PCR chip, kit and application thereof

Abstract

The application relates to the fields of molecular biology and environmental biotechnology, and discloses a PCR chip, a kit and application thereof. The PCR chip comprises a chip plate, primer pairs for specifically amplifying bvcA genes, cfrA genes, mbrA genes, pceA genes, pteA genes, tceA genes, vcrA genes and internal reference genes are loaded on the chip plate, and a gradient dilution template of an internal reference gene for drawing a standard curve is arranged on the chip plate; the gradient dilution template of the internal reference gene is obtained by gradient dilution of internal reference gene template DNA and addition of internal reference gene primers. The PCR chip can detect absolute quantification of chlorinated hydrocarbon microbial degradation related reduction dehalogen functional genes through qPCR technology, so that the PCR chip can be used for accurately detecting the copy number of the chlorinated hydrocarbon microbial degradation related reduction dehalogen functional genes, thereby the feasibility of chlorinated hydrocarbon pollution bioremediation can be explored, the chlorinated hydrocarbon pollution bioremediation process can be evaluated, and the chlorinated hydrocarbon pollution bioremediation method can be further assisted and guided.

Description

Technical Field

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

[0002] Chlorinated hydrocarbons are compounds in which the hydrogen atoms of hydrocarbon molecules 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, and are also a common type of pollutant in the environment. Chlorinated hydrocarbons and their degradation products contain acute toxins, carcinogens, mutagens, and endocrine disruptors, seriously endangering human health and ecological safety. Their entry into the environment mainly includes: production processes; entry during use as chemical intermediates; entry as impurities from downstream products; sudden accidents or improper handling and disposal, such as waste incineration and its dumping grounds; and entry through direct use, such as chlorobenzene and hexachlorobenzene entering the environment directly as pesticides. Low-chlorinated hydrocarbon pollutants can also be naturally generated 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.

[0003] For contaminated groundwater, it is particularly important to adopt appropriate remediation technologies. Commonly used remediation technologies include physical, chemical, and biological remediation techniques.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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 allows for the identification of different genera and species within a microbial community and the assessment of 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 enables the clustering and analysis of functional genes. qPCR absolute quantification technology utilizes changes in fluorescence signals to detect changes in the amount of amplification products in each cycle of the PCR amplification reaction, performing absolute quantitative analysis by measuring the molecular data of the target gene in the sample.

[0008] Compared to qPCR, which has a clearly defined target gene, 16S rRNA sequencing may face the challenge of failing to detect results due to low abundance of the genus / species in the community. Furthermore, 16S rRNA sequencing primarily targets community species and is insufficient for functional gene analysis, while metagenomic sequencing focuses on community DNA, detecting a massive number of genes. For functional genes, individual searching and analysis are required, which can easily waste sequencing resources if only functional genes are identified.

[0009] Patent application CN 109913563 A discloses a method for assessing the natural degradation of chlorobenzene-contaminated sites through anaerobic microbial degradation. This method uses real-time quantitative PCR (qPCR) to detect groundwater samples from contaminated sites, obtaining the absolute quantities of all known chlorobenzene-reducing dehalogenating bacteria (including Dehalobacter, Dehalogenimonas, and Dehalococcoides). This allows for a rapid and economical determination of whether chlorobenzene pollutants in the site can undergo natural degradation through anaerobic microbial degradation. However, this method detects the absolute quantity of chlorobenzene-reducing dehalogenating bacteria. While it can confirm the presence and trends of Dehalobacter, Dehalogenimonas, and Dehalococcoides, it inevitably faces the problem of not being able to detect the content of other bacteria with reducing dehalogenation capabilities and the existence of corresponding reducing dehalogenation genes. Summary of the Invention

[0010] The purpose of this invention is to overcome the problems existing in the prior art and provide a PCR chip, a kit and its application. The PCR chip can be used for absolute quantitative experiments and calculations of the copy number of each gene, and has reliability in assessing the biodegradability of chlorinated hydrocarbon contaminated sites, studying the degradation process and verifying the remediation effect.

[0011] To achieve the above objectives, the present invention provides a PCR chip, which includes a chip plate loaded with primer pairs for specifically amplifying the bvcA gene, cfrA gene, mbrA gene, pceA gene, pteA gene, tceA gene, vcrA gene and internal reference gene, respectively, and a gradient dilution template loaded with the internal reference gene for plotting a standard curve.

[0012] The gradient dilution template of the internal reference gene is obtained by gradient dilution of the internal reference gene template DNA and the addition of primer pairs specifically for amplifying the internal reference gene.

[0013] Primer pairs for specific amplification of the bvcA gene, the nucleotide sequences of which are shown in SEQ ID NO.1-2;

[0014] Primer pairs for specific amplification of the cfrA gene, the nucleotide sequences of which are shown in SEQ ID NO.3-4;

[0015] Primer pairs for specific amplification of the mbrA gene, the nucleotide sequences of which are shown in SEQ ID NO.5-6;

[0016] Primer pairs for specific amplification of the pceA gene, the nucleotide sequences of which are shown in SEQ ID NO.7-8;

[0017] Primer pairs for specific amplification of the pteA gene, the nucleotide sequences of which are shown in SEQ ID NO.9-10;

[0018] Primer pairs for specific amplification of the tceA gene, the nucleotide sequences of which are shown in SEQ ID NO.11-12;

[0019] Primer pairs for specific amplification of the vcrA gene, the nucleotide sequences of which are shown in SEQ ID NO.13-14;

[0020] The primer pairs for specifically amplifying the internal reference gene have nucleotide sequences shown in SEQ ID NO.15-16.

[0021] A second aspect of the present invention provides a kit comprising a PCR chip as described above.

