Method for predicting existence of pseudomonas aeruginosa pollution or hazardous substances thereof in packaged drinking water

Abstract

The invention provides a method for predicting the existence of pseudomonas aeruginosa pollution or harmful substances thereof in packaged drinking water, which is used for judging whether the packaged drinking water is polluted or not by detecting the existence of pseudomonas aeruginosa metabolites in the packaged drinking water. The metabolite is prepared from 4-hydroxy-2-heptyl quinolinone, Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4 (1H)-ketone, 2, 4-dihydroxyquinoline and phenazine-1-formamide, and the metabolite is prepared from the following components: 2-hydroxy-2-heptyl quinolinone, Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4 (1H)-ketone, 2, 4-dihydroxyquinoline and According to the method disclosed by the invention, whether the packaged drinking water is polluted by the pseudomonas aeruginosa or not can be confirmed within one hour, the detection work can be directly finished in a common laboratory, and the requirements on experimental hardware, personnel quality and the like of microbiological detection are reduced; the confirmation result is intuitive, and the professional skill requirement on the research and judgment personnel of the microorganism result is reduced. The method established by the invention can be popularized as a common triple quadrupole mass spectrometry method, and is beneficial to large-area rapid screening of the condition that the packaged drinking water is polluted by pseudomonas aeruginosa.

Description

Technical Field

[0001] The invention relates to a method for predicting the presence of Pseudomonas aeruginosa contamination or harmful substances thereof in packaged drinking water, and belongs to the technical field of detection. Background Art

[0002] Pseudomonas aeruginosa is a widespread foodborne and waterborne opportunistic pathogen. It is also known as Pseudomonas aeruginosa because it synthesizes a green, water-soluble pigment during growth, resulting in the appearance of green pus at the site of infection. Because it can survive in low-nutrient water, it is widely distributed in various water bodies. Furthermore, it possesses and transfers drug-resistant genes, making it more difficult to eradicate than common Gram-negative bacteria.

[0003] According to the "National Food Safety Standard for Packaged Drinking Water" (GB 19298-2014), purified drinking water is packaged drinking water produced using water from a public water supply (commonly known as tap water) through distillation, electrodialysis, ion exchange, reverse osmosis, and other appropriate water purification processes. For example, the "distilled water" commonly found in supermarkets is purified drinking water. If packaged drinking water contains Pseudomonas aeruginosa, and if consumed in large quantities by patients or infants with damaged gastrointestinal mucosa, the endotoxins, exotoxins, hemolysins, and other pathogenic factors produced by the bacteria can infect the body and cause secondary diseases such as acute gastroenteritis, meningitis, and sepsis, and in severe cases, death.

[0004] The US FDA has established clear limits for Pseudomonas aeruginosa in medical devices and pharmaceuticals; the EU has stringently regulated testing requirements for cosmetics, drinking water, and other sectors; and China has also detailed regulations for the detection and control of this bacterium in national standards such as the "Sanitary Standards for Drinking Water." However, monitoring data indicates that excessive levels of Pseudomonas aeruginosa are still detected in some drinking water or purified water, and incidents of Pseudomonas aeruginosa poisoning occur occasionally. Therefore, timely, reliable, and rapid detection methods are crucial for monitoring this bacterium.

[0005] Currently, the detection methods for Pseudomonas aeruginosa are becoming increasingly diverse. Traditional methods rely on isolation and culture using selective media, with identification based on morphological observation of suspected colonies and associated physiological and biochemical reactions. While this method is highly accurate, it is also highly dependent on the expertise of the tester for interpretation. Furthermore, the corresponding toxin detection experiments are complex, requiring a minimum of three days for testing, and the test results are subject to significant lags. Molecular biology techniques such as PCR, ELISA, and gene chip technology are becoming increasingly popular due to their rapid and sensitive nature. However, these methods have drawbacks such as high laboratory requirements and personnel requirements, expensive equipment, and complex operational procedures.

