Method for identifying direct discharge of domestic sewage and application thereof

Abstract

The application discloses a method for identifying direct discharge of domestic sewage and application thereof. The method comprises the following steps: detecting the copy number concentration of bacteriophage marker genes and Bacteroides marker genes of adjacent two days of a water body to be detected respectively, calculating the ratio of the copy number concentration of the bacteriophage marker genes and the copy number concentration of the Bacteroides marker genes per day, and calculating the ratio between the two days of the water body to be detected, and combining a specific ratio threshold to determine whether the direct discharge of domestic sewage appears in the water body to be detected, and the day when the direct discharge of domestic sewage appears can be accurately determined, so that the direct discharge of domestic sewage can be traced and controlled; wherein the bacteriophage marker gene is CPQ_064, and the Bacteroides marker gene is BacHum, BacH, HuBac and / or HF183.

Description

Technical Field

[0001] This application relates to the field of water pollution monitoring technology, and in particular to a method for identifying direct discharge of domestic sewage and its application. Background Technology

[0002] Water environment safety is the cornerstone of maintaining ecological balance, protecting public health, and promoting sustainable social development. However, with rapid urbanization and industrialization, water pollution problems are becoming increasingly serious, among which the discharge of domestic sewage is one of the main sources of eutrophication and pathogenic microbial contamination in water bodies.

[0003] In urban water environment management, the collection and treatment of domestic sewage relies on a vast and complex sewage pipe network system. Ideally, all sewage should enter sewage treatment plants through the pipe network for treatment to meet standards before being discharged. However, in actual operation, due to pipe network damage, combined sewer overflows, and illegal discharge outlets, untreated domestic sewage is often directly discharged into rivers, lakes, and other natural water bodies (i.e., "direct discharge of domestic sewage"). Such incidents are characterized by their instantaneous nature, concealment, and high pollution load, which can severely impact local aquatic ecosystems and represent a key focus and challenge in water environment supervision. Therefore, quickly and accurately identifying and locating the sources of direct sewage discharge is of paramount importance for water pollution prevention and control.

[0004] In practical environmental monitoring, researchers use automated monitoring stations at monitoring sites to perform high-frequency automatic monitoring of water quality parameters (such as ammonia nitrogen, dissolved oxygen, and COD). However, industrial wastewater and agricultural non-point source pollution can both cause these indicators to rise, making it impossible to clearly point the pollution source to domestic sewage, thus hindering source tracing and solving the problem of direct sewage discharge. Furthermore, under conditions of large water flow, direct-flow sewage is rapidly diluted, resulting in weak signal response and a high risk of missed detections. While detecting unique artificial chemicals in the water (such as caffeine and pharmaceutical components) can also trace domestic sewage, this technology is limited by its complex detection process, high cost, reliance on large instruments such as liquid chromatography-mass spectrometry, and the limited range of substances that can be detected, making it unsuitable for rapid, large-scale screening.

[0005] Microbial source tracking (MST) technology is widely used for detecting biological wastewater. This technology effectively determines the presence of human-specific molecular marker genes in water bodies by detecting these genes. A significant advantage of this method compared to traditional methods is its extremely high source specificity, clearly mapping pollution "fingerprints" to human activities. However, MST, using qPCR detection, can only determine the presence and extent of human-origin pollution in the water body; it cannot effectively determine the time period during which domestic wastewater was discharged, thus hindering source tracing and control.

[0006] Therefore, there is a need for a method that can effectively determine whether there is active domestic sewage input in water bodies, so as to achieve accurate source tracing and effective prevention and control. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for identifying direct discharge of domestic sewage and its application.

[0008] The primary objective of this invention is to provide products for detecting bacteriophage marker genes and Bacteroidetes marker genes for the identification of direct discharge of domestic sewage.

[0009] A second objective of this invention is to provide a detection kit.

[0010] A third objective of this invention is to provide a method for identifying direct discharge of domestic sewage.

[0011] A fourth objective of this invention is to provide the application of any of the methods described above in identifying direct discharge of domestic sewage.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013] This invention seeks protection for the application of products that detect phage marker genes and Bacteroidetes marker genes in identifying direct discharge of domestic sewage, wherein the phage marker gene is CPQ_064 and the Bacteroidetes marker genes are BacHum, BacH, HuBac and / or HF183.

[0014] The GeneBank accession number of CPQ_064 is JQ995537.1, the GeneBank accession number of BacHum is AF233411.1, the GeneBank accession number of BacH is AF233408.1, the GeneBank accession number of Hubac is AF233409.1, and the GeneBank accession number of HF183 is AF294909.1.

[0015] Preferably, the identification of direct discharge of domestic sewage includes identifying whether direct discharge of domestic sewage occurs in the water body and / or determining the time of direct discharge of domestic sewage in the water body.

[0016] Preferably, the product is a detection reagent and / or a detection kit.

[0017] Preferably, the direct discharge of domestic sewage is direct discharge of fecal sewage.

[0018] The present invention also claims protection for a detection kit containing detection primers for detecting bacteriophage marker genes and Bacteroidetes marker genes;

[0019] The phage marker gene is CPQ_064, and the Bacteroidetes marker genes are BacHum, BacH, HuBac and / or HF183.

[0020] The GeneBank accession number of CPQ_064 is JQ995537.1, the GeneBank accession number of BacHum is AF233411.1, the GeneBank accession number of BacH is AF233408.1, the GeneBank accession number of Hubac is AF233409.1, and the GeneBank accession number of HF183 is AF294909.1.

[0021] The detection primers for CPQ_064 are CPQ_064F with nucleotide sequences as shown in SEQ ID NO: 1 and CPQ_064R with nucleotide sequences as shown in SEQ ID NO: 2.