[0022] The third aspect of the present invention provides the application of the PCR chip or the kit described above in detecting the copy number of functional genes related to chlorinated hydrocarbon degradation and reductive dehalogenation.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] A seventh aspect of the present invention provides a method for detecting the copy number of functional genes related to reductive dehalogenation of chlorinated hydrocarbons, the method comprising the following steps:

[0027] (1) Collect bacterial cells from the sample to be tested and extract total DNA from the bacterial cells;

[0028] (2) Using the total DNA as a template, a qPCR reaction is performed using the PCR chip described in any one of claims 1-4 to obtain the Ct value of each detection well on the PCR chip;

[0029] (3) Plot a standard curve based on the relationship between the copy number of the internal reference gene and its Ct value, and then determine the copy number of the functional gene based on the standard curve.

[0030] The PCR chip provided by this invention is loaded with primer pairs that 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 hydrocarbons, and vcrA and bvcA genes related to the dehalogenation degradation of low-halogenated hydrocarbons). It also contains an internal control gradient dilution template for absolute quantification, enabling the detection of absolute quantification of reductive dehalogenation functional genes related to the microbial degradation of chlorinated hydrocarbons using qPCR technology. Therefore, the PCR chip described in this invention can accurately detect the copy number of reductive dehalogenation functional genes related to the microbial degradation of chlorinated hydrocarbons, explore the feasibility of bioremediation of chlorinated hydrocarbon pollution, assess the bioremediation process of chlorinated hydrocarbon pollution, and further assist in guiding the improvement of bioremediation methods for chlorinated hydrocarbon pollution. Attached Figure Description

[0031] Figure 1 It shows the arrangement of primer pairs and gradient dilution templates of internal reference genes loaded on the PCR chip plate;

[0032] Figure 2 This is a standard curve plotted based on the detection results of the internal reference gene gradient dilution template in Example 1;

[0033] Figure 3 This is the amplification curve of the internal reference gene gradient dilution template in Example 1;

[0034] Figure 4 This is a melting curve of the internal reference gene template in Example 1, obtained by gradient dilution.

[0035] Figure 5 It is the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants after 1 day of enrichment culture on LB medium.

[0036] Figure 6 This is a melting curve diagram of each gene after amplification in Example 1;

[0037] Figure 7 This refers to the amount of reductive dehalogenation functional genes related to chlorinated hydrocarbon degradation in water samples collected from petrochemical plants and enriched in LB medium for 5 days.

[0038] Figure 8 This refers to the amount of reductive dehalogenation functional genes related to chlorinated hydrocarbon degradation in water samples collected from petrochemical plants and enriched in LB medium for 17 days.

[0039] Figure 9 It is the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants after 30 days of enrichment culture on LB medium.

[0040] Figure 10It is the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants and enriched in LB medium for 60 days;

[0041] Figure 11 It is the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants and enriched in TAP medium for 1 day;

[0042] Figure 12 It is the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants and enriched in TAP medium for 5 days;

[0043] Figure 13 It is the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants and enriched in TAP medium for 17 days;

[0044] Figure 14 It is the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants after 30 days of enrichment culture in TAP medium.

[0045] Figure 15 It refers to the amount of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in water samples collected from petrochemical plants and enriched in TAP medium for 60 days. Detailed Implementation

[0046] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0047] 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.

[0048] The present invention provides a PCR chip, which includes a chip plate loaded with primer pairs for specifically amplifying the bvcA gene, cfrA gene, mbrA gene, pceA gene, pteA gene, tceA gene, vcrA gene and internal reference gene, respectively, and a gradient dilution template loaded with the internal reference gene for plotting a standard curve.

[0049] The gradient dilution template of the internal reference gene is obtained by gradient dilution of the internal reference gene template DNA and the addition of primer pairs specifically for amplifying the internal reference gene.

[0050] Primer pairs for specific amplification of the bvcA gene, the nucleotide sequences of which are shown in SEQ ID NO.1-2;

[0051] Primer pairs for specific amplification of the cfrA gene, the nucleotide sequences of which are shown in SEQ ID NO.3-4;

[0052] Primer pairs for specific amplification of the mbrA gene, the nucleotide sequences of which are shown in SEQ ID NO.5-6;

[0053] Primer pairs for specific amplification of the pceA gene, the nucleotide sequences of which are shown in SEQ ID NO.7-8;

[0054] Primer pairs for specific amplification of the pteA gene, the nucleotide sequences of which are shown in SEQ ID NO.9-10;

[0055] Primer pairs for specific amplification of the tceA gene, the nucleotide sequences of which are shown in SEQ ID NO.11-12;

[0056] Primer pairs for specific amplification of the vcrA gene, the nucleotide sequences of which are shown in SEQ ID NO.13-14;

[0057] The primer pairs for specifically amplifying the internal reference gene have nucleotide sequences shown in SEQ ID NO.15-16.

[0058] This invention integrates functional genes related to the dehalogenation degradation of chlorinated hydrocarbons (CHPs) onto a single chip, enabling the acquisition of data on the dehalogenation and reductive dehalogenation of CHPs at multiple sampling points or time intervals in a single experiment, thereby reflecting the degradation status of CHPs. The PCR chip of this invention includes a built-in internal reference gene (16S rRNA) for serially diluted samples, allowing simultaneous amplification of other CHP-related dehalogenation degradation functional genes to obtain a standard amplification curve and calculate the copy number of the target degradation functional gene. By carefully selecting CHP-related reductive dehalogenation functional genes (i.e., the target gene), the chip exhibits high sensitivity, is suitable for high-throughput amplification / screening, and is rapid and highly reproducible. This PCR chip can achieve quantitative detection of CHP-related reductive dehalogenation functional genes in various environmental media and cultures.

[0059] In a specific implementation, the gradient dilution template of the internal reference gene is prepared by pre-preparing the internal reference gene template DNA, performing gradient dilution, and preparing multiple gradient standard curve wells. The standard curve wells contain different concentrations of internal reference gene template DNA and primer pairs (internal reference gene primers) for specifically amplifying the internal reference gene.

[0060] Furthermore, the prepared PCR plate can also contain qPCR premix in the standard curve wells. In this way, when performing the test, the qPCR premix can be removed and the qPCR reaction can be performed simultaneously with the gene to be tested to obtain the Ct value (cycle threshold), and a standard curve can be plotted for the calculation of the copy number of the gene to be tested.

