specific implementation, in step (1), the treated water sample can be enriched and cultured to increase the microbial content.

[0108] In a preferred embodiment, step (2) includes the following steps: mixing the total RNA, reverse transcription reaction mixture, gDNA remover, and RNase-free water to form a reverse transcription reaction system, performing a reverse transcription reaction, and obtaining cDNA.

[0109] In one specific implementation, the reverse transcription reaction is performed using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit from TransGen Biotech, where the reverse transcription mixture refers to TransScript All-in-One SuperMix for qPCR.

[0110] In a preferred embodiment, the conditions for the reverse transcription reaction in step (2) include: incubation at 40-45°C for 12-18 min and heating at 82-88°C for 4-6 s.

[0111] In a specific implementation, step (3) includes: adding cDNA, qPCR reaction mixture, reference dye and nuclease-free water to each detection well of a PCR chip plate containing primer pairs to form a reaction system, and then performing a qPCR reaction.

[0112] In a preferred embodiment, the conditions for the qPCR reaction in step (3) include: heating at 90-95°C for 5-10 seconds, followed by reaction at 55-65°C for 20-60 seconds, and repeating 40-60 times.

[0113] In a specific implementation, 2 is adopted. -ΔΔCT The relative fold increase of the target gene is calculated using the above method. Therefore, in this embodiment, the method further includes: detecting the Ct values ​​of the target gene and the internal reference gene in the reference sample (control sample) using the above method.

[0114] The present invention does not limit the specific selection of the control sample, and it can be selected according to the actual sample to be tested. In one specific embodiment, the control sample is laboratory zebrafish culture water.

[0115] 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.

[0116] The preparation method of the culture medium used in the following examples is as follows:

[0117] Preparation of TAP Inorganic Salt Medium: Each 1L of medium contains 25mL of salt solution #1, 0.375mL of salt solution #2, 1mL of salt solution #3, 2.42g / L Tris, and 1mL of glacial acetic acid, with the remainder being water. Prepare the medium according to the above formula, then adjust the pH to 7.0 and sterilize at 121℃ for 20 minutes. Specifically, salt solution #1 contains: 15g / L NH4Cl, 4g / L MgSO4·7H2O, and 2g / L CaCl2·2H2O; salt solution #2 contains: 28.8g / L K2HPO4 and 14.4g / L KH2PO4; salt solution #3 contains: 200g / L sodium EDTA, 220g / L ZnSO4·7H2O, 57g / L H3BO3, 101.2g / L MnCl2·4H2O, and 6H2O CoCl2·6H2O. 32.2g / L, CuSO4·5H2O 31.4g / L, (NH4)6Mo7O 24 ·4H2O 22g / L, FeSO4·7H2O 99.8g / L.

[0118] Preparation of LB medium: Each 1L of medium contains 10g / L tryptone, 5g / L yeast extract, 10g / L NaCl, and the remainder is water. Prepare the medium according to the above formula, then adjust the pH to 7.0 and sterilize at 121℃ for 20 minutes.

[0119] Example 1

[0120] This embodiment illustrates a method for detecting the expression levels of functional genes related to microbial degradation of chlorinated hydrocarbons in a test sample (a petrochemical water sample) using the PCR chip described in this invention.

[0121] (1) Collect the bacterial flora and extract total RNA from the test samples and control samples.

[0122] Collect bacterial flora from test samples and control samples.

[0123] A sampling point was selected at a petrochemical plant, and water samples (containing 1,2-dichloropropene at a concentration of 300 ppb) were collected using clean sampling bottles. Samples were sealed and stored at 4°C and transported to the laboratory for refrigeration at 4°C within 24 hours. Glass filter bottles were treated with DEPC water and then autoclaved for later use. Millipore 0.22 μm filter membranes were autoclaved for later use. Before testing, the water samples were removed from the refrigerator and filtered. One filter membrane was collected for every 1 L of water. The sterile edge of the filter membrane was removed, and the membrane was divided into four equal pieces and frozen at -80°C for later use.

[0124] Before RNA extraction, rinse a bacterial filter membrane with enzyme-free water to ensure complete rinsing, and then centrifuge at 7000 rpm / min to collect the bacterial cells and obtain the bacterial community in the water sample.

[0125] Using laboratory zebrafish culture water as a control, the bacterial flora in the laboratory zebrafish culture water was collected according to the above method.

[0126] RNA extraction

[0127] Total RNA was extracted from bacterial colonies on the filter membrane 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 centrifuging, 100 μL of sterile PBS solution or enzyme-free water was added to the bacterial cells, and the cells were dispersed and mixed homogeneously by vortexing or pipetting. Then, 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 13000g 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 30 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.