[0022] Preferably, the detection primers for BacHum are BacHum-160-F with nucleotide sequences as shown in SEQ ID NO: 3 and BacHum-241-R with nucleotide sequences as shown in SEQ ID NO: 4;

[0023] The detection primers for BacH are BacH-F with nucleotide sequences as shown in SEQ ID NO: 5 and BacH-R with nucleotide sequences as shown in SEQ ID NO: 6;

[0024] The detection primers for HuBac are HuBac-F with the nucleotide sequence shown in SEQ ID NO: 7 and HuBac-R with the nucleotide sequence shown in SEQ ID NO: 8;

[0025] The detection primers for HF183 are HF183-F with nucleotide sequences as shown in SEQ ID NO: 9 and HF183-R with nucleotide sequences as shown in SEQ ID NO: 10.

[0026] This invention also claims protection for a method for identifying direct discharge of domestic sewage, comprising the following steps:

[0027] S1. Obtain water samples from the test body on day n and day n-1, respectively, and detect the copy number (GC) of the phage marker gene in the water samples from day n and day n-1, respectively. 噬菌体 Copy number of Bacteroidetes marker genes (GC) 拟杆菌 ;

[0028] Where n is an integer ≥1; the phage marker gene is CPQ_064, and the Bacteroidetes marker genes BacHum, BacH, HuBac and / or HF183 are mentioned.

[0029] S2. Based on step S1, obtain the copy number (GC) of the phage marker gene in the water samples to be tested on day n and day n-1. 噬菌体 Copy number of Bacteroidetes marker genes (GC) 拟杆菌 Calculate the gene ratio of the water sample to be tested on day n according to formula I. n Ratio and gene ratio on day n-1 Day （n-1） ratio;

[0030] Formula I: Day ratio = (Log 10 GC phage / 100mL) / (Log 10 GC Bacteroides / 100mL);

[0031] S3. Gene ratio of the tested water sample on day n based on step S2. n Ratio and gene ratio on day n-1 Day （n-1） The ratio is calculated according to Formula II, which represents the ratio of the water sample tested on day n to day n-1.

[0032] Formula II: Ratio = (Day n Ratio - Day (n-1) (ratio) / Day n ratio;

[0033] S4. If the ratio of the water sample to be tested on day n to day n-1 obtained in step S3 is <-0.1, it indicates that the water sample to be tested was directly discharged into the water body between day n and day n-1.

[0034] If the ratio of the water sample to be tested on day n to day n-1 obtained in step S3 is ≥-0.1, it indicates that no direct discharge of sewage occurred in the water sample between day n and day n-1.

[0035] Preferably, in step S1, the copy number (GC) of the phage marker gene is detected using qPCR. 噬菌体 Copy number of Bacteroidetes marker genes (GC) 拟杆菌 .

[0036] More preferably, the copy number of Bacteroidetes marker genes is detected by GC. 拟杆菌 When the Bacteroides marker gene is BacHum, it is detected by qPCR using the combination of nucleotide sequences BacHum-160-F (SEQ ID NO: 3) and BacHum-241-R (SEQ ID NO: 4).

[0037] The Bacteroidetes marker gene is BacH, which is detected by qPCR using the combination of nucleotide sequences as shown in SEQ ID NO: 5 (BacH-F) and nucleotide sequences as shown in SEQ ID NO: 6 (BacH-R).

[0038] The Bacteroidetes marker gene is HuBac, which is detected by qPCR using HuBac-F with nucleotide sequences as shown in SEQ ID NO: 7 and HuBac-R with nucleotide sequences as shown in SEQ ID NO: 8.

[0039] The Bacteroides marker gene is HF-183, which is detected by qPCR using HF183-F with nucleotide sequences as shown in SEQ ID NO: 9 and HF183-R with nucleotide sequences as shown in SEQ ID NO: 10.

[0040] The method described in this invention uses the concentrations of Bacteroidetes marker genes and bacteriophage marker genes in the water body to be tested as indicators. It calculates the copy number concentration of marker genes in the water body each day and calculates the ratio of the copy number concentrations of marker genes. Then, based on the ratio between two consecutive days in the water body to be tested, combined with a specific ratio threshold, it determines whether there is direct discharge of domestic sewage in the water body to be tested, and can accurately determine on which day the direct discharge of domestic sewage occurred, thereby realizing the source tracing and control of direct discharge of domestic sewage.

[0041] This invention also claims protection for the application of any of the methods described above in identifying direct discharge of domestic sewage.

[0042] Preferably, identifying direct discharge of domestic sewage includes identifying whether direct discharge of domestic sewage occurs in the water body and / or determining the time of direct discharge of domestic sewage in the water body.

[0043] More preferably, the direct discharge of domestic sewage is direct discharge of fecal sewage.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention provides the application of products that detect phage marker genes and Bacteroidete marker genes in identifying direct discharge of domestic sewage. The phage marker gene is CPQ_064, and the Bacteroidete marker genes are BacHum, BacH, HuBac, and / or HF183. This invention also claims protection for a method for identifying direct discharge of domestic sewage. This method involves detecting the copy number concentrations of phage marker genes and Bacteroidete marker genes in a test water body over two consecutive days, calculating the ratio of the daily phage marker gene copy number concentration to the daily Bacteroidete marker gene copy number concentration, and calculating the ratio between the two days in the test water body. Combined with a specific ratio threshold, this method determines whether direct discharge of domestic sewage has occurred in the test water body and can accurately determine the day on which the direct discharge occurred, thereby enabling the tracing and control of direct discharge of domestic sewage. Attached Figure Description

[0046] Figure 1 The results show the copy number of the HuBac gene in the three simulated aquatic reactors for each day in Test Example 1; A shows the copy number of the HuBac gene in simulated aquatic reactor A; B shows the copy number of the HuBac gene in simulated aquatic reactor B; C shows the copy number of the HuBac gene in the simulated aquatic reactors.