[0061] In one specific embodiment, the chip plate has multiple detection wells, and each primer pair and each concentration of gradient dilution template of the standard curve are individually loaded into the detection well.

[0062] In one specific embodiment, the chip board is a 96-hole board or a 384-hole board, preferably a 96-hole board.

[0063] In a preferred embodiment, the concentration of the primers is 0.1–0.5 μM.

[0064] This invention targets the microbial degradation process of chlorinated hydrocarbons (CHPs) in the environment. Through literature review and testing of actual polluted water samples, seven functional genes related to microbial reductive dehalogenation were screened to characterize the reductive dehalogenation process of microorganisms. Furthermore, this invention designed and verified the specificity of primers for these genes, using universal 16S rRNA primers as internal controls. By rationally arranging these eight genes into a PCR chip for absolute quantification of functional genes related to the reductive dehalogenation of CHP degradation, absolute quantification experiments and calculations of the copy number of each gene were performed using a 16S rRNA gradient dilution template loaded on the chip. This method demonstrates reliability in assessing the biodegradation capacity of CHP-contaminated sites, studying the degradation process, and verifying remediation effects.

[0065] In this invention, the design process of primer pairs for specifically amplifying functional genes related to the biodegradation of chlorinated hydrocarbons includes the following steps:

[0066] Through research, dehalogenation functional genes related to the stepwise dehalogenation degradation process of different chlorinated hydrocarbons were screened, and the status and endpoint information of the designed genes were verified by literature. 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 problems, 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 were screened. Primer pairs with sharp single peak melting curves were screened by qPCR preliminary experiments, and primers with single band PCR products were screened by gel electrophoresis experiments. Primer pairs that specifically amplified functional genes related to the bioreduction dehalogenation of chlorinated hydrocarbons were obtained. The primer sequences, product lengths, annealing temperatures, and sequence numbers of the seven bioreduction dehalogenation-related functional genes and one internal reference gene (16S rRNA) involved in this invention are detailed in Table 1.

[0067] Table 1

[0068]

[0069] To achieve absolute quantification of the target gene and select a suitable internal reference gene, qPCR amplification was first performed to confirm the internal reference gene as a sharp single peak, thus preliminarily confirming its usability. Gel electrophoresis and gel imaging were then used to confirm the internal reference gene as a bright single band, further confirming its usability. The internal reference gene band was recovered through purification of the amplified product or gel extraction, and its band monotony was confirmed again by gel electrophoresis. Finally, the concentration of the purified product was determined using Nanodrop, and serially diluted samples were designed for qPCR amplification. A logarithmic standard curve of Ct value versus gene copy number was plotted to calculate the copy number of the target gene. 0.8 μL of the forward and reverse primers for the dehalogenase gene and the internal reference gene were added to each qPCR reaction tube, along with an additional set of serially diluted internal reference gene samples and their forward and reverse primers. The well openings of the plate were temporarily sealed with a heat-sensitive sealing film and stored at -80℃ for later use.

[0070] The PCR chip of this invention is loaded with primer pairs for functional genes related to the bioreduction and dehalogenation of chlorinated hydrocarbons (including pceA, cfrA, pteA, tceA, and mbrA genes related to the dehalogenation degradation of high-halogenated hydrocarbons, as well as vcrA and bvcA genes related to the dehalogenation of low-halogenated hydrocarbons). By loading primer pairs targeting the above seven functional genes (target genes), the PCR chip has high sensitivity and reliability for detecting the biodegradation of chlorinated hydrocarbon pollution.

[0071] Please refer to the following: Figure 1 In one specific implementation, qPCR experiments with three biological replicates of three test samples can be performed simultaneously on a 96-well plate. The samples are serially diluted using internal reference genes loaded on the plate to plot a standard curve. The absolute copy number of seven functional genes related to bioreduction and dehalogenation can be obtained at one time through this PCR chip, which can accurately determine the pollution or degradation status of chlorinated hydrocarbons and analyze their degradation capacity.

[0072] The PCR chip provided by this invention is simple to operate and features seven highly sensitive target genes. It is loaded with an internal control gradient dilution template for absolute quantification calculations, enabling absolute quantification of each gene in a single experiment. This makes 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.

[0073] The present invention also proposes a kit comprising the PCR chip described above.

[0074] In a specific embodiment, the kit may also contain reagents for performing qPCR reactions. In one specific embodiment, the reagents for performing qPCR reactions may be a passive reference dye, qPCR premix, and water.

[0075] In one specific implementation, the qPCR premix includes DNA polymerase, dNTPs, and buffer.

[0076] In one specific implementation, the water used is nuclease-free water.

[0077] This invention also proposes the application of the PCR chip or kit described above in detecting the copy number of reductive dehalogenation functional genes related to chlorinated hydrocarbon degradation. The PCR chip of this invention carries a serially diluted 16S rRNA gene sample, which can be simultaneously amplified with other functional genes related to chlorinated hydrocarbon dehalogenation degradation to obtain a standard amplification curve, thereby enabling the calculation of the copy number of reductive dehalogenation functional genes related to chlorinated hydrocarbon degradation.

[0078] This invention also proposes the application of the PCR chip or kit described above in the detection of chlorinated hydrocarbon-degrading bacteria, wherein chlorinated hydrocarbon-degrading bacteria refer to microorganisms capable of degrading chlorinated hydrocarbons. Because the PCR chip described in this invention can detect the copy number of reductive dehalogenation functional genes related to chlorinated hydrocarbon degradation, it can more comprehensively reflect the presence of chlorinated hydrocarbon-degrading bacteria in contaminated sites.

[0079] 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.

[0080] 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.