[0128] (2) Reverse transcription

[0129] Using the extracted total RNA as a template, reverse transcription was performed using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit from TransGen. The reverse transcription reaction system is shown in Table 2 below (total RNA can be added according to the actual amount extracted, and then RNase-free water is used to make the total reaction system 20 μL). This kit can simultaneously complete gDNA elimination and reverse transcription. The reaction conditions are: incubation at 42°C for 15 minutes and heating at 85°C for 5 seconds.

[0130] Table 2

[0131]

[0132]

[0133] (3) qPCR reaction

[0134] Using cDNA obtained from reverse transcription as a template, qPCR reaction was performed using a PCR chip;

[0135] The primer pairs used for specific amplification of each gene are arranged in the following order on the PCR chip: Figure 1 As shown in Table 1, the primer sequences for the functional genes and internal reference genes are shown in Table 1. Each detection well of the PCR chip plate contains 20 μL of qPCR reaction system.

[0136] The qPCR reaction system was prepared according to the instructions using the PerfectStart Green qPCR SuperMix kit from TransGen Biotech AQ601. The reaction system is shown in Table 3 below.

[0137] This experiment used the Roche LC480 qPCR instrument. The qPCR amplification program was as follows: heating at 95℃ for 3 minutes, quantitative detection (heating at 94℃ for 5 seconds, followed by reaction at 60℃ for 30 seconds, for a total of 45 cycles), and melting curve (heating at 95℃ for 15 seconds, reaction at 65℃ for 1 minute, immediately increasing the temperature to 95℃, and reaction at 40℃ for 10 seconds).

[0138] Table 3

[0139] Reagent composition volume cDNA template Variables (as needed) Forward Primer (10μM) 0.4μL (0.2μM Final Concentration) Reverse Primer (10μM) 0.4μL (0.2μM Final Concentration) 2×PerfectStart Green qPCR SuperMix 10μL Passive Reference Dye (50×) 0.4μL Nuclease-free Water Variables (as needed) Total volume 20μL

[0140] (4) Data Analysis

[0141] After the experiment, the Analysis method was selected, and the Abs Quant / 2nd Der analysis method was used to calculate the Ct value of each detection well. The laboratory zebrafish culture water was used as a reference (control) without chlorinated hydrocarbon pollution. The relative expression fold of the dehalogenation functional gene relative to the internal reference gene 16S rRNA was calculated by the Ct value.

[0142] The formula and method for calculating relative expression multiples are as follows:

[0143] ΔCt=Ct 目的基因 -Ct 内参基因

[0144] ΔΔCt=ΔCt 待测样品 -ΔCt 参比(对照)

[0145] Relative expression ratio = 2 -ΔΔCt

[0146] A relative fold increase of >1 indicates activation of gene expression, with gene expression increasing / upregulated; a relative fold increase of <1 indicates inhibition of gene expression, with gene expression decreasing / downregulated.

[0147] The test results of relative expression fold are as follows Figure 2As shown, the melting curves of the genes involved (a total of 8 genes including the target gene and the internal reference gene) are as follows. Figure 3 As shown.

[0148] Depend on Figure 2 It can be seen that pceA, cfrA, tceA, vcrA, and bvcA were not detected, while mbrA and pteA showed trace increases in expression. This indicates that the water sample from the petrochemical plant contains genes capable of dehalogenation degradation. It also suggests that the water sample was recently contaminated with chlorinated hydrocarbons and that the contaminated flora contained species capable of degrading chlorinated hydrocarbons.

[0149] Depend on Figure 3 As can be seen, the melting curve temperature range of the genes involved in the detection is normal, and the curve peaks are sharp single peaks, confirming the specificity of gene amplification. Under normal conditions without inhibitor interference, if there is only one copy of the target nucleic acid in the detection well, the Ct value is approximately 38. However, in this experiment, some genes had low expression levels, while other primer pairs showed normal amplification. Considering that this water sample was collected from water sources, there are many uncontrollable factors such as microbial content, types, and inhibitors. Therefore, the experimental sensitivity can be considered high.

[0150] Example 2

[0151] This embodiment illustrates a method for detecting the expression levels of functional genes related to microbial degradation of chlorinated hydrocarbons in a test sample (a water sample enriched and cultured for 1 day from a petrochemical plant) using the PCR chip described in this invention.