[0047] Figure 2 The results show the copy number of the BacHum gene in the three simulated aquatic reactors for each day in Test Example 1; A shows the copy number of the BacHum gene in simulated aquatic reactor A; B shows the copy number of the BacHum gene in simulated aquatic reactor B; C shows the copy number of the BacHum gene in the simulated aquatic reactors.

[0048] Figure 3 The results show the copy number of the BacH gene in the three simulated aquatic reactors for each day in Test Example 1; A shows the copy number of the BacH gene in simulated aquatic reactor A; B shows the copy number of the BacH gene in simulated aquatic reactor B; C shows the copy number of the BacH gene in the simulated aquatic reactors.

[0049] Figure 4 The figures show the copy number of the HF183 gene in three simulated aquatic reactors for each day in Test Example 1; A shows the copy number of the HF183 gene in simulated aquatic reactor A; B shows the copy number of the HF183 gene in simulated aquatic reactor B; and C shows the copy number of the HF183 gene in the simulated aquatic reactors.

[0050] Figure 5The figures show the copy number of the CPQ_064 gene in three simulated aquatic reactors for each day in Test Example 1; A shows the copy number of the CPQ_064 gene in simulated aquatic reactor A; B shows the copy number of the CPQ_064 gene in simulated aquatic reactor B; and C shows the copy number of the CPQ_064 gene in the simulated aquatic reactors.

[0051] Figure 6 The graph shows the ratio and proportion results of CPQ_064 and BacHum in Test Example 1;

[0052] Figure 7 The graph shows the ratio and proportion results of CPQ_064 and BacH in Test Example 1;

[0053] Figure 8 The graph shows the ratio and proportion results of CPQ_064 and Hubac in Test Example 1;

[0054] Figure 9 The graph shows the ratio and proportion results of CPQ_064 and HF183 in Test Example 1;

[0055] Figure 10 The graph shows the ratio and proportion results of CPQ_064 and BacHum in Test Example 2;

[0056] Figure 11 The graph shows the ratio and proportion results of CPQ_064 and BacH in Test Example 2;

[0057] Figure 12 The graph shows the ratio and proportion results of CPQ_064 and Hubac in Test Example 2;

[0058] Figure 13 The graph shows the ratio and proportion results of CPQ_064 and HF183 in Test Example 2. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0060] Example 1: A method for identifying direct discharge of domestic sewage

[0061] A method for identifying direct discharge of domestic sewage includes the following steps:

[0062] S1. Obtain water samples for testing on day n and day n-1, and then use qPCR amplification technology to detect the copy number (GC) of CPQ_064 (phage marker gene, GeneBank: JQ995537.1) in the water samples on day n and day n-1, respectively. 噬菌体 Copy number GC of BacHum (Bacteroidetes marker gene, GeneBank: AF233411.1) BacHum (GC) 拟杆菌 );

[0063] Taking the detection of gene copy number in a water sample on day n as an example, the specific detection method is as follows:

[0064] S11. The water sample to be tested on day n is filtered with sterile gauze, then particulate matter is removed by a 500-mesh sieve, and then filtered by a 0.22μm filter membrane to collect the filter membrane containing microorganisms.

[0065] S12. Cut the filter membrane containing microorganisms obtained in step S11 into small pieces and put it into a 2mL sterile centrifuge tube. Extract the DNA (DNA template) from the filter membrane containing microorganisms using a water DNA extraction kit (Magen, model: D3145). Amplify the DNA using a qPCR instrument (Roche LightCycler 480II) according to the amplification system shown in Table 1. Calculate the copy number of the CPQ_064 gene based on the amplified Cq value and the CPQ_064 standard curve.

[0066] Table 1 Amplification System

[0067]

[0068] The amplification program was: 95℃, 5 min; 95℃, 5 s, 60℃, 30 s, 40 cycles.

[0069] The CPQ_064 standard curve is obtained by measuring the gene concentration of the standard plasmid containing the CPQ_064 gene synthesized by BGI Genomics using an ultra-micro spectrophotometer and performing a 10-fold serial dilution, as shown in Formula III.

[0070] Formula III (CPQ_064 Standard Curve): Y CPQ_064 =-3.3489X CPQ_064 +4.2457;

[0071] Where Y CPQ_064 X is the copy number of the CPQ_064 gene. CPQ_064 The Cq value amplified for CPQ_064;

[0072] Replace CPQ-064F (SEQ ID NO: 1) and CPQ-064R (SEQ ID NO: 2) in the amplification system shown in Table 1 with equal amounts of BacHum-160-F (SEQ ID NO: 3) and BacHum-241-R (SEQ ID NO: 4), perform amplification according to the amplification procedure shown above, and calculate the copy number of the BacHum gene using the BacHum standard curve shown in Formula IV;

[0073] Formula IV (BacHum Standard Curve): Y BacHum =-3.2168X BacHum +4.0765;

[0074] Where Y BacHum X represents the copy number of the BacHum gene. BacHum The Cq value is the value amplified by BacHum;

[0075] S2. Based on step S1, the copy number (GC) of CPQ_064 (phage marker gene) in the water sample to be tested on day n and day n-1 is calculated. 噬菌体 Copy number GC of BacHum (Bacteroidetes marker gene) BacHum (GC) 拟杆菌 ), calculate the gene ratio of the water sample to be tested on day n according to formula I. n The ratio and the gene ratio of the water sample to be tested on day n-1. （n-1） ratio;

[0076] Formula I: Day ratio = (Log 10 GC 噬菌体 / 100mL) / (Log 10 GC 拟杆菌 / 100mL);

[0077] S3. Gene ratio of the water sample to be tested on day n, obtained from step S2. n The ratio and the gene ratio of the water sample to be tested on day n-1. （n-1） The ratio is calculated according to Formula II, which represents the ratio of the water sample tested on day n to day n-1.