[0081] This invention also proposes a method for detecting the copy number of functional genes related to reductive dehalogenation of chlorinated hydrocarbons, the method comprising the following steps:

[0082] (1) Collect bacterial cells from the sample to be tested and extract total DNA from the bacterial cells;

[0083] (2) Using the total DNA as a template, a qPCR reaction is performed using the PCR chip as described above to obtain the Ct value of each detection well on the PCR chip;

[0084] (3) Plot a standard curve based on the relationship between the copy number of the internal reference gene and its Ct value, and then determine the copy number of the functional gene based on the standard curve.

[0085] In one specific implementation, in step (1), the sample to be tested is an environmental sample of the contaminated site or its culture. Specifically, it can be a water sample from the contaminated site or a culture of the water sample from the contaminated site. By enriching the water sample, the content of microorganisms in the water sample can be increased. The water sample culture can be obtained by artificially adding corresponding nutrients and stress pollutants (such as organic components such as beef extract, peptone, yeast powder, inorganic salt ions, electron donors such as acetic acid and sodium acetate, and chlorinated hydrocarbon components such as 1,2-dichloroethane) and enriching it at about 30°C for 1 to 60 days.

[0086] In a preferred embodiment, step (2) of the qPCR reaction procedure includes three stages:

[0087] The conditions for the first stage include: reacting at 90–95°C for 2–5 minutes;

[0088] The conditions for the second stage include: heating at 90-95℃ for 5-20 seconds, reacting at 60-65℃ for 40-50 seconds, and reacting at 70-80℃ for 20-50 seconds; repeating this cycle 40-60 times.

[0089] The conditions for the third stage include: reacting at 90-95℃ for 10-20 seconds, reacting at 60-65℃ for 1-3 minutes, reacting at 90-95℃ (the gradual increase in temperature at this temperature is used to end the detection and obtain the melting curve, therefore, there is no need to set the reaction time), and reacting at 30-50℃ for 5-20 seconds.

[0090] In this invention, primer pairs for specific gene amplification are individually present in each detection well of the PCR chip plate, so that specific amplification of each gene is carried out in different detection wells. In a specific embodiment, step (2) includes: adding DNA, qPCR premix, reference dye and nuclease-free water to each detection well of the PCR chip plate loaded with primer pairs for specific amplification of the target gene and internal reference gene to form a qPCR reaction system, and then performing a qPCR reaction.

[0091] 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 products.

[0092] The preparation method of the culture medium used in the following examples is as follows:

[0093] 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, and MnCl2·4H2O. 101.2g / L, CoCl2·6H2O32.2g / L, CuSO4·5H2O 31.4g / L, (NH4)6Mo7O24 ·4H2O 22g / L, FeSO4·7H2O 99.8g / L.

[0094] 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.

[0095] Example 1

[0096] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in LB medium for 1 day) using the PCR chip described in this invention.

[0097] (1) Collect bacterial cells from the sample to be tested and extract total DNA from the bacterial cells.

[0098] 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. 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. 10 ml of the water sample was added to 20 mL of sterile water, 40 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin. The samples were then cultured at 30°C for 1 day (24 hours). After 1 day, the liquid changed from an initial pale blue to an orange-red color, indicating the formation of a micro-anaerobic environment in the sealed culture bottle. The samples were centrifuged at 7000 rpm, the supernatant was discarded, and the bacterial cells were collected and frozen at -80°C for later use.

[0099] Total DNA was extracted from bacterial cells using the QIAGEN QIAamp DNA Mini Kit via centrifugation column extraction. The experiment was conducted at room temperature, using RNase-free pipette tips and EP tubes to ensure no contamination or degradation of the extracted DNA. 180 μL of ATL solution and 20 μL of proteinase K were added to the frozen centrifuged bacterial cells, mixed thoroughly, and vortexed to disperse the cells into a homogeneous solution. The solution was then incubated at 56 °C for 3 h until complete lysis, with occasional vortexing. Next, 200 μL of AL buffer was added, vortexed for 15 s, and incubated at 70 °C for 10 min. Then, 200 μL of anhydrous ethanol (96-100%) was added, and vortexed for 15 s. The liquid was transferred to QIAamp Mini Spin tubes, centrifuged at 6000 × g for 1 min, and the liquid inside the outer tube was discarded along with the outer tube. Transfer the QIAamp Mini Spin tube into a new 2mL outer tube, add 500μL of AW1 liquid to the inner tube, centrifuge at 6000×g for 1 min, and discard the liquid in the outer tube along with the outer tube. Transfer the QIAamp Mini Spin tube into a new 2mL outer tube, add 500μL of AW2 liquid to the inner tube, centrifuge at 20000×g for 3 min, and discard the liquid in the outer tube along with the outer tube. Finally, transfer the QIAamp Mini Spin tube into a new 1.5mL outer tube (i.e., a 1.5mL EP tube), add 200μL of AE liquid, incubate at room temperature for 1 min, centrifuge at 6000×g for 1 min, collect the DNA in the EP tube, and store at -80℃ for later use. Measure the concentration and quality of the extracted DNA using a full-wavelength microplate reader. When the OD... 260 / OD 280 When the ratio is around 1.8, it indicates that the extracted DNA is of good quality and can be used for subsequent experiments.

[0100] (2) qPCR reaction

[0101] for Figure 1 The PCR chip plate shown (pre-filled with gradient dilutions of primer pairs and internal reference genes for standard curves) contains wells for the target gene (the functional gene to be tested). The extracted DNA is used as a template for qPCR. The reaction is performed using the PerfectStart Green qPCR SuperMix kit. The reaction system is shown in Table 2 below (template DNA can be added according to the actual extraction amount, and then the entire reaction system is made up to 20 μL with nuclease-free water). Specifically, the target gene wells contain the extracted sample DNA template and reaction mixture (i.e., 2×PerfectStart Green qPCR SuperMix and Passive Reference Dye).

[0102] For the internal reference gene serially diluted template wells, since all reactants (internal reference gene primers, internal reference gene serially diluted DNA templates, and qPCR reaction mixture) have already been added when preparing the PCR chip plate, the qPCR reaction can be carried out directly.