[0006] For example, CN201910582662.7, titled "A Sensitive Detection Method for Microorganisms in Packaged Drinking Water," discloses a sensitive detection method for microorganisms in packaged drinking water, particularly suitable for detecting Pseudomonas aeruginosa in packaged drinking water. The present invention effectively separates potential microorganisms from a test sample through membrane separation, centrifugation, and other methods, and inoculates them into a liquid culture medium. Combined with a constant temperature oscillation culture method, the sensitivity of the detection is increased. After 8-24 hours of culture, optical detection methods can be used to objectively identify differences in the turbidity of the culture medium, thereby making a preliminary screening judgment and assessing the microbial contamination risk of the product. CN201711126058.0, Invention Title: Quadruple Fluorescent PCR Primer Set, Probe Set, Kit, and Method for Detecting Four Pathogenic Bacteria in Drinking Water, discloses a quadruple fluorescent PCR primer set for detecting four pathogenic bacteria in drinking water. The set includes primers for coliform bacteria, Streptococcus faecalis, Pseudomonas aeruginosa, and Clostridium perfringens. This method belongs to the field of genetic engineering detection technology. The primers and probes do not interfere with each other and can only amplify and emit fluorescent signals for specific target sequences, while non-target sequences are not amplified or emit fluorescent signals. This method can accurately detect the four pathogenic bacteria in drinking water, exhibiting advantages such as good specificity, small standard error, short detection time, and reagent cost savings. It is suitable for detecting pathogenic bacteria indicators in drinking water standards. CN202411597075.2, Invention Title: Primers, Probes, Kit, and Applications for Rapid Detection of Pseudomonas aeruginosa in Drinking Water, relates to RPA detection primers and probes for rapid detection of Pseudomonas aeruginosa in drinking water. The primers and probes exhibit good inclusiveness and exclusivity. Also provided is a rapid detection method for Pseudomonas aeruginosa based on the detection primers and probes, which can be used for rapid detection using a lateral flow test strip. Summary of the Invention

[0007] The present invention uses liquid chromatography-QTOF high-resolution mass spectrometry to discover and confirm markers in packaged drinking water under Pseudomonas aeruginosa stress, and establishes a rapid prediction method based on mass spectrometry-marker technology.

[0008] The present invention provides a method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water. The method determines whether the drinking water is contaminated by detecting the presence of Pseudomonas aeruginosa metabolites in the packaged drinking water. The metabolites include:

[0009] Pseudane V, 2-nonylquinolin-4(1H)-one, 2,4-dihydroxyquinoline.

[0010] The metabolites also contain 4-hydroxy-2-heptylquinolinone, phenazine-1-carboxylic acid, pyocyanin, phenazine-1-carboxamide and Pseudomonas aeruginosa chelating protein.

[0011] Wherein, the detection method is a mass spectrometry-biomarker detection method.

[0012] Wherein, the detection method comprises the following steps:

[0013] a. Sample preparation;

[0014] b. Preparation of standard solution:

[0015] Accurately weigh appropriate amounts of 4-hydroxy-2-heptylquinolinone, Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4(1H)-one, pyocyanin, 2,4-dihydroxyquinoline, phenazine-1-carboxamide, and Pseudomonas aeruginosa chelatin standards into a 10 mL volumetric flask. Dissolve the standards in 50% methanol-water, dilute to the mark, and shake well.

[0016] c. Liquid chromatography-QTOF mass spectrometry detection;

[0017] d. Judgment of test results: If the sample contains 4-hydroxy-2-heptylquinolinone,

[0018] Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4(1H)-one, pyocyanin, 2,4-dihydroxyquinoline, phenazine-1-carboxamide and Pseudomonas aeruginosa chelatin can be used to determine that packaged drinking water is contaminated with Pseudomonas aeruginosa.