[0078] Formula II: Ratio = (Day n Ratio - Day (n-1) (ratio) / Day n ratio;

[0079] S4. If the ratio of the water sample to be tested on day n to day n-1 obtained in step S3 is <-0.1, it indicates that the water sample to be tested was directly discharged into the water body between day n and day n-1.

[0080] If the ratio of the water sample to be tested on day n to day n-1 obtained in step S3 is ≥-0.1, it indicates that no direct discharge of sewage occurred in the water sample between day n and day n-1.

[0081] Example 2: A method for identifying direct discharge of domestic sewage

[0082] A method for identifying direct discharge of domestic sewage, the difference between this method and the method shown in Example 1 is that BacHum (Bacteroides marker gene, GeneBank: AF233411.1) is replaced with BacH (Bacteroides marker gene, GeneBank: AF233408.1), and BacHum-160-F (SEQ ID NO: 3) and BacHum-241-R (SEQ ID NO: 4) in step S12 are replaced with equal amounts of BacH-F (SEQ ID NO: 5) and BacH-R (SEQ ID NO: 6), and the BacH standard curve is shown in Formula V. The remaining steps are the same, and then the ratio of the water sample to be tested on day n to day n-1 is calculated. Then, according to step S4 of Example 1, it is determined whether direct discharge of sewage occurs between day n and day n-1.

[0083] Formula V (BacH standard curve): Y BacH =-3.2243X BacH +3.5746;

[0084] Where Y BacH X represents the copy number of the BacH gene. BacH The Cq value is the result of BacH amplification.

[0085] Example 3: A method for identifying direct discharge of domestic sewage

[0086] A method for identifying direct discharge of domestic sewage, the difference between this method and the method shown in Example 1 is that BacHum (Bacteroides marker gene, GeneBank: AF233411.1) is replaced with HuBac (Bacteroides marker gene, GeneBank: AF233409.1), and BacHum-160-F (SEQ ID NO: 3) and BacHum-241-R (SEQ ID NO: 4) in step S12 are replaced with equal amounts of HuBac-F (SEQ ID NO: 7) and HuBac-R (SEQ ID NO: 8), and the HuBac standard curve is shown in Formula VI. The remaining steps are the same, and then the ratio of the water sample to be tested on day n to day n-1 is calculated. Then, according to step S4 of Example 1, it is determined whether direct discharge of sewage occurs between day n and day n-1.

[0087] Formula VI (HuBac Standard Curve): Y HuBac =-3.1879X HuBac +4.9771;

[0088] Where Y HuBac X represents the copy number of the Hubac gene. HuBac The Cq value for Hubac amplification.

[0089] Example 4: A method for identifying direct discharge of domestic sewage

[0090] A method for identifying direct discharge of domestic sewage, the difference between this method and the method shown in Example 1 is that BacHum (Bacteroides marker gene, GeneBank: AF233411.1) is replaced with HF183 (Bacteroides marker gene, GeneBank: AF294909.1), and BacHum-160-F (SEQ ID NO: 3) and BacHum-241-R (SEQ ID NO: 4) in step S12 are replaced with equal amounts of HF183-F (SEQ ID NO: 9) and HF183-R (SEQ ID NO: 10), and the HF183 standard curve is shown in Formula VII. The remaining steps are the same, and then the ratio of the water sample to be tested on day n to day n-1 is calculated. Then, according to step S4 of Example 1, it is determined whether direct discharge of sewage occurs between day n and day n-1.

[0091] Formula VII (HF183 Standard Curve): Y HF183 =-3.2739X HF183 +5.0142;

[0092] Where Y HF183X represents the copy number of the HF183 gene. HF183 The Cq value is the result of HF183 amplification.

[0093] Test Example 1: Testing a method for identifying direct discharge of domestic sewage

[0094] I. Attenuation Test of Bacteriophage Marker Genes and Bacteroidetes Marker Genes

[0095] 1. Experimental Methods

[0096] Weigh 30g of fresh human feces (fecal samples include those from healthy individuals and those from patients) and dissolve them in 3L of deionized water. After thorough mixing, filter the solution using sterile gauze and then remove particulate matter using a 500-mesh sieve. Collect the fecal filtrate and transfer 1L of the filtrate to an aquatic environment simulation reactor (a cylindrical container made of 0.5cm thick plexiglass, 26cm in diameter and 25cm in height). Make up the volume to 10L with pure water and keep the water in the reactor stirred at 240r / min for the reaction (recorded as day 0). On day 6 and day 13 of the reaction, add fecal filtrate to the reactor at a rate of 10g / L.

[0097] The aquatic environment simulation reactor reacted for a total of 22 days, with days 0-6 designated as the first stage, days 7-13 as the second stage, and days 14-22 as the third stage. Three identical aquatic environment simulation reactors were prepared in parallel, forming a triple-replication, triple-stage parallel experimental group. The three aquatic environment simulation reactors were designated as A, B, and C, respectively. Taking the testing of bacteriophage marker genes and Bacteroidetes marker genes in aquatic environment simulation reactor A as an example, the specific details are as follows:

[0098] During the reaction process in the water environment simulation reactor, 100 mL of water sample was taken from the water environment simulation reactor every day and filtered using a 0.22 μm filter membrane to collect the filter membrane containing microorganisms each day; the water samples on the 6th and 13th days were taken before the addition of fecal filtrate.