[0103] The qPCR method was performed in three steps, with a total of 45 cycles: 95℃ for 3 min, 45 cycles for quantitative detection (95℃ for 10 s, 61℃ for 45 s, 80℃ for 30 s), and the melting curve segment (95℃ for 15 s, 65℃ for 1 min, 95℃, 40℃ for 10 s).

[0104] Table 2

[0105]

[0106]

[0107] (3) Data Analysis

[0108] After the qPCR experiment, select Analysis and use the Abs Quant / 2nd Der analysis method to calculate the Ct value (cycle threshold) for each well. Then, use the Ct value of the internal reference gene (16S rRNA 10^) after serial dilution. -3 Up to 16S rRNA 10^ -9 Concentration), calculate the copy number of the internal reference gene for the standard curve according to the following formula, and plot the standard curve ( Figure 2 At the same time, check whether the spacing between the amplification curves of the internal reference gene at each gradient dilution is uniform and the amplification effect of parallel samples. Figure 3 And whether the melting curves of the internal reference genes at each gradient dilution are sharp single peaks and their overlap ( Figure 4 ( ) to confirm that the results are available.

[0109]

[0110] OD 260 OD of internal reference gene template DNA 260 The results were obtained using a full-wavelength microplate reader.

[0111] Base number: The number of bases in the template DNA of the internal reference gene, obtained by NCBI-Blast alignment;

[0112] A linear equation (y = -3.5643x + 31.628, amplification efficiency E > 90%) was obtained using a standard curve. Substituting the Ct values ​​of each internal reference gene and the target gene, the absolute copy numbers of the 16S rRNA of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the internal reference gene were calculated based on the Ct values ​​of each internal reference gene and the target gene after 1 day of enrichment culture in LB medium for petrochemical water samples. A plot was then drawn based on the calculation results. Figure 5The melting curves of the genes involved (a total of 8 genes including the target gene and the internal reference gene) are shown in the figure below. Figure 6 As shown.

[0113] The results show ( Figure 5 The copy numbers of the tceA, bvcA, and pteA genes were relatively high, with tceA having the highest copy number at 3.9 × 10⁻⁶. 3 The copy number per L of water was lower than that of other reductive dehalogenase genes, indicating that the petrochemical plant's water sample, after being cultured in LB medium for 1 day, contained genes capable of dehalogenation degradation and had an impact on the indigenous microbial community, which contained species capable of degrading chlorinated hydrocarbons.

[0114] Example 2

[0115] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in LB medium for 5 days from a petrochemical plant) using the PCR chip described in this invention.

[0116] The method described in Example 1 was implemented, except that in step (1), the step of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant, and collecting water (1,2-dichloropropene content of 300 ppb) from the sampling point in a clean sampling bottle. The sample was sealed and stored at 4℃, and transported to the laboratory for refrigeration at 4℃ within 24 hours. The water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample, and enriching and culturing at 30℃ for 5 days; after 5 days, the liquid was observed to be orange-red, indicating that a micro-anaerobic environment had been formed in the sealed culture bottle, centrifuged at 7000 rpm / min, discarded the supernatant, and the bacterial cells were collected and frozen at -80℃ for later use.

[0117] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from the petrochemical water samples collected in this embodiment after enrichment culture in LB medium for 5 days were calculated, and plotted based on the calculation results. Figure 7 .

[0118] The results show ( Figure 7 Similar to Example 1, the highest copy number of the tceA gene among the detected reductive dehalogenase genes was 3.4 × 10⁻⁶. 3 The copy number was around 16S rRNA / L water, but compared to Example 1, after 5 days of cultivation, the microbial content in the water sample increased significantly, with the 16S rRNA copy number reaching 2.4 × 10⁻⁶. 9The copy number was around 1 / L of water, and more reductive dehalogenase genes were detected, including bvcA, pceA, pteA, and cfrA. This indicates that after the water sample from the petrochemical plant was cultured in LB medium for 5 days, the copy number of genes capable of dehalogenation degradation in the water sample increased, and the number of bacteria capable of degrading chlorinated hydrocarbons in the indigenous bacterial community also increased.

[0119] Example 3

[0120] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in LB medium for 17 days from a petrochemical plant) using the PCR chip described in this invention.

[0121] The method described in Example 1 was implemented, except that in step (1), the step of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant, and using a clean sampling bottle to collect water (1,2-dichloropropene content of 300 ppb) from the sampling point in the petrochemical plant; the sample was sealed and stored at 4℃, and transported to the laboratory for refrigeration at 4℃ within 24 hours. The water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample, and enriched and cultured at 30℃ for 17 days; after 17 days, the liquid was observed to be pale yellow like LB and turbid, indicating that an anaerobic environment had been formed in the sealed culture bottle and a large number of bacteria had been generated. The supernatant was discarded by centrifugation at 7000 rpm / min, and the bacterial cells were collected and frozen at -80℃ for later use.

[0122] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from petrochemical water samples enriched in LB medium for 17 days were calculated, and plotted based on the calculation results. Figure 8 .

[0123] The results show ( Figure 8 Similar to Example 2, the reductive dehalogenase genes tceA, bvcA, pceA, pteA, and cfrA were detected in water samples cultured for 17 days. However, unlike Example 2, the gene with the highest copy number was not tceA, but bvcA, with a copy number of 3.0 × 10⁻⁶. 3 The copy number was approximately 1 / L of water. After 17 days of cultivation, the microbial community in the water sample changed. The number of species carrying the bvcA gene increased, and the copy number of this gene also increased significantly. This indicates that after 17 days of cultivation in LB medium, the water sample from the petrochemical plant contained an increase in the copy number of genes capable of dehalogenation degradation, and the dominant species in the indigenous microbial community capable of degrading chlorinated hydrocarbons changed.

[0124] Example 4

[0125] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in LB medium for 30 days from a petrochemical plant) using the PCR chip described in this invention.

