Molecular targets for identification of pseudomonas aeruginosa and method for quantitative detection thereof

Abstract

The application discloses a molecular target for identifying pseudomonas aeruginosa and a detection method thereof, and belongs to the technical field of microorganisms. The molecular target for identifying pseudomonas aeruginosa is shown in any one of SEQ ID NO. 1 to SEQ ID NO. 4. The molecular target can detect more pseudomonas aeruginosa, has a larger coverage of pseudomonas aeruginosa, and enhances practicability. The detection method has the advantages of simple operation, easy result determination, short detection time, strong specificity, low cost, and good stability.

Description

[0001] This application is a divisional application of CN114107532A (application date: December 20, 2021; application number: 2021115607002; invention title: Molecular Target for Identification of Pseudomonas aeruginosa and Quantitative Detection Method thereof). Technical Field

[0002] This invention belongs to the field of microbial technology, specifically relating to molecular targets for the identification of Pseudomonas aeruginosa and their detection methods. Background Technology

[0003] Pseudomonas aeruginosa, commonly known as Pseudomonas aeruginosa, is widely distributed in nature and is one of the most common bacteria in freshwater and soil. It can be found in the air, on animal skin, in the intestines, and in the respiratory tract. This bacterium can produce various exotoxins and endotoxins, and can be transmitted through water sources, soil, tools, and contact. It is highly resistant to disinfectants, drying, ultraviolet light, and other physical and chemical factors, as well as adverse environmental conditions, and is a complete pathogen causing acute intestinal diseases and skin inflammation. In recent years, reports of contamination of packaged drinking water by this bacterium have increased significantly, indicating a serious contamination situation. Early studies found that the detection rate of *Pseudomonas aeruginosa* in commercially available natural mineral water in my country could reach 23.6%, and in bottled purified drinking water, it could reach 11.6%. In 2018, a water sample inspection in Guangdong Province showed that the contamination rate of *P. aeruginosa* in packaged drinking water and natural mineral water reached 8.36% (23 / 275). Internationally, a survey in Germany found *P. aeruginosa* in 1.2%-10.2% of packaged drinking water samples; Greece found a *P. aeruginosa* contamination rate of 5.9% (90 / 1527) in sampled mineral water; and an Australian study found a contamination rate of 10%. 5 CFU / mL.

[0004] Furthermore, the contamination of ready-to-eat vegetables and fruits by *Pseudomonas aeruginosa* has consistently posed a threat to consumer health. While ready-to-eat vegetables have gained popularity in recent years due to their perceived health benefits, they are also a common source of foodborne illnesses. Jamaican researchers have found widespread *P. aeruginosa* contamination in retail markets and supermarkets, with a higher frequency of contamination in market-sourced produce. Their research on *P. aeruginosa* resistance and virulence factors in fresh vegetable substrates revealed that this bacterium is a major contaminant in fresh vegetables and may be a source of infection for susceptible populations in the community. Early studies found that *P. aeruginosa* contamination rates in vegetable salads could reach 64.5%. With improved sanitation, in 2015, researchers found that the incidence of *P. aeruginosa* contamination in tomato or bell pepper products was as high as 5%. *Pseudomonas aeruginosa* can contaminate ready-to-eat vegetables and fruits at many stages. For example, before harvest, *P. aeruginosa* populations can colonize growing crops. After harvest, the risk can be amplified through further direct contamination or the spread of existing populations during processing and post-harvest handling. Water contaminated with *P. aeruginosa* can form chains of contamination into fields, including runoff from nearby animal pastures and irrigation from contaminated sources. *P. aeruginosa* has low requirements for organic nutrients and is a dominant bacteria in the spoilage of vegetables and fruits such as lettuce, tomatoes, and cucumbers, seriously affecting the transportation and storage of ready-to-eat vegetables and fruits. If infection is caused by ingestion of *P. aeruginosa*, a healthy person needs more than 10... 5 The bacterial concentration of CFU / g, but even if it is less than 10 3 Even at concentrations as low as CFU / g, Pseudomonas aeruginosa can colonize the intestines of susceptible individuals, potentially leading to gastrointestinal infections, bacteremia, and bloodborne transmission. Infants may also develop Pseudomonas aeruginosa sepsis, characterized by necrotizing intestinal lesions with a history of diarrhea. Therefore, rapid identification and accurate quantification of Pseudomonas aeruginosa are crucial for preventing contamination in food safety, drinking water safety, and clinical treatment.

[0005] The development of molecular biology has facilitated the rapid identification of *Pseudomonas aeruginosa*. The current standard SN / T2206.12-2014, "Microbiological Examination Methods for Cosmetics Part 12: PCR Method for *Pseudomonas aeruginosa*", uses the conserved exotoxin A gene fragment of *P. aeruginosa* as the target fragment, designs PCR amplification primers, and performs rapid detection of *P. aeruginosa* in cosmetics. Furthermore, there are some reports on molecular detection targets and primers for *P. aeruginosa* using PCR detection methods both domestically and internationally. Common specific genes include SyrB, toxA, I6S-23S, l6S rDNA, oprl, fliC, ecfX, ecfX+gyrB, ETA, opr, exoU, and exoS. Among methods for detecting *P. aeruginosa*, the traditional culture method is the gold standard. However, the biochemical identification in the traditional method requires approximately 48 hours of enrichment, followed by 24–48 hours of chromogenic culture and further biochemical identification. The testing cycle takes about a week or longer, making the process cumbersome and time-consuming. Meanwhile, traditional methods of determining whether a strain is *Pseudomonas aeruginosa* based on whether it produces a copper-green pigment can lead to missed or false positives. For example, the current Chinese national standard GB8538—2016, "National Food Safety Standard: Test Methods for Drinking Natural Mineral Water," identifies *P. aeruginosa* based on the production of blue / green colonies. However, *P. putrefactive bacteria* also produces blue / green under the same conditions, so counting suspected colonies based on pigment production may result in false positives. This standard also classifies colonies other than those producing blue / green, fluorescence, or reddish-brown pigments as non-*P. aeruginosa*, but studies have shown that some *P. aeruginosa* strains do not produce pigment, which could lead to inaccurate positive results. Furthermore, the application of