[0099] Each filter membrane containing microorganisms was cut into small pieces and placed in a 2 mL sterile centrifuge tube. DNA (DNA template) was extracted from each filter membrane using a water DNA extraction kit (Magen, model: D3145). The DNA was amplified according to the methods described in Examples 1-4. The daily copy numbers of the CPQ_064, BacHum, BacH, HuBac, and HF183 genes in the aquatic environment simulation reactor were calculated using the standard curves shown in Formulas III-VII. The copy numbers were then converted to logarithmic values. 10The concentration in 100 mL of water was calculated in the form of CPQ_064 gene, which is a phage marker gene, and BacHum, BacH, HuBac and HF183 genes are all Bacteroidetes marker genes.

[0100] Next, for each gene in the three stages of the reaction process in the aquatic environment simulation reactor, the copy number was fitted according to the first-order kinetic decay model shown in Formula VIII to obtain the decay constant of each gene in the three stages. Then, the time T required for each gene to decay to 90% in the three stages was calculated according to Formula IX. 90 ;

[0101] Formula VIII: C t =C0*e^ (-kt) Where C0 is the copy number of the gene on day 0; C t Let t be the copy number of the corresponding gene in the water environment simulation reactor on day t, where t is an integer from 1 to 22;

[0102] Formula IX: T 90 =-ln(0.1) / k; where k The decay constant of this gene is calculated using formula VIII.

[0103] Next, the T values ​​of each gene in the three stages were analyzed. 90 The average value is taken to obtain the time required for the gene to decay to 90% in the water environment simulated reactor.

[0104] Following the method described above, the same treatment was applied to water environment simulation reactors B and C. The copy numbers of the CPQ_064, BacHum, BacH, HuBac, and HF183 genes were calculated daily in the water environment simulation reactors, and the average time required for each gene to decay to 90% in the water environment simulation reactors was calculated.

[0105] 2. Experimental Results

[0106] (1) The daily copy number of the HuBac gene in three simulated aquatic reactors is as follows: Figure 1 As shown, Figure 1 In the figure, A represents the copy number of the HuBac gene in the water environment simulation reactor A. Figure 1 In the figure, B represents the copy number of the HuBac gene in the water environment simulation reactor B. Figure 1 In the figure, C represents the copy number results of the HuBac gene in the water environment simulation reactor; where Figure 1a1, b1, and c1 represent the first stage in the water environment simulation reactor, a2, b2, and c2 represent the second stage, and a3, b3, and c3 represent the third stage.

[0107] The attenuation parameters of the HuBac gene in a simulated aquatic reactor are shown in Table 2.

[0108] Table 2. Attenuation parameters of the HuBac gene in an aquatic environment simulation reactor.

[0109]

[0110] The results showed that the average time required for the HuBac gene to decay to 90% in aquatic environment simulation reactor A was 1.24 days, the average time required for decay to 90% in aquatic environment simulation reactor B was 1.91 days, and the average time required for decay to 90% in aquatic environment simulation reactor C was 1.60 days.

[0111] (2) The daily copy number of the BacHum gene in the three aquatic environment simulation reactors is as follows: Figure 2 As shown, Figure 2 In the figure, A represents the copy number of the BacHum gene in the water environment simulation reactor A. Figure 2 In the figure, B represents the copy number of the BacHum gene in the water environment simulation reactor B. Figure 2 In the figure, C represents the copy number results of the BacHum gene in the water environment simulation reactor; where Figure 2 a1, b1, and c1 represent the first stage in the water environment simulation reactor, a2, b2, and c2 represent the second stage, and a3, b3, and c3 represent the third stage.

[0112] The attenuation parameters of the BacHum gene in the aquatic environment simulation reactor are shown in Table 3.

[0113] Table 3. Attenuation parameters of the BacHum gene in an aquatic environment simulation reactor.

[0114]

[0115] The results showed that the average time required for the BacHum gene to decay to 90% in aquatic environment simulation reactor A was 1.69 days, the average time required for decay to 90% in aquatic environment simulation reactor B was 1.72 days, and the average time required for decay to 90% in aquatic environment simulation reactor was 1.79 days.

[0116] (3) The daily copy number of the BacH gene in the three aquatic environment simulation reactors is as follows: Figure 3 As shown, Figure 3 In the figure, A represents the copy number of the BacH gene in the water environment simulation reactor A. Figure 3 In the figure, B represents the copy number of the BacH gene in the water environment simulation reactor B. Figure 3 In the figure, C represents the copy number results of the BacH gene in the water environment simulation reactor; where... Figure 3 a1, b1, and c1 represent the first stage in the water environment simulation reactor, a2, b2, and c2 represent the second stage, and a3, b3, and c3 represent the third stage.

[0117] The attenuation parameters of the BacH gene in the aquatic environment simulation reactor are shown in Table 4.

[0118] Table 4. Attenuation parameters of the BacH gene in an aquatic environment simulation reactor.

[0119]

[0120] The results showed that the average time required for the BacH gene to decay to 90% in aquatic environment simulation reactor A was 1.71 days, the average time required for decay to 90% in aquatic environment simulation reactor B was 1.81 days, and the average time required for decay to 90% in aquatic environment simulation reactor was 1.56 days.

[0121] (4) The daily copy number of the HF183 gene in the three aquatic environment simulation reactors is as follows: Figure 4 As shown, Figure 4 In the figure, A represents the copy number of the HF183 gene in the water environment simulation reactor A. Figure 4 In the figure, B represents the copy number of the HF183 gene in the water environment simulation reactor B. Figure 4 In the figure, C represents the copy number results of the HF183 gene in the water environment simulation reactor; where Figure 4 a1, b1, and c1 represent the first stage in the water environment simulation reactor, a2, b2, and c2 represent the second stage, and a3, b3, and c3 represent the third stage.

[0122] The attenuation parameters of the HF183 gene in the aquatic environment simulation reactor are shown in Table 5.