[0152] The method described in Example 1 was followed, except that the steps for collecting the microbial community in the test sample and control sample included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloropropene content of 300 ppb) from that sampling point using a clean sampling bottle; sealing and storing the samples at 4°C and transporting them to the laboratory for refrigeration at 4°C within 24 hours; enriching the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L resazurine sodium salt to 10 mL of water sample and enriching at 30°C for 1 day to obtain the test sample. Using the water sample filter membrane from Example 1 as a control, the expression changes of functional genes related to chlorinated hydrocarbon biodegradation were analyzed in the water samples cultured with the added medium. After 24 hours, the liquid changed from light blue to orange-red, indicating that a micro-anaerobic environment had been formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (sample 2.1 PPT), pH (sample 7.1), and ORP (sample 63) were measured. The collected bacterial cells were frozen at -80℃ for later use.

[0153] The test results of this embodiment are as follows: Figure 4As shown, the results indicated that the expression levels of the pceA, cfrA, tceA, vcrA, bvcA, mbrA, and pteA genes all changed to varying degrees, suggesting that the water sample cultured with appropriate culture medium contained bacteria capable of dehalogenation degradation. Except for the decreased expression level of tceA, the expression levels of the other six genes increased. This is because the results of Example 1 indicated that the water sample was recently contaminated with chlorinated hydrocarbons, affecting the indigenous bacterial community, which contains species capable of degrading chlorinated hydrocarbons. The color change of the added resazan after 1 day of enrichment culture suggests that microorganisms are rapidly consuming oxygen, forming a micro-anaerobic environment. The significantly increased content of dehalogenation-degrading bacteria in the water sample with added culture medium indicates that these bacteria can produce oxygen-consuming growth and rapidly carry out degradation.

[0154] Example 3

[0155] This embodiment illustrates a method for detecting the expression levels of functional genes related to microbial degradation of chlorinated hydrocarbons in a test sample (a water sample collected from a petrochemical plant and enriched for 5 days) using the PCR chip described in this invention.

[0156] The method described in Example 1 was followed, except that the steps for collecting the microbial community in the test sample and control sample included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloropropene content of 300 ppb) from that point using a clean sampling bottle. The samples were sealed and stored at 4°C and transported to the laboratory for refrigeration at 4°C within 24 hours. Enrichment culture was performed on the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L resazurine sodium salt to 10 mL of the water sample. Enrichment culture was carried out at 30°C for 5 days to obtain the test sample. Using the water sample filter membrane from Example 1 as a control, the expression changes of functional genes related to chlorinated hydrocarbon biodegradation in the water samples cultured with the added culture medium were analyzed. After 5 days, the liquid changed from the initial light blue to orange-red and then showed a very weak red color at 5 days. It was slightly darker and more turbid than the original culture medium, indicating that a strong micro-anaerobic environment or near-anaerobic environment had been formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (sample 2.0 PPT), pH (sample 7.2), and ORP (sample -77) were measured. The collected bacterial cells were frozen at -80℃ for later use.

[0157] The test results of this embodiment are as follows: Figure 5As shown, the results indicated that the expression levels of the pceA, cfrA, tceA, vcrA, bvcA, mbrA, and pteA genes all changed to varying degrees, indicating that the water sample cultured with appropriate culture medium contained bacteria capable of dehalogenation degradation. Except for tceA, whose expression level remained unchanged, the expression levels of the other six genes increased. This is because the results of Example 1 already suggested that the water sample was recently contaminated with chlorinated hydrocarbons, affecting the indigenous bacterial community, which contains species capable of degrading chlorinated hydrocarbons. In Example 2, after 1 day of enrichment culture, the color change of the added azurite indicated that microorganisms were rapidly consuming oxygen, forming a micro-anaerobic environment. In this example, after 5 days of culture, the anaerobic environment was further enhanced, indicating that the expression of relevant degradation genes by dehalogenation degrading bacteria in the water sample with added culture medium was more significantly increased. These bacteria are capable of oxygen-consuming growth and can rapidly carry out degradation.

[0158] Example 4

[0159] This embodiment illustrates a method for detecting the expression levels of functional genes related to microbial degradation of chlorinated hydrocarbons in a test sample (a water sample enriched and cultured for 10 days from a petrochemical plant) using the PCR chip described in this invention.

[0160] The method described in Example 1 was followed, except that the steps for collecting the bacterial communities in the test samples and control samples included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloropropene content of 300 ppb) from that point using a clean sampling bottle; sealing and storing the samples at 4°C and transporting them to the laboratory for refrigeration at 4°C within 24 hours; enriching the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of water sample and enriching at 30°C for 10 days to obtain the test samples. Using the water sample filter membrane from Example 1 as a control, the expression changes of functional genes related to chlorinated hydrocarbon biodegradation in the water samples cultured with the added medium were analyzed. After 10 days, the liquid changed from the initial light blue to orange-red and finally to a turbid light yellow, indicating that a strong micro-anaerobic environment or basically anaerobic environment had been formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (sample 2.1 PPT), pH (sample 7.2), and ORP (sample -188) were measured. The collected bacterial cells were frozen at -80℃ for later use.