[0126] The method described in Example 1 was implemented, except that step (1) of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant, and collecting water (1,2-dichloropropene content of 300 ppb) from the sampling point in a clean sampling bottle. The sample was sealed and stored at 4℃ and transported to the laboratory for refrigeration at 4℃ within 24 hours. The water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample. The enrichment culture was carried out at 30℃ for 30 days. After 30 days, the liquid was observed to be pale yellow like LB medium and turbid, indicating that an anaerobic environment had been formed in the sealed culture bottle and a large number of bacteria had been generated. The supernatant was discarded by centrifugation at 7000 rpm / min, and the bacterial cells were collected and frozen at -80℃ for later use.

[0127] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from petrochemical water samples enriched in LB medium for 30 days were calculated, and plotted based on the calculation results. Figure 9 .

[0128] The results show ( Figure 9 Similar to Example 2, the reductive dehalogenase genes tceA, bvcA, pceA, pteA, and cfrA were detected in water samples cultured for 30 days. However, unlike Example 2, the tceA gene had the highest copy number, reaching 2.0 × 10⁻⁶. 3 The copy number was around 1 / L of water, followed by bvcA. After 30 days of cultivation, the microbial community in the water sample underwent subtle changes. The population carrying the tceA gene was again the most abundant, and the copy number of this gene also increased significantly. This indicates that after 30 days of cultivation in LB medium, the water sample from the petrochemical plant contained an increase in the copy number of genes capable of dehalogenation degradation. Furthermore, the dominant population in the indigenous microbial community capable of degrading chlorinated hydrocarbons was changing, but the strains containing tceA and bvcA remained the most dominant strains under these cultivation conditions.

[0129] Example 5

[0130] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in LB medium for 60 days from a petrochemical water sample) using the PCR chip described in this invention.

[0131] The method described in Example 1 was implemented, except that in step (1), the step of collecting bacteria from the sample to be tested included: selecting a sampling point in a petrochemical plant, and using a clean sampling bottle to collect water (1,2-dichloropropene content of 300 ppb) from the sampling point in the petrochemical plant. The sample was sealed and stored at 4℃, and transported to the laboratory for refrigeration at 4℃ within 24 hours; the water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile LB medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample, and enriched and cultured at 30℃ for 60 days; after 60 days, the liquid was observed to be pale yellow like LB and turbid, indicating that an anaerobic environment had been formed in the sealed culture bottle and a large number of bacteria had been generated. The supernatant was discarded by centrifugation at 7000 rpm / min, and the bacteria were collected and frozen at -80℃ for later use.

[0132] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from petrochemical water samples enriched in LB medium for 60 days were calculated, and plotted based on the calculation results. Figure 10 .

[0133] The results show ( Figure 10 Similar to the previous results, after 60 days of culture, the reductive dehalogenase genes detectable in the water sample were tceA, bvcA, pteA, and cfrA. However, unlike the previous results, the gene with the highest copy number was again bvcA, with a copy number of 2.0 × 10⁻⁶. 3 The number of copies per liter of water was around 100, followed by tecA. The microbial community in the water sample underwent subtle changes, but the strains containing tceA and bvcA remained the dominant strains under these culture conditions. The two strains worked together to degrade chlorinated hydrocarbon pollutants in the culture medium under these conditions.

[0134] Example 6

[0135] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in TAP medium for 1 day from a petrochemical water sample) using the PCR chip described in this invention.

[0136] The method described in Example 1 was implemented, except that step (1) of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant, and collecting water (1,2-dichloropropene content of 300 ppb) from the sampling point in a clean sampling bottle. The sample was sealed and stored at 4°C, and transported to the laboratory for refrigeration at 4°C within 24 hours. The water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile TAP medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample, and enriching and culturing at 30°C for 1 day; after 1 day, the liquid was observed to be slightly pale blue, centrifuged at 7000 rpm / min, the supernatant was discarded, and the bacterial cells were collected and frozen at -80°C for later use.

[0137] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from petrochemical water samples enriched in TAP medium for 1 day were calculated, and plotted based on the calculation results. Figure 11 .

[0138] The results show ( Figure 11 Among all the reductive dehalogenase genes detected, tceA, bvcA, and pteA had relatively high copy numbers, with tceA having the highest at 3.0 × 10⁻⁶. 5 The copy number is around 4.7 × 10⁻⁶ copies / L of water, while the 16S rRNA copy number is around 4.7 × 10⁻⁶ copies / L. 8 The copy number per L of water is around 100, indicating that there are indeed a large number of bacteria in this culture system, as well as microorganisms capable of reductive dehalogenation degradation. However, unlike Examples 1-5, the proportion of bacteria performing reductive dehalogenation degradation in this inorganic salt culture medium is much higher than that in LB culture conditions.

[0139] Example 7

[0140] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing a petrochemical water sample in TAP medium for 5 days) using the PCR chip described in this invention.

[0141] The method described in Example 1 was implemented, except that in step (1), the step of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant, and using a clean sampling bottle to collect water (1,2-dichloropropene content of 300 ppb) from the sampling point in the petrochemical plant. The sample was sealed and stored at 4℃, and transported to the laboratory for refrigeration at 4℃ within 24 hours; the water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile TAP medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample, and enriched and cultured at 30℃ for 5 days; after 5 days, the liquid was observed to be slightly light pink, indicating the formation of a micro-anaerobic environment, centrifuged at 7000 rpm / min, the supernatant was discarded, and the bacterial cells were collected and frozen at -80℃ for later use.

[0142] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from the petrochemical water samples collected in this embodiment after enrichment culture in TAP medium for 5 days were calculated, and plotted based on the calculation results. Figure 12 .

[0143] The results show ( Figure 12 Similar to Example 6, the copy numbers of tceA, bvcA, pteA, and vcrA genes were high in all detected reductive dehalogenase genes, with tceA having the highest copy number of 3.0 × 10⁻⁶. 6 The copy number was around 1 / L water, followed by bvcA, indicating that there were indeed a large number of bacteria in this culture system, as well as microorganisms capable of reductive dehalogenation degradation. Similar to Examples 1-5, tceA and bvcA remained the most dominant genes for reductive dehalogenation degradation in this inorganic salt culture medium.