[0123] Table 5. Attenuation parameters of the HF183 gene in a simulated aquatic reactor.

[0124]

[0125] The results showed that the average time required for the HF183 gene to decay to 90% in water environment simulation reactor A was 1.52 days, the average time required for decay to 90% in water environment simulation reactor B was 1.83 days, and the average time required for decay to 90% in water environment simulation reactor C was 1.20 days.

[0126] (5) The daily copy number results of the CPQ_064 gene in the three aquatic environment simulation reactors are as follows: Figure 5 As shown, Figure 5 In the figure, A represents the copy number of the CPQ_064 gene in the water environment simulation reactor A. Figure 5 In the figure, B represents the copy number of the CPQ_064 gene in the water environment simulation reactor B. Figure 5 In the figure, C represents the copy number results of the CPQ_064 gene in the water environment simulation reactor; where Figure 5 a1, b1, and c1 represent the first stage in the water environment simulation reactor, a2, b2, and c2 represent the second stage, and a3, b3, and c3 represent the third stage.

[0127] The attenuation parameters of the CPQ_064 gene in the aquatic environment simulation reactor are shown in Table 6.

[0128] Table 6. Attenuation parameters of the CPQ_064 gene in a simulated aquatic reactor.

[0129]

[0130] The results showed that the average time required for the CPQ_064 gene to decay to 90% in aquatic environment simulation reactor A was 3.51 days, in aquatic environment simulation reactor B it was 3.50 days, and in aquatic environment simulation reactor C it was 3.22 days.

[0131] In summary, Bacteroidetes marker genes (BacHum, BacH, HuBac, and HF183) exhibited rapid decay characteristics in water, with their copy number concentration decreasing by more than an order of magnitude within 24 to 48 hours. Furthermore, the decay rates (time required to decay to 90%) among different Bacteroidetes marker genes showed no significant difference. In contrast, the bacteriophage marker gene CPQ_064 required nearly 4 days (96 hours) to decay to 90% in water. This indicates a significant difference in the decay characteristics between the bacteriophage marker gene CPQ_064 and the Bacteroidetes marker genes.

[0132] II. Method Testing

[0133] 1. Experimental Methods

[0134] As shown in "I. Attenuation Test of Phage Marker Genes and Bacteroidetes Marker Genes", the daily copy numbers of CPQ_064, BacHum, BacH, HuBac, and HF183 genes in water environment simulation reactors A, B, and C were determined and calculated, and the copy numbers were converted into logarithmic values. 10 The concentration in 100 mL of water is calculated in the form of [formula missing].

[0135] Next, following the methods shown in Examples 1 to 4, the ratio of phage marker genes to Bacteroidetes marker genes in each water environment simulation reactor and the ratio of adjacent two days were calculated according to Formula I and Formula II. The average value of the results (ratios and ratios) of the same day in the three water environment simulation reactors was taken as the test result.

[0136] 2. Experimental Results

[0137] (1) The ratio and proportion of the phage marker gene (CPQ_064) and the Bacteroidetes marker gene (BacHum) calculated according to the method shown in Example 1 are shown in the figure below. Figure 6 As shown in Table 7, the ratio and proportion of the phage marker gene (CPQ_064) and the Bacteroidetes marker gene (BacHum) are presented.

[0138] Table 7. Ratio and percentage results of CPQ_064 and BacHum

[0139]

[0140] New fecal samples were added to the water environment simulation reactor on days 6 and 13 (i.e., direct discharge of domestic sewage occurred). When calculating the ratio of CPQ_064 gene to BacHum gene according to the method shown in Example 1, there was no regular change in the time before and after the direct discharge of domestic sewage (i.e., before and after days 6 and 13) compared with other time periods. This indicates that the results of the ratio of CPQ_064 gene to BacHum gene cannot accurately determine on which day the direct discharge of domestic sewage (i.e., the addition of new feces) occurred.

[0141] When calculating the ratio between two adjacent days according to Formula II, the results show that the ratio on day 7 is -0.37 < -0.1, and the ratio on day 14 is -0.69 < -0.1, indicating that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation; while the ratios for other time periods are all ≥ -0.1, indicating no misjudgment. The results show that the method shown in Example 1 can accurately identify direct discharge of domestic sewage.

[0142] (2) The ratio and proportion of the phage marker gene (CPQ_064) and the Bacteroidetes marker gene (BacH) calculated according to the method shown in Example 2 are shown in the figure below. Figure 7 As shown in Table 8, the ratio and proportion of the phage marker gene (CPQ_064) and the Bacteroidetes marker gene (BacH) are presented.

[0143] Table 8. Ratio and percentage results of CPQ_064 and BacH

[0144]

[0145] When calculating the ratio of the CPQ_064 gene to the BacH gene using the method shown in Example 2, the ratio of the two genes did not change regularly compared to other times before and after the direct discharge of domestic sewage (i.e., around day 6 and day 13). This indicates that the results of the ratio of the CPQ_064 gene to the BacH gene cannot accurately determine which day the direct discharge of domestic sewage occurred.

[0146] When calculating the ratio between two adjacent days according to Formula II, the results show that the ratio on day 7 is -0.31 and the ratio on day 14 is -0.48, indicating that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation; while the ratios for other time periods are all ≥-0.1, and there will be no misjudgment. The results show that the method shown in Example 2 can accurately identify direct discharge of domestic sewage.

[0147] (3) The ratio and proportion of the phage marker gene (CPQ_064) and the Bacteroidetes marker gene (HuBac) calculated according to the method shown in Example 3 are shown in the figure below. Figure 8 As shown in Table 9, the ratio and proportion of phage marker gene (CPQ_064) and Bacteroidetes marker gene (HuBac) are presented.