[0161] The test results of this embodiment are as follows: Figure 6As shown, the results indicated that, except for tceA, the expression levels of pceA, cfrA, vcrA, bvcA, mbrA, and pteA genes were all elevated to varying degrees, indicating that the water sample cultured with appropriate culture medium contained bacteria capable of dehalogenation degradation. Since the results of Example 1 already suggested that the water sample was recently contaminated with chlorinated hydrocarbons and that this contamination affected the indigenous bacterial community, which contained species capable of degrading chlorinated hydrocarbons, the color changes of the added azurite in Examples 2 and 3, through enrichment culture, suggested that microorganisms were rapidly consuming oxygen, gradually forming a near-anaerobic environment. In this example, after 10 days of culture, the anaerobic environment was further enhanced, showing a turbid pale yellow color, indicating that the expression of relevant degradation genes by the dehalogenation-degrading bacteria in the water sample with added culture medium was more significantly elevated. These bacteria are capable of oxygen-consuming growth and can rapidly carry out degradation.

[0162] Example 5

[0163] This embodiment illustrates a method for detecting changes in the expression levels of functional genes related to chlorinated hydrocarbon degradation in a test sample (a water sample enriched and cultured with 50 mg / L dichloroethane for 1 day) using the PCR chip described in this invention.

[0164] The method described in Example 1 was followed, except that the steps for collecting the bacterial communities in the test samples and control samples included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloroethane content 300 ppb) from that point using clean sampling bottles; sealing and storing the samples at 4°C and transporting them to the laboratory for refrigeration at 4°C within 24 hours; enriching the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, 250 μg / L sodium resazurin, and 10 mg / L or 50 mg / L 1,2-dichloroethane to 10 mL of the water sample, and enriching at 30°C for 1 day. The bacterial culture enriched with 10 mg / L 1,2-dichloroethane was used as a control, and the expression changes of functional genes related to chlorinated hydrocarbon biodegradation were analyzed in the water sample enriched with 50 mg / L 1,2-dichloroethane. One day later, the liquid changed from light blue to orange-red, indicating that a micro-anaerobic environment had been formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (control sample 2.0 PPT, test sample 2.1 PPT), pH (control sample 7.1, test sample 7.1), and ORP (control sample 80, test sample 77) were measured. The collected bacterial cells were frozen and stored at -80℃ for later use.

[0165] The test results of this embodiment are as follows: Figure 7As shown, the results indicated that the pceA, cfrA, tceA, vcrA, bvcA, mbrA, and pteA genes all exhibited varying degrees of high expression, suggesting that the addition of 50 mg / L 1,2-dichloroethane significantly promoted the expression of dehalogenation-related functional genes in the water sample compared to the addition of 10 mg / L 1,2-dichloroethane. This is because the results of Example 1 suggest that the water sample was recently contaminated with chlorinated hydrocarbons, affecting the indigenous microbial community, which contains species capable of degrading chlorinated hydrocarbons. After 1 day of enrichment culture, the color change of the added azure indicated that microorganisms were consuming oxygen, creating a micro-anaerobic environment. Combined with the qPCR results, it can be concluded that the content of dehalogenation-degrading bacteria (chlorinated hydrocarbon-degrading bacteria) in the water sample with 50 mg / L 1,2-dichloroethane was also significantly increased, indicating that these bacteria not only tolerate this concentration of dichloroethane but can also rapidly degrade it.

[0166] Example 6

[0167] This embodiment illustrates a method for detecting changes in the expression levels of functional genes related to chlorinated hydrocarbon degradation in a test sample (a water sample enriched and cultured with 50 mg / L dichloroethane for 5 days) using the PCR chip described in this invention.

[0168] The method described in Example 1 was followed, except that the steps for collecting the bacterial communities in the test samples and control samples included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloroethane content ≥300 ppb) from that point using clean sampling bottles; sealing and storing the samples at 4°C and transporting them to the laboratory for refrigeration at 4°C within 24 hours; enriching the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, 250 μg / L sodium resazurin, and 10 mg / L or 50 mg / L 1,2-dichloroethane to 10 mL of the water sample, and enriching at 30°C for 5 days. The bacterial culture enriched with 10 mg / L 1,2-dichloroethane was used as a control, and the expression changes of functional genes related to chlorinated hydrocarbon biodegradation were analyzed in the water sample enriched with 50 mg / L 1,2-dichloroethane. Five days later, the liquid changed from an initial pale blue to a slightly darker pale yellow, almost the same as LB itself, indicating that an anaerobic environment had been almost formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (control sample 2.0 PPT, test sample 1.7 PPT), pH (control sample 7.1, test sample 6.9), and ORP (control sample -80, test sample -79) were measured. The collected bacterial cells were frozen at -80℃ for later use.