[0144] Example 8

[0145] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in TAP medium for 17 days from a petrochemical plant) using the PCR chip described in this invention.

[0146] The method described in Example 1 was implemented, except that in step (1), the step of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant, and using a clean sampling bottle to collect water (1,2-dichloropropene content of 300 ppb) from the sampling point in the petrochemical plant. The sample was sealed and stored at 4℃, and transported to the laboratory for refrigeration at 4℃ within 24 hours. The water sample was enriched and cultured. 10 ml of the water sample was added with 20 mL of sterile water, 40 mL of sterile TAP medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin, and enriched and cultured at 30℃ for 17 days. After 17 days, the liquid was observed to be colorless, but slightly turbid, and black fine sand-like particles were produced at the bottom, indicating that a large number of bacterial cells had grown and that there might be an oxidation-reduction effect on the metals in the medium, resulting in the precipitation of some metals. The supernatant was discarded by centrifugation at 7000 rpm / min, and the bacterial cells were collected and frozen at -80℃ for later use.

[0147] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from the petrochemical water samples enriched in TAP medium for 17 days in this embodiment were calculated, and plotted based on the calculation results. Figure 13 .

[0148] The results show ( Figure 13 Similar to Example 6, the copy numbers of tceA, bvcA, pteA, and vcrA genes were high in all the detected reductive dehalogenase genes, with tceA having the highest copy number, followed by bvcA. This indicates that a large number of bacteria are indeed present in this culture system, and there are microorganisms capable of reductive dehalogenation degradation. Moreover, similar to Examples 1-5, tceA and bvcA remain the most dominant genes for reductive dehalogenation degradation in this inorganic salt culture medium.

[0149] Example 9

[0150] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing in TAP medium for 30 days from a petrochemical plant) using the PCR chip described in this invention.

[0151] The method described in Example 1 was implemented, except that step (1) of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant and collecting water (1,2-dichloropropene content of 300 ppb) from the sampling point using a clean sampling bottle. The sample was sealed and stored at 4°C and transported to the laboratory for refrigeration at 4°C within 24 hours; the water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile TAP medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample and enriching and culturing at 30°C for 30 days; after 30 days, the liquid was observed to be colorless but slightly turbid, and black fine sand-like particles were produced at the bottom, indicating that a large number of bacterial cells had grown and that there might be an oxidation-reduction effect on the metals in the culture medium, resulting in the precipitation of some metals. The supernatant was discarded by centrifugation at 7000 rpm / min, and the bacterial cells were collected and frozen at -80°C for later use.

[0152] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water samples obtained from petrochemical water samples enriched in TAP medium for 30 days were calculated, and plotted based on the calculation results. Figure 14 .

[0153] The results show ( Figure 14 Among all the reductive dehalogenase genes detected, tceA, bvcA, pteA, and vcrA had relatively high copy numbers, with tceA having the highest at 4.0 × 10⁻⁶. 6 The copy number was around 1 / L water, followed by bvcA, indicating that there were indeed a large number of bacteria in this culture system, as well as microorganisms capable of reductive dehalogenation degradation. Similar to Examples 1-5, tceA and bvcA remained the most dominant genes for reductive dehalogenation degradation in this inorganic salt culture medium.

[0154] Example 10

[0155] This embodiment illustrates a method for detecting the copy number of chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes in a test sample (a water sample obtained by enriching and culturing a petrochemical water sample in TAP medium for 60 days) using the PCR chip described in this invention.

[0156] The method described in Example 1 was implemented, except that step (1) of collecting the bacterial cells in the sample to be tested included: selecting a sampling point in a petrochemical plant and collecting water (1,2-dichloropropene content of 300 ppb) from the sampling point using a clean sampling bottle. The sample was sealed and stored at 4℃ and transported to the laboratory for refrigeration at 4℃ within 24 hours. The water sample was enriched and cultured by adding 20 mL of sterile water, 40 mL of sterile TAP medium, 100 mg / L sodium acetate, and 250 μg / L sodium resazurin to 10 mL of the water sample and enriching and culturing at 30℃ for 60 days. After 60 days, the liquid was observed to be colorless but slightly turbid, and black fine sand-like particles were produced at the bottom, indicating that a large number of bacterial cells had grown and that there might be an oxidation-reduction reaction with the metals in the culture medium, resulting in the precipitation of some metals. The supernatant was discarded by centrifugation at 7000 rpm / min, and the bacterial cells were collected and frozen at -80℃ for later use.

[0157] Based on the standard curve and the Ct values ​​of the internal reference gene and the target gene obtained from the detection, the absolute copy numbers of the chlorinated hydrocarbon degradation-related reductive dehalogenation functional genes and the 16S rRNA of the internal reference gene in the water sample obtained from the petrochemical water sample collected in TAP medium after 60 days of enrichment culture were calculated, and plotted based on the calculation results. Figure 15 .

[0158] The results show ( Figure 15 Among all the reductive dehalogenase genes detected, tceA, bvcA, pteA, vcrA, and pecA had high copy numbers, with tceA having the highest, followed by bvcA, and then pteA. This indicates that a large number of bacteria were indeed present in the culture system, and that microorganisms capable of reductive dehalogenation degradation were present. Moreover, similar to Examples 1-5, tceA and bvcA remained the most dominant genes for reductive dehalogenation degradation in this inorganic salt culture medium. The bacterial species containing these two genes were the key bacterial species for the biodegradation of chlorinated hydrocarbons in this water sample.

[0159] As can be seen from the above, through Examples 1-5, it is evident that within 60 days of enrichment culture in LB medium, there were significant changes in various chlorinated hydrocarbon degrading microorganisms and chlorinated hydrocarbon degradation-related functional genes within the culture system. With the extension of culture time, changes in the color and turbidity of the culture medium indicate a gradual shift towards an anaerobic environment within the culture system, and an increase in the microbial content. The 16S rRNA gene copy number obtained by qPCR confirms the increase in bacterial content within the system. Simultaneously, chlorinated hydrocarbon degradation-related functional genes also changed with the extension of culture time, from primarily tceA in the early stages to higher copy numbers of both tceA and bvcA in the later stages, reflecting subtle changes in the microbial population in the water sample. The strains containing tceA and bvcA are likely the most dominant strains under these culture conditions, and their combined action degrades chlorinated hydrocarbon pollutants in the culture medium under these conditions.