[0148] Table 9. Ratio and percentage results of CPQ_064 and Hubac

[0149]

[0150] When calculating the ratio of the CPQ_064 gene to the HuBac gene using the method shown in Example 3, the ratio of the two genes did not show a regular change compared to other times before and after the direct discharge of domestic sewage (i.e., around day 6 and day 13). This indicates that the results of the ratio of the CPQ_064 gene to the HuBac gene cannot accurately determine which day the direct discharge of domestic sewage occurred.

[0151] When calculating the ratio between two adjacent days according to Formula II, the results show that the ratio on day 7 is -0.23 and the ratio on day 14 is -0.18, both < -0.1. This indicates that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation. The ratios for other time periods are all ≥ -0.1, indicating no misjudgment. The results demonstrate that the method shown in Example 3 can accurately identify direct discharge of domestic sewage.

[0152] (4) The ratio and proportion of the phage marker gene (CPQ_064) and the Bacteroidetes marker gene (HF183) calculated according to the method shown in Example 4 are shown in the figure below. Figure 9 As shown in Table 10, the ratio and proportion of phage marker gene (CPQ_064) and Bacteroidetes marker gene (HF183) are presented.

[0153] Table 10: Ratio and percentage results of CPQ_064 and HF183

[0154]

[0155] When calculating the ratio between two adjacent days according to Formula II, the results show that the ratio on day 7 is -0.34 and the ratio on day 14 is -0.55, both < -0.1. This indicates that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation. The ratios for other time periods are all ≥ -0.1, indicating no misjudgment. The results demonstrate that the method shown in Example 4 can accurately identify direct discharge of domestic sewage.

[0156] In summary, the methods described in Examples 1 to 4 can accurately identify direct discharge of domestic sewage and accurately determine the time when direct discharge of domestic sewage occurs.

[0157] Test Example 2: Application of a method for identifying direct discharge of domestic sewage

[0158] I. Experimental Methods

[0159] The 1L of filtrate obtained from Test Example 1 was transferred to an aquatic environment simulation reactor (a cylindrical container made of 0.5cm thick plexiglass, 26cm in diameter and 25cm in height), and replenished to 10L using domestic sewage (collected from the sewage outlet of the Donghuicheng residential area in Huangpu District), resulting in aquatic environment simulation reactor A'. The water in aquatic environment simulation reactor A' was stirred at a rate of 240r / min for the reaction (recorded as day 0) for a total of 22 days. On day 6 and day 13 of the reaction, 10g / L of fecal filtrate was added to the aquatic environment simulation reactor, respectively.

[0160] Next, as shown in Test Example 1, the copy numbers of CPQ_064, BacHum, BacH, HuBac, and HF183 genes in the water environment simulation reactor A' were measured and calculated daily, and the copy numbers were converted into log10 form to calculate the concentration per 100 mL of water.

[0161] Next, following the methods shown in Examples 1 to 4, the ratio of bacteriophage marker genes to each Bacteroidetes marker gene and the ratio of adjacent two days in the aquatic environment simulation reactor A' were calculated according to Formula I and Formula II.

[0162] II. Experimental Results

[0163] (1) The ratio and proportion of CPQ_064 and BacHum in the water environment simulation reactor A' (with actual sewage added), calculated according to the method shown in Example 1, are shown in the figure below. Figure 10 As shown in Table 11, the ratio and proportion results of CPQ_064 and BacHum are presented.

[0164] Table 11. Ratio and percentage results of CPQ_064 and BacHum

[0165]

[0166] In the water environment simulation reactor A', new fecal samples were added on days 6 and 13 (i.e., direct discharge of domestic sewage occurred). The results showed that the ratio of the two genes did not change regularly before and after the direct discharge of domestic sewage (i.e., before and after days 6 and 13) compared with other times. This indicates that the ratio of the CPQ_064 gene and the BacHum gene cannot accurately determine on which day direct discharge of domestic sewage (i.e., the addition of new feces) occurred in the actual water body.

[0167] When calculating the ratio of two consecutive days in the water environment simulation reactor A' containing actual wastewater according to Formula II, the ratio on day 7 is -0.16 and the ratio on day 14 is -0.28, both < -0.1. This indicates that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation. The ratios at other times are all ≥ -0.1, so there will be no misjudgment.

[0168] (2) The ratio and proportion of CPQ_064 and BacH in the water environment simulation reactor A' (with actual sewage added), calculated according to the method shown in Example 2, are shown in the figure below. Figure 11 As shown in Table 12, the ratio and proportion results of CPQ_064 and BacH are presented.

[0169] Table 12: Ratio and percentage results of CPQ_064 and BacH

[0170]

[0171] When calculating the ratio of the CPQ_064 gene to the BacH gene in the water environment simulation reactor A' with added actual sewage according to the method shown in Example 2, the ratio of the two genes did not change regularly compared with other times before and after the direct discharge of domestic sewage (i.e., around day 6 and day 13). This indicates that the results of the ratio of the CPQ_064 gene to the BacH gene cannot accurately determine on which day the direct discharge of domestic sewage (i.e., the addition of new feces) occurred in the actual water body.

[0172] When calculating the ratio of adjacent days in the water environment simulation reactor A' containing actual wastewater according to Formula II, the ratio on day 7 is -0.33 and the ratio on day 14 is -0.46, both < -0.1. This indicates that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation. The ratios at other times are all ≥ -0.1, so there will be no misjudgment.

[0173] (3) The ratio and proportion of CPQ_064 and HuBac in the water environment simulation reactor A' calculated according to the method shown in Example 3 are shown in the figure below. Figure 12 As shown in Table 13, the ratio and proportion results of CPQ_064 and Hubac are presented.