[0169] The test results of this embodiment are as follows: Figure 8As shown, the results indicated that the pceA, cfrA, tceA, vcrA, bvcA, mbrA, and pteA genes all exhibited varying degrees of high expression, suggesting that compared to the addition of 10 mg / L 1,2-dichloroethane, 50 mg / L 1,2-dichloroethane significantly promoted the expression of dehalogenation-related functional genes in the water sample, with vcrA showing significantly high expression. This is because the results of Example 1 indicated that the water sample was recently contaminated with chlorinated hydrocarbons, impacting the indigenous microbial community, which contains species capable of degrading chlorinated hydrocarbons. After 5 days of enrichment culture, the color change of the added resazurite indicated that microorganisms were consuming oxygen, creating a near-anaerobic environment. Based on the qPCR results, it can be concluded that after culturing water samples with 50 mg / L 1,2-dichloroethane for 5 days, bacteria capable of dehalogenation and degradation proliferated in large numbers. Among them, vcrA, which performs vinyl chloride dehalogenation and degradation, was highly expressed. This indicates that these dehalogenation-degrading bacteria can not only tolerate this concentration of dichloroethane, but also rapidly proliferate and express vcrA to degrade dichloroethane.

[0170] Example 7

[0171] This embodiment illustrates a method for detecting changes in the expression levels of functional genes related to chlorinated hydrocarbon degradation in a test sample (a water sample enriched and cultured with 50 mg / L dichloroethane for 10 days) using the PCR chip described in this invention.

[0172] The method described in Example 1 was followed, except that the steps for collecting the bacterial communities in the test samples and control samples included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloroethane content 300 ppb) from that point using clean sampling bottles; sealing and storing the samples at 4°C and transporting them to the laboratory for refrigeration at 4°C within 24 hours; enriching the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, 250 μg / L sodium resazurin, and 10 mg / L or 50 mg / L 1,2-dichloroethane to 10 mL of the water sample and enriching for 10 days at 30°C. The bacterial culture enriched with 10 mg / L 1,2-dichloroethane was used as a control, and the expression changes of functional genes related to chlorinated hydrocarbon biodegradation were analyzed in the water sample enriched with 50 mg / L 1,2-dichloroethane. After 10 days, the liquid changed from an initial light blue to a turbid light yellow, indicating that an anaerobic environment had been formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (control sample 2.0 PPT, test sample 1.9 PPT), pH (control sample 7.1, test sample 6.9), and ORP (control sample -192, test sample -200) were measured. The collected bacterial cells were frozen at -80℃ for later use.

[0173] The test results of this embodiment are as follows: Figure 9 As shown, the results indicated that the pceA, cfrA, tceA, vcrA, bvcA, mbrA, and pteA genes all exhibited varying degrees of high expression, suggesting that the addition of 50 mg / L 1,2-dichloroethane significantly promoted the expression of dehalogenation-related functional genes in the water sample compared to the addition of 10 mg / L 1,2-dichloroethane. Similar to Example 6, vcrA showed extremely high expression, while the expression levels of pceA, cfrA, bvcA, mbrA, and pteA genes also significantly increased. Although the expression level of tceA increased, the change was not significant compared to Example 5. This is because the results of Example 1 suggested that the water sample was recently contaminated with chlorinated hydrocarbons, affecting the indigenous microbial community, which contained species capable of degrading chlorinated hydrocarbons. The color change of the added resazan after 10 days of enrichment culture indicated that microorganisms were consuming oxygen, creating an anaerobic environment. Based on the qPCR results, it can be concluded that after culturing water samples with 50 mg / L 1,2-dichloroethane for 10 days, the bacteria capable of dehalogenation degradation proliferated in large numbers, and the expression levels of genes related to vinyl chloride dehalogenation degradation increased. This indicates that these dehalogenation-degrading bacteria can not only tolerate this concentration of dichloroethane, but also rapidly proliferate and express the corresponding degradation genes to degrade dichloroethane.

[0174] Example 8

[0175] This embodiment illustrates a method for detecting changes in the expression levels of functional genes related to chlorinated hydrocarbon degradation in a test sample (a water sample enriched and cultured with 50 mg / L tetrachloroethane for 1 day) using the PCR chip described in this invention.