[0019] Preferably, the sample preparation method in step a is: accurately aspirate 1 mL of water sample, accurately add 1 mL of chromatographic acetonitrile, vortex at high speed for 5 minutes to inactivate bacteria, centrifuge at 12000 rpm / min for 5 minutes at 5°C; and aspirate the supernatant for later use.

[0020] The liquid chromatography conditions for liquid chromatography-QTOF mass spectrometry detection in step c are:

[0021] Chromatographic column: Luna Omega Polar C18 column (2.1×100 mm, 1.6 μm); mobile phase: A: 2 mmol / L ammonium acetate solution (containing 0.1% formic acid), B: acetonitrile (containing 2 mM ammonium acetate and 0.1% formic acid, containing 5% water); flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 2 μL; gradient elution conditions: 0.0-4.0 min, 95%-70% mobile phase A;

[0022] 4.0-9.0 min, 70% to 5% mobile phase A; 9.0-14.0 min, 5% mobile phase A; 14.1-16.5 min, 95% mobile phase A;

[0023] The QTOF mass spectrometry conditions were as follows: ion source type: ESI source, curtain gas (nitrogen) flow rate: 30 psi; spray gas (nitrogen) flow rate: 50 psi; auxiliary heating gas (nitrogen) flow rate: 55 psi; ion source temperature: 500°C; ionization voltage: 5000 V / -4500 V.

[0024] After the liquid chromatography-QTOF mass spectrometry detection, data acquisition was performed using the TOFMS-IDA-TOFMSMS mode, wherein the TOF MS acquired an m / z range of 100-1300 with an accumulation time of 0.13 s; the TOF MS simultaneously acquired product secondary ion fragments of 10 compounds with an accumulation time of 0.05 s; the secondary mass range was MS / MS (CID), m / z 50–1300; the declustering voltage was 80 V / -80, and the collision voltage was 35 V±15 V / -35 V±15 V.

[0025] The metabolite spectrum is as follows Figure 11 As shown, the retention times of the metabolites are: 8.37 min; 7.22 min; 7.10 min; 9.43 min; 3.18 min; 4.91 min; 6.57 min; 7.48 min, with a retention time fluctuation of 10%.

[0026] This invention innovatively uses mass spectrometry-biomarker technology to establish a method for predicting contamination of packaged drinking water with Pseudomonas aeruginosa. The present invention discovered eight novel compounds remaining in water contaminated with Pseudomonas aeruginosa. Three of these substances, Pseudane V, 2-nonylquinolin-4(1H)-one, and 2,4-dihydroxyquinoline, are newly discovered biomarkers for Pseudomonas aeruginosa. Furthermore, 4-hydroxy-2-heptylquinolinone, phenazine-1-carboxylic acid, pyocyanin, and phenazine-1-carboxamide Pseudomonas aeruginosa chelatin have been detected in packaged drinking water for the first time. Because this method is the first to use mass spectrometry-biomarker technology to detect contamination of water with Pseudomonas aeruginosa, and given the lack of previous reports of using this technology to detect this bacterium in packaged drinking water, the present invention can utilize these eight biomarkers for diagnosis.

[0027] The beneficial effects of the present invention are:

[0028] The method established by the present invention can confirm whether packaged drinking water is contaminated with Pseudomonas aeruginosa within one hour. Compared with the current GB 8538-2022 (Pseudomonas aeruginosa) test cycle of three days or more, this greatly shortens the test cycle and improves test efficiency. Compared with the methods disclosed in patents CN201910582662.7, CN201711126058.0, and CN202411597075.2, this method also shortens the test cycle and reduces the requirements for laboratory hardware and personnel.