[0174] Table 13: Ratio and percentage results of CPQ_064 and Hubac

[0175]

[0176] When calculating the ratio of the CPQ_064 gene to the HuBac gene in the water environment simulation reactor A' with actual sewage added according to the method shown in Example 3, the ratio of the two genes did not change regularly compared with other times before and after the direct discharge of domestic sewage (i.e. around day 6 and day 13). This indicates that the results of the ratio of the CPQ_064 gene to the HuBac gene cannot determine on which day the direct discharge of domestic sewage occurred in the actual water body.

[0177] When calculating the ratio of adjacent days in the water environment simulation reactor A' containing actual wastewater according to Formula II, the ratio on day 7 is -0.34 and the ratio on day 14 is -0.31, both < -0.1. This indicates that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation. The ratios at other times are all ≥ -0.1, so there will be no misjudgment.

[0178] (4) The ratio and proportion of CPQ_064 and HF183 in the water environment simulation reactor A' calculated according to the method shown in Example 4 are shown in the figure below. Figure 13 As shown in Table 14, the ratio and proportion results of CPQ_064 and HF183 are presented.

[0179] Table 14: Ratio and percentage results of CPQ_064 and HF183

[0180]

[0181] When calculating the ratio of the CPQ_064 gene to the HF183 gene in a water environment simulation reactor A' with added actual sewage according to the method shown in Example 4, the ratio of the two genes did not change regularly compared with other times before and after the direct discharge of domestic sewage (i.e., around day 6 and day 13). This indicates that the results of the ratio of the CPQ_064 gene to the HF183 gene cannot determine on which day the direct discharge of domestic sewage occurred in the actual water body.

[0182] When calculating the ratio of adjacent days in the water environment simulation reactor A' containing actual wastewater according to Formula II, the ratio on day 7 is -0.22 and the ratio on day 14 is -0.44, both < -0.1. This indicates that direct discharge of domestic sewage occurred between day 6 and day 7, and between day 13 and day 14, which is consistent with the actual situation. The ratios at other times are all ≥ -0.1, so there will be no misjudgment.

[0183] In summary, the methods described in Examples 1 to 4 can identify direct discharge of domestic sewage (containing numerous microorganisms) when performing the identification of direct discharge of domestic sewage, and can accurately determine the time of occurrence of direct discharge of domestic sewage without misjudgment or omission.

[0184] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A test kit, characterized in that, It contains detection primers for detecting bacteriophage marker genes and Bacteroidetes marker genes; The phage marker gene is CPQ_064, and the Bacteroidetes marker gene is BacH; The GeneBank accession number of CPQ_064 is JQ995537.1, and the GeneBank accession number of BacH is AF233408.1; The detection primers for CPQ_064 are CPQ_064F with nucleotide sequences as shown in SEQ ID NO: 1 and CPQ_064R with nucleotide sequences as shown in SEQ ID NO: 2; The detection primers for BacH are BacH-F with nucleotide sequences as shown in SEQ ID NO: 5 and BacH-R with nucleotide sequences as shown in SEQ ID NO:

6.

2. A method for identifying direct discharge of domestic sewage, characterized in that, Includes the following steps: S1. Obtain water samples from the test body on day n and day n-1, respectively, and detect the copy number (GC) of the phage marker gene in the water samples from day n and day n-1, respectively. 噬菌体 Copy number of Bacteroidetes marker genes (GC) 拟杆菌 ; Where n is an integer ≥1; the phage marker gene is CPQ_064, and the Bacteroidetes marker gene is BacH; S2. Based on step S1, obtain the copy number (GC) of the phage marker gene in the water samples to be tested on day n and day n-1. 噬菌体 Copy number of Bacteroidetes marker genes (GC) 拟杆菌 Calculate the gene ratio of the water sample to be tested on day n according to formula I. n Ratio and gene ratio on day n-1 Day （n-1） ratio; Formula I: Day ratio = (Log 10 GC 噬菌体 / 100mL) / (Log 10 GC 拟杆菌 / 100mL); S3. Gene ratio of the tested water sample on day n based on step S2. n Ratio and gene ratio on day n-1 Day （n-1） The ratio is calculated according to Formula II, which represents the ratio of the water sample tested on day n to day n-1. Formula II: Ratio = (Day n Ratio - Day (n-1) (ratio) / Day n ratio; S4. If the ratio of the water sample to be tested on day n to day n-1 obtained in step S3 is <-0.1, it indicates that the water sample to be tested was directly discharged into the water body between day n and day n-1. If the ratio of the water sample to be tested on day n to day n-1 obtained in step S3 is ≥-0.1, it indicates that no direct discharge of sewage occurred in the water sample to be tested between day n and day n-1. The direct discharge of domestic sewage refers to the direct discharge of fecal sewage.

3. The method according to claim 2, characterized in that, In step S1, the copy number (GC) of the phage marker gene is detected using qPCR. 噬菌体 Copy number of Bacteroidetes marker genes (GC) 拟杆菌 .

4. The method according to claim 3, characterized in that, GC detection of phage marker genes 噬菌体 At that time, qPCR was used to detect the nucleotide sequence CPQ_064F as shown in SEQ ID NO: 1 and the nucleotide sequence CPQ_064R as shown in SEQ ID NO:

2.

5. The method according to claim 3, characterized in that, GC detection of Bacteroidetes marker gene copy number 拟杆菌 When the Bacteroides marker gene is BacH, it is detected by qPCR using nucleotide sequences BacH-F as shown in SEQ ID NO: 5 and BacH-R as shown in SEQ ID NO:

6.

6. The application of the method according to any one of claims 2 to 5 in identifying direct discharge of domestic sewage, characterized in that, The direct discharge of domestic sewage refers to the direct discharge of fecal sewage.

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