[0176] The method described in Example 1 was followed, except that the steps for collecting the bacterial communities in the test samples and control samples included: selecting a sampling point in a petrochemical plant, collecting water samples (1,2-dichloropropene content of 300 ppb) from that sampling point using a clean sampling bottle; sealing and storing the samples at 4°C, and transporting them to the laboratory for refrigeration at 4°C within 24 hours. Enrichment culture was performed on the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, 250 μg / L resazurin sodium salt, and 10 mg / L or 50 mg / L tetrachloroethane, and enriching at 30°C for 1 day. Using the bacterial culture enriched with 10 mg / L tetrachloroethane as a control, the expression of functional genes related to chlorinated hydrocarbon biodegradation was analyzed in the water sample enriched with 50 mg / L tetrachloroethane. One day later, the liquid changed from light blue to orange-yellow, indicating that a micro-anaerobic environment had been formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (control sample 2.0 PPT, test sample 2.1 PPT), pH (test control 7.1, control sample 7.1), and ORP (control sample 70, test sample 62) were measured. The collected bacterial cells were frozen and stored at -80℃ for later use.

[0177] The test results of this embodiment are as follows: Figure 10 As shown, the results indicated that, except for pceA, the gene expression levels of cfrA, tceA, vcrA, bvcA, mbrA, and pteA all changed to varying degrees. Among them, the expression levels of mbrA and pteA increased significantly, with pteA expression being higher than mbrA. This suggests that the water sample with added 50 mg / L tetrachloroethane contained bacteria capable of dehalogenation degradation. This is because the results of Example 1 indicated that the water sample was recently contaminated with chlorinated hydrocarbons, affecting the indigenous microbial community, which contained species capable of degrading chlorinated hydrocarbons. After 1 day of enrichment culture, the color change of the added azurite indicated that microorganisms were rapidly consuming oxygen, forming a micro-anaerobic environment. Furthermore, this demonstrates that compared to the 10 mg / L tetrachloroethane addition, the content of dehalogenation-degrading bacteria in the 50 mg / L tetrachloroethane addition water sample was significantly increased. qPCR results showed that these bacteria mainly degrade tetrachloroethane and trichloroethane, indicating that they not only tolerated this concentration of tetrachloroethane but also rapidly carried out degradation.

[0178] Example 9

[0179] This embodiment illustrates a method for detecting changes in the expression levels of functional genes related to chlorinated hydrocarbon degradation in a test sample (a water sample enriched and cultured with 50 mg / L tetrachloroethane for 5 days) using the PCR chip described in this invention.

[0180] The method described in Example 1 was followed, except that the steps for collecting the bacterial communities in the test samples and control samples included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloropropene content of 300 ppb) from that point using clean sampling bottles; sealing and storing the samples at 4°C and transporting them to the laboratory for refrigeration at 4°C within 24 hours; enriching the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, 250 μg / L sodium resazurin, and 10 mg / L or 50 mg / L tetrachloroethane to 10 mL of water sample and incubating at 30°C for 5 days. Using the bacterial culture enriched with 10 mg / L tetrachloroethane as a control, the expression of functional genes related to chlorinated hydrocarbon biodegradation was analyzed in the water sample enriched with 50 mg / L tetrachloroethane. Five days later, the liquid changed from its initial pale blue to a pale yellow almost like LB itself, indicating that an anaerobic environment had been basically formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (control sample 2.0 PPT, test sample 1.7 PPT), pH (control sample 7.1, test sample 6.9), and ORP (control sample -82, test sample -76) were measured. The collected bacterial cells were frozen at -80℃ for later use.

[0181] The test results of this embodiment are as follows: Figure 11 As shown, the results indicated that the gene expression levels of pceA, cfrA, tceA, vcrA, bvcA, mbrA, and pteA all changed to varying degrees. Among them, the expression levels of mbrA and pteA increased significantly, with mbrA expression being higher than pteA. This suggests that the water sample with added 50 mg / L tetrachloroethane contained bacteria capable of dehalogenation degradation. This is because the results of Example 1 indicated that the water sample was recently contaminated with chlorinated hydrocarbons, affecting the indigenous bacterial community, which contained species capable of degrading chlorinated hydrocarbons. After 5 days of enrichment culture, the color change of the added resazurite indicated that microorganisms were rapidly consuming oxygen, essentially forming an anaerobic environment. Meanwhile, it was shown that compared with the addition of 10 mg / L tetrachloroethane, the content of dehalogenating bacteria in the water sample with the addition of 50 mg / L tetrachloroethane was significantly increased, and the qPCR results showed that these bacteria mainly degraded tetrachloroethane and trichloroethane. Unlike Example 8, the expression level of mbrA was higher than that of pteA in this example, indicating that after several days of cultivation, the tetrachloroethane content in the water sample decreased while the trichloroethane content increased, suggesting that the bacterial community in the water sample was also undergoing corresponding changes. At the same time, these bacteria were not only able to tolerate this concentration of tetrachloroethane, but also able to carry out rapid degradation.