[0160] Similar to Examples 1-5, Examples 6-10 clearly show significant changes in various chlorinated hydrocarbon degrading microorganisms and chlorinated hydrocarbon degradation-related functional genes within the culture system after 60 days of enrichment culture in TAP medium. With prolonged culture time, changes in the color and turbidity of the culture medium indicate a gradual shift towards an anaerobic environment, and an increase in the microbial content. The 16S rRNA gene copy number obtained by qPCR confirms this increase in bacterial content. Simultaneously, chlorinated hydrocarbon degradation-related functional genes also change with prolonged culture time, shifting from primarily tceA in the early stages to higher copy numbers of both tceA and bvcA in the later stages. This reflects subtle changes in the microbial population in the water sample. The strains containing tceA and bvcA are likely the dominant strains under these culture conditions, working together to degrade chlorinated hydrocarbon pollutants in the culture medium.

[0161] The difference between LB and TAP media lies in the fact that TAP media is almost entirely inorganic, resulting in a slower formation of the anaerobic environment compared to LB. However, due to the absence of organic matter, it is more favorable for the growth of dehalogenation-reducing anaerobic bacteria, leading to a higher tceA gene copy number and lower complexity of the gene or microbial community. Various low-copy-number functional genes were observed to change only in the LB culture system. However, considering that the original contaminated water source, culture time, culture temperature, and added contaminants were consistent between the LB and TAP culture systems, the overall trend of changes in genes related to the dehalogenation-reducing degradation of chlorinated hydrocarbons was similar, demonstrating the sensitivity and specificity of this detection method. Furthermore, in qPCR reactions, 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. In this experiment, even with 10^... -9 The concentration of 16S rRNA also showed relatively normal amplification, indicating that the detection method has high sensitivity.

[0162] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including 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, characterized in that, The PCR chip includes a chip plate loaded with primer pairs for specifically amplifying the bvcA gene, cfrA gene, mbrA gene, pceA gene, pteA gene, tceA gene, vcrA gene and internal reference gene, respectively, and a gradient dilution template loaded with the internal reference gene for plotting a standard curve. The gradient dilution template of the internal reference gene is obtained by gradient dilution of the internal reference gene template DNA and the addition of primer pairs specifically for amplifying the internal reference gene. Primer pairs for specific amplification of the bvcA gene, the nucleotide sequences of which are shown in SEQ ID NO.1-2; Primer pairs for specific amplification of the cfrA gene, the nucleotide sequences of which are shown in SEQ ID NO.3-4; Primer pairs for specific amplification of the mbrA gene, the nucleotide sequences of which are shown in SEQ ID NO.5-6; Primer pairs for specific amplification of the pceA gene, the nucleotide sequences of which are shown in SEQ ID NO.7-8; Primer pairs for specific amplification of the pteA gene, the nucleotide sequences of which are shown in SEQ ID NO.9-10; Primer pairs for specific amplification of the tceA gene, the nucleotide sequences of which are shown in SEQ ID NO.11-12; Primer pairs for specific amplification of the vcrA gene, the nucleotide sequences of which are shown in SEQ ID NO.13-14; The primer pairs for specifically amplifying the internal reference gene have nucleotide sequences shown in SEQ ID NO.15-16.

2. The PCR chip according to claim 1, characterized in that, The chip plate has multiple detection wells, and each primer pair and each concentration of gradient dilution template of the standard curve are individually loaded into the detection well.

3. The PCR chip according to claim 1, characterized in that, The chip board is a 96-hole board or a 384-hole board.

4. The PCR chip according to claim 1, characterized in that, The concentration of primers was 0.1–0.5 μM.

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

6. The reagent kit according to claim 5, characterized in that, The kit also includes a reference dye, qPCR premix, and water.

7. The use of the PCR chip according to any one of claims 1-4 or the kit according to claim 5 or 6 in detecting the copy number of functional genes related to chlorinated hydrocarbon degradation and reductive dehalogenation.

8. The use of the PCR chip according to any one of claims 1-4 or the kit according to claim 5 or 6 in the detection of chlorinated hydrocarbon degrading bacteria.

9. The use of the PCR chip according to any one of claims 1-4 or the kit according to claim 5 or 6 in evaluating the microbial degradation capacity of chlorinated hydrocarbon contaminated sites.

10. The use of the PCR chip according to any one of claims 1-4 or the kit according to claim 5 or 6 in assessing the feasibility of microbial remediation of chlorinated hydrocarbon contaminated sites.

11. A method for detecting the copy number of functional genes related to reductive dehalogenation of chlorinated hydrocarbons, characterized in that, The method includes the following steps: (1) Collect bacterial cells from the sample to be tested and extract total DNA from the bacterial cells; (2) Using the total DNA as a template, a qPCR reaction is performed using the PCR chip described in any one of claims 1-4 to obtain the Ct value of each detection well on the PCR chip; (3) Plot a standard curve based on the relationship between the copy number of the internal reference gene and its Ct value, and then determine the copy number of the functional gene based on the standard curve.

12. The method according to claim 11, characterized in that, In step (2), the qPCR reaction procedure includes three stages: The conditions for the first stage include: reacting at 90–95°C for 2–5 minutes; The conditions for the second stage include: heating at 90-95℃ for 5-20 seconds, reacting at 60-65℃ for 40-50 seconds, and reacting at 70-80℃ for 20-50 seconds; repeating this cycle 40-60 times. The conditions for the third stage include: reaction at 90-95℃ for 10-20 seconds, reaction at 60-65℃ for 1-3 minutes, reaction at 90-95℃, and reaction at 30-50℃ for 5-20 seconds.

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