[0029] The method developed by this invention can be performed directly in a common laboratory, reducing the requirements for experimental hardware and personnel quality for microbial testing. Its intuitive confirmation results reduce the professional skills required for interpreting microbial results. The method developed by this invention can be extended to a common triple quadrupole mass spectrometry method, facilitating large-scale rapid screening of packaged drinking water for Pseudomonas aeruginosa contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 TIC graph and BPC graph in positive ion mode;

[0031] Figure 2 TIC graph and BPC graph in negative ion mode;

[0032] Figure 3 Chromatogram, mass spectrum, and secondary fragmentation pattern of 4-hydroxy-2-heptylquinolinone;

[0033] Figure 4 Chromatogram, mass spectrum, and secondary fragmentation pattern of Pseudane V;

[0034] Figure 5 Chromatogram, mass spectrum, and secondary fragmentation pattern of phenazine-1-carboxylic acid;

[0035] Figure 6 Chromatogram, mass spectrum, and secondary fragmentation pattern of 2-nonylquinolin-4(1H)-one;

[0036] Figure 7 Chromatogram, mass spectrum, and secondary fragmentation pattern of pyocyanin;

[0037] Figure 8 Chromatogram, mass spectrum, and secondary fragmentation pattern of 2,4-dihydroxyquinoline;

[0038] Figure 9 Chromatogram, mass spectrum, and secondary fragmentation pattern of phenazine-1-carboxamide;

[0039] Figure 10 Chromatogram, mass spectrum and secondary fragmentation pattern of Pseudomonas aeruginosa ferritin;

[0040] Figure 11 Prediction model for Pseudomonas aeruginosa contamination of water bodies;

[0041] Figure 12 Negative sample spectrum. DETAILED DESCRIPTION

[0042] Example 1 Method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water

[0043] 1 Materials and Methods

[0044] 1.1 Materials

[0045] Standards of 4-hydroxy-2-heptylquinolinone (CAS No.: 611-36-9), Pseudane V (CAS No.: 109072-26-6), phenazine-1-carboxylic acid (CAS No.: 2538-68-3), 2-nonylquinolin-4(1H)-one (CAS No.: 55396-45-7), pyocyanin (CAS No.: 85-66-5), 2,4-dihydroxyquinoline (CAS No.: 86-95-3), phenazine-1-carboxamide (CAS No.: 550-89-0), and Pseudomonas aeruginosa ferritin (CAS No.: 79236-62-7) were purchased from Tianjin Alta Technology Co., Ltd.

[0046] Pseudomonas aeruginosa strains were purchased from the ATCC Biological Standard Resource Center in the United States. The strains were activated and diluted to 10 -7 stand-by.

[0047] Mass spectrometry grade methanol and mass spectrometry grade acetonitrile (Merck, Germany), mass spectrometry grade formic acid and mass spectrometry grade ammonium acetate (Sigma-Aldrich, Germany); all laboratory water is real-time prepared first-class water; a certain brand of packaged drinking water is used as simulated water for the experiment.

[0048] 1.2 Instruments and Equipment

[0049] Liquid chromatography-QTOF high-resolution time-of-flight mass spectrometer (SCIEX, USA); Centrifuge 5810R high-speed centrifuge (Eppendorf, Germany); Milli-Q ultrapure water instrument (Millipore, USA); BP211D balance (Sartorius, Germany); ME203 balance (Mettler Toledo, Switzerland).

[0050] 1.3 Methods

[0051] 1.3.1 Preparation of standard solution

[0052] Accurately weigh appropriate amounts of 4-hydroxy-2-heptylquinolinone, Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4(1H)-one, pyocyanin, 2,4-dihydroxyquinoline, phenazine-1-carboxamide, and Pseudomonas aeruginosa chelatin standards into a 10 mL volumetric flask. Dissolve the standards in 50% methanol-water, dilute to the mark, and shake well for qualitative confirmation.

[0053] 1.3.2 Sample processing

[0054] 1.3.2.1 Preparation of positive samples

[0055] Accurately pipette 20mL of negative packaged drinking water (negative according to GB 8538-2022 test) and add 10 -7 After adding 1 mL of bacterial solution, it was cultured at 25°C for 3 days. This process simulates the entire chain of traditional packaged water production, processing, transportation and storage.