[0182] Example 10

[0183] This embodiment illustrates a method for detecting changes in the expression levels of functional genes related to chlorinated hydrocarbon degradation in a test sample (a water sample enriched and cultured with 50 mg / L tetrachloroethane for 10 days) using the PCR chip described in this invention.

[0184] The method described in Example 1 was followed, except that the steps for collecting the bacterial communities in the test samples and control samples included: selecting a sampling point in a petrochemical plant and collecting water samples (1,2-dichloropropene content of 300 ppb) from that point using clean sampling bottles; sealing and storing the samples at 4°C and transporting them to the laboratory for refrigeration at 4°C within 24 hours; enriching the water samples by adding 20 mL of sterile water, 20 mL of sterile TAP inorganic salt medium, 20 mL of sterile LB medium, 100 mg / L sodium acetate, 250 μg / L sodium resazurin, and 10 mg / L or 50 mg / L tetrachloroethane to 10 mL of water sample and enriching at 30°C for 10 days. Using the bacterial culture enriched with 10 mg / L tetrachloroethane as a control, the expression of functional genes related to chlorinated hydrocarbon biodegradation was analyzed in the water sample enriched with 50 mg / L tetrachloroethane. After 10 days, the liquid changed from an initial light blue to a turbid light yellow, indicating that an anaerobic environment had been formed in the sealed culture bottle. The supernatant was collected by centrifugation at 7000 rpm / min, and the salinity (control sample 2.0 PPT, test sample 1.9 PPT), pH (control sample 7.1, test sample 6.9), and ORP (control sample -220, test sample -201) were measured. The collected bacterial cells were frozen at -80℃ for later use.

[0185] The test results of this embodiment are as follows: Figure 12As shown, the results indicated that, except for tceA, the gene expression levels of pceA, cfrA, vcrA, bvcA, mbrA, and pteA all changed to varying degrees. Among them, the expression levels of mbrA and pteA increased significantly, with mbrA expression being higher than pteA. This suggests that the water sample with added 50 mg / L tetrachloroethane contained bacteria capable of dehalogenation degradation, as the results of Example 1 indicated that the water sample was recently contaminated with chlorinated hydrocarbons, affecting the indigenous bacterial community, which contained species capable of degrading chlorinated hydrocarbons. The color change of the added resazan after 10 days of enrichment culture suggested that microorganisms were rapidly consuming oxygen, creating an anaerobic environment. Meanwhile, it was shown that compared with the addition of 10 mg / L tetrachloroethane, the content of dehalogenating bacteria in the water sample with the addition of 50 mg / L tetrachloroethane was significantly increased, and the qPCR results showed that these bacteria mainly degraded tetrachloroethane and trichloroethane. Similar to Example 9, the expression level of mbrA was higher than that of pteA in this example, indicating that after several days of cultivation, the tetrachloroethane content in the water sample decreased while the trichloroethane content increased. Therefore, the corresponding degrading bacteria were induced to express the mbrA gene, which can degrade trichloroethane. At the same time, these bacteria not only tolerated this concentration of tetrachloroethane, but also were able to carry out degradation rapidly.

[0186] As can be seen from the above, Example 1 clearly shows an increase in the expression levels of the two target genes, indicating that these two genes do indeed show changes in expression in the water sample, confirming the presence of these two genes and the bacteria carrying them. Examples 2-4, 5-7, and 8-10 show changes in the color of resveratrol sodium salt in the culture medium, gradual turbidity of the medium, and a continuous decrease in ORP with prolonged culture time. These all indicate that the culture environment gradually becomes anaerobic, suggesting the survival and proliferation of tolerant or degrading bacteria. Simultaneously, PCR chip detection results confirm the changes in the target genes with prolonged culture time; the overall trend is that the longer the culture time, the higher the gene expression level, indicating that with prolonged culture time, the corresponding target genes and the bacteria carrying them transcribe and express the corresponding genes. In particular, for Examples 5-7, in culture media with different concentrations of dichloroethane, the expression level of the vcrA gene was observed to increase with... The expression levels of vcrA, a gene related to the dehalogenation of low-halogenated compounds, gradually increased with prolonged culture time. The increase in vcrA expression was directly related to the high concentration of dichloroethane in the culture medium. For Examples 10-12, in cultures with different concentrations of dichloroethane, the expression levels of mbrA and pteA genes gradually increased with prolonged culture time. mbrA and pteA, genes related to the dehalogenation of high-halogenated compounds, showed an increase in expression directly related to the high concentration of tetrachloroethane in the culture medium. These data illustrate the role of these genes in pollutant resistance or degradation. However, the upregulation of some target genes was not significant, indicating that these genes and the bacteria carrying them may not play a crucial degradation role in this culture system. Therefore, by using the PCR chip provided by this invention to detect the expression of seven target genes, the feasibility and progress of bioremediation of contaminated sites for which water samples were collected can be assessed, and further guidance can be provided for improving bioremediation methods for chlorinated hydrocarbon pollution.