[0056] Accurately draw 1 mL of water sample, add 1 mL of chromatographic acetonitrile, vortex at high speed for 5 minutes to inactivate bacteria, and centrifuge at 12000 rpm / min for 5 minutes at 5°C. Accurately draw the supernatant for UHPLC-QTOF analysis

[0057] 1.3.3 Liquid chromatography-QTOF mass spectrometry analysis

[0058] 1.3.3.1 Liquid chromatography conditions

[0059] Chromatographic column: Luna Omega Polar C18 column (2.1×100 mm, 1.6 μm); mobile phase: A is 2 mmol / L ammonium acetate solution (containing 0.1% formic acid), B is acetonitrile (containing 2 mM ammonium acetate and 0.1% formic acid, containing 5% water); flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0060] The gradient elution conditions of the chromatography were as follows: 0.0-4.0 min, 95% to 70% mobile phase A; 4.0-9.0 min, 70% to 5% mobile phase A; 9.0-14.0 min, 5% mobile phase A; 14.1-16.5 min, 95% mobile phase A;

[0061] 1.3.3.2 QTOF mass spectrometry conditions

[0062] Ion source type: ESI source, curtain gas (nitrogen) flow rate: 30 psi; nebulizer gas (nitrogen) flow rate: 50 psi; auxiliary heater gas (nitrogen) flow rate: 55 psi; ion source temperature: 500°C; ionization voltage: 5000 V / -4500 V. Data acquisition was performed using TOFMS-IDA-TOFMSMS mode: 1. TOFMS acquisition was performed over the m / z range of 100-1300, with an accumulation time of 0.13 s. 2. TOFMS simultaneously acquired secondary fragment ions of 10 compounds, with an accumulation time of 0.05 s. 3. Secondary mass range: MS / MS (CID), m / z 50–1300. 4. DBS dynamic background subtraction was enabled throughout the analysis to automatically eliminate background interference and remove invalid information, thereby ensuring the validity of the secondary data. 5. Declustering voltage: 80 V / -80 V, collision voltage: 35 V ± 15 V / -35 V ± 15 V.

[0063] Mass spectrometry data were collected and analyzed using SCIEX OS2.2 and Peak View 2.2.

[0064] 2 Results and Analysis

[0065] 2.1 Pre-processing process

[0066] Since packaged drinking water is relatively pure and contains less impurities, the study used methanol, acetonitrile, and salt to inactivate bacteria. Acetonitrile can effectively precipitate proteins and is better adapted to sample detection using mass spectrometry than salt. In the present invention, by comparing the precipitation effect of the acetonitrile to sample ratio, it was determined that adding 1 mL of acetonitrile to 1 mL of water inactivated bacteria.

[0067] 2.1 Marker identification based on non-targeted technology

[0068] like Figure 1 and Figure 2 As shown, the chromatographic conditions and mass spectrometry conditions used in the present invention obtained the TIC diagram in the positive and negative ion modes, and the strongest ion intensity at each time point in the TIC diagram was continuously depicted to obtain a BPC diagram. It can be seen from the BPC diagram that the compound distribution profile obtained by this method is uniform and the peak rows are symmetrical, thereby achieving efficient separation and data acquisition of Pseudomonas aeruginosa markers.

[0069] Peak View 2.2 was used to analyze the above data. It is mainly based on the mass deviation, isotope distribution and molecular formula fitting of the primary mass spectrum and the secondary mass spectrum of the high-resolution mass spectrometer. The compounds identified above were identified in combination with databases such as Mycotoxin HR-MS / MS Spectral Library 1.0, Accurate Mass Metabolite HR-MS / MS Spectral Library 2.0, SCIEX All-in-one with NIST 2017, and ChemSpider. The list of identified compounds is shown in Table 2

[0070] Table 2 Information of compounds identified by non-targeted methods

[0071]

[0072]

[0073]