[0187] 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 PCR chip for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons, characterized in that, The PCR chip includes primer pairs for specifically amplifying the dehalogenated functional genes bvcA, cfrA, mbrA, pceA, pteA, tceA and vcrA, respectively, and primer pairs for specifically amplifying the internal reference gene. The nucleotide sequences of the upstream and downstream primers of the primer pair used for specific amplification of the bvcA gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The primer pair used for specific amplification of the cfrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The primer pair used for specific amplification of the mbrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The primer pair used for specific amplification of the pceA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The primer pair used for specific amplification of the pteA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. The primer pair used for specific amplification of the tceA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively. The primer pair used for specific amplification of the vcrA gene has the nucleotide sequences of its upstream and downstream primers as shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

2. The PCR chip according to claim 1, characterized in that, The primer pair used for specific amplification of the internal reference gene has the sequences of its upstream and downstream primers as shown in SEQ ID NO.15 and SEQ ID NO.

16.

3. The PCR chip according to claim 1 or 2, characterized in that, The PCR chip also includes a chip plate, with each primer pair existing individually in the detection well of the chip plate.

4. A reagent kit, characterized in that, The kit includes the PCR chip as described in any one of claims 1-3.

5. The use of the PCR chip according to any one of claims 1-3 or the kit according to claim 4 in detecting the expression levels of seven dehalogenation functional genes related to the microbial degradation of chlorinated hydrocarbons.

6. The use of the PCR chip according to any one of claims 1-3 or the kit according to claim 4 in the detection of chlorinated hydrocarbon degrading bacteria.

7. The application of the PCR chip according to any one of claims 1-3 or the kit according to claim 4 in evaluating the microbial degradation capacity of chlorinated hydrocarbon contaminated sites.

8. The application of the PCR chip according to any one of claims 1-3 or the kit according to claim 4 in assessing the feasibility of microbial remediation of chlorinated hydrocarbon contaminated sites.

9. A method for detecting the expression levels of functional genes related to the microbial degradation of chlorinated hydrocarbons, characterized in that, The method includes the following steps: A1. Collect the bacterial community in the sample to be tested and extract the total RNA of the bacterial community; A2. Using the total RNA as a template, reverse transcription was performed to obtain cDNA; A3. Using the cDNA as a template, qPCR reaction is performed using the PCR chip described in any one of claims 1-3 to obtain the Ct values ​​of the dehalogenated functional gene and the internal reference gene. A4. Calculate the relative expression fold of the dehalogenation functional gene relative to the internal reference gene based on the Ct value.

10. A method for assessing the microbial degradation capacity of chlorinated hydrocarbon contaminated sites, characterized in that, The method includes the following steps: (1) Collect water samples from the contaminated site and collect the microbial community in the water samples or treated water samples; (2) Extract total RNA from the bacterial community and use the total RNA as a template for reverse transcription to obtain cDNA; (3) Using the cDNA as a template, qPCR reaction is performed using the PCR chip described in any one of claims 1-3 to obtain the Ct values ​​of the dehalogenated functional gene and the internal reference gene; (4) Calculate the relative expression fold of the dehalogenation functional gene relative to the internal reference gene based on the Ct value, and evaluate the degradation ability of microorganisms in the contaminated site for chlorinated hydrocarbons by the relative expression fold.

11. The method according to claim 9 or 10, characterized in that, The reverse transcription process includes: mixing the total RNA, reverse transcription reaction mixture, gDNA removal agent and RNase-free water to form a reverse transcription reaction system, and carrying out the reverse transcription reaction to obtain cDNA.

12. The method according to any one of claims 9-11, characterized in that, The conditions for the reverse transcription reaction include: incubation at 40–45°C for 12–18 min, followed by heating at 82–88°C for 4–6 s.

13. The method according to any one of claims 9-12, characterized in that, The conditions for qPCR reaction include: heating at 90–95℃ for 5–10 seconds, followed by reaction at 55–65℃ for 20–60 seconds, for 40–60 cycles.

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