[0074] 2.2 Qualitative confirmation

[0075] The compounds with high absolute response and good peak shape in Table 2, such as 4-hydroxy-2-heptylquinolinone, Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4(1H)-one, pyocyanin, 2,4-dihydroxyquinoline, phenazine-1-carboxamide, and Pseudomonas aeruginosa chelatin, were selected as the main markers and qualitative confirmation was performed by purchasing relevant standard substances. The standard solution prepared in 1.3.1 and the sample solution treated in 1.3.2 were collected and compared according to the method shown in 1.3.3 to perform qualitative confirmation of the compounds. The results are shown in Figure 1. Figure 3-10 shown.

[0076] Comprehensive comparison of the chromatograms, mass spectra and secondary fragmentation patterns of the standard solutions confirmed that the above eight compounds were specific biomarkers produced after drinking water was contaminated by Pseudomonas aeruginosa.

[0077] 2.3 Specific Information of the 8 Biomarkers

[0078] 4-Hydroxy-2-heptylquinolinone: also known as Pseudane-VII, this compound is a signal molecule produced by Pseudomonas aeruginosa. This compound is directly related to the reproduction and metabolism of Pseudomonas aeruginosa.

[0079] Pseudane V: This type of bacterial secondary metabolite was first discovered in the study of metabolites of marine Pseudomonas aeruginosa and was first found in drinking water contaminated by Pseudomonas aeruginosa.

[0080] Phenazine-1-carboxylic acid: also known as Tubermycin B, a secondary metabolite widely found in microorganisms such as Pseudomonas and Streptomyces.

[0081] Pseudane IX: also known as 2-nonylquinolin-4(1H)-one, this compound was previously extracted only from a small number of plants such as rue leaves. It was first discovered in drinking water contaminated by Pseudomonas aeruginosa.

[0082] Pyocyanin: This compound is a characteristic biomarker of Pseudomonas aeruginosa. This type of compound has certain toxicity. It is irritating to the skin and mucous membranes and may cause allergic reactions.

[0083] 2,4-Dihydroxyquinoline: also known as Quinoline-2,4-diol, was first discovered in drinking water contaminated by Pseudomonas aeruginosa.

[0084] Phenazine-1-carboxamide: also known as shenqinmycin, is a very important natural product that exists in microorganisms such as Streptomyces and Pseudomonas aeruginosa.

[0085] Pseudomonas aeruginosa ferritin: also known as Pyochelin, is an iron carrier of Pseudomonas aeruginosa and an important biomarker and virulence factor of Pseudomonas aeruginosa.

[0086] 2.4 Prediction model based on eight biomarkers

[0087] Refer to 1.3.2 and 1.3.3 to build a prediction model for predicting Pseudomonas aeruginosa contamination of water bodies. Figure 11 , relative to the negative sample spectrum ( Figure 12 ), the chromatographic peak signals of the 8 markers are obvious and intuitive. This method can simultaneously detect the above 8 compounds to determine that the packaged drinking water is contaminated by Pseudomonas aeruginosa.

[0088] Among them, the source of negative samples is: accurately transfer 20mL of negative packaged drinking water (using GB

[0089] 8538-2022 tested negative), a dilution of 10 -7 After adding 1 mL of bacterial solution, it was cultured at 25°C for 3 days. This process simulates the entire chain of production, processing, transportation and storage of traditional packaged drinking water.

[0090] Accurately pipette 1 mL of water sample, add 1 mL of chromatographic acetonitrile, vortex at high speed for 5 minutes to inactivate bacteria, and centrifuge at 12,000 rpm / min for 5 minutes at 5°C. Accurately pipette the supernatant for UHPLC-QTOF analysis.

[0091] In summary, the eight compounds used in the present invention as markers for Pseudomonas aeruginosa contamination of packaged drinking water have high credibility.

Claims

1. A method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water, characterized by: It determines whether packaged drinking water is contaminated by detecting the presence of Pseudomonas aeruginosa metabolites in the packaged drinking water. The metabolites include: Pseudane V, 2-nonylquinolin-4(1H)-one, 2,4-dihydroxyquinoline.

2. The method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water according to claim 1, characterized in that: The metabolites also contain 4-hydroxy-2-heptylquinolinone, phenazine-1-carboxylic acid, pyocyanin, phenazine-1-carboxamide and Pseudomonas aeruginosa chelating protein.

3. The method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water according to claim 1 or 2, characterized in that: The detection method is a mass spectrometry-biomarker detection method.

4. The method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water according to claim 3, characterized in that: The detection method comprises the following steps: a. Sample preparation; b. Preparation of standard solution: Accurately weigh appropriate amounts of 4-hydroxy-2-heptylquinolinone, Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4(1H)-one, pyocyanin, 2,4-dihydroxyquinoline, phenazine-1-carboxamide, and Pseudomonas aeruginosa chelatin standards into a 10 mL volumetric flask. Dissolve the standards in 50% methanol-water, dilute to the mark, and shake well. c. Liquid chromatography-QTOF mass spectrometry detection; d. Interpretation of test results: If the sample contains 4-hydroxy-2-heptylquinolinone, Pseudane V, phenazine-1-carboxylic acid, 2-nonylquinolin-4(1H)-one, pyocyanin, 2,4-dihydroxyquinoline, phenazine-1-carboxamide and Pseudomonas aeruginosa chelatin, it can be determined that the packaged drinking water is contaminated with Pseudomonas aeruginosa.

5. The method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water according to claim 4, characterized in that: The sample preparation method described in step a is as follows: accurately aspirate 1 mL of water sample, accurately add 1 mL of chromatographic acetonitrile, vortex at high speed for 5 minutes to inactivate bacteria, centrifuge at 12000 rpm / min at 5°C for 5 minutes; and aspirate the supernatant for later use.

6. The method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water according to claim 4, characterized in that: The liquid chromatography conditions for liquid chromatography-QTOF mass spectrometry detection in step c are: Chromatographic column: Luna Omega Polar C18 column (2.1×100 mm, 1.6 μm); mobile phase: A: 2 mmol / L ammonium acetate solution (containing 0.1% formic acid), B: acetonitrile (containing 2 mM ammonium acetate and 0.1% formic acid, containing 5% water); flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 2 μL; gradient elution conditions: 0.0-4.0 min, 95%-70% mobile phase A; 4.0-9.0 min, 70% to 5% mobile phase A; 9.0-14.0 min, 5% mobile phase A; 14.1-16.5 min, 95% mobile phase A; The QTOF mass spectrometry conditions were as follows: ion source type: ESI source, curtain gas (nitrogen) flow rate: 30 psi; spray gas (nitrogen) flow rate: 50 psi; auxiliary heating gas (nitrogen) flow rate: 55 psi; ion source temperature: 500°C; ionization voltage: 5000 V / -4500 V.

7. The method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water according to claim 4, characterized in that: After the liquid chromatography-QTOF mass spectrometry detection, data acquisition was performed using the TOFMS-IDA-TOFMSMS mode, wherein the TOF MS acquired an m / z range of 100-1300 with an accumulation time of 0.13 s; the TOF MS simultaneously acquired product secondary ion fragments of 10 compounds with an accumulation time of 0.05 s; the secondary mass range was MS / MS (CID), m / z 50–1300; the declustering voltage was 80 V / -80, and the collision voltage was 35 V±15 V / -35 V±15 V.

8. The method for predicting the presence of Pseudomonas aeruginosa contamination or its harmful substances in packaged drinking water according to any one of claims 1 to 4, characterized in that: The spectrum of the metabolites is shown in FIG11 , wherein the retention times of the metabolites are: 8.37 min; 7.22 min; 7.10 min; 9.43 min; 3.18 min; 4.91 min; 6.57 min; 7.48 min, and the retention time fluctuates by 10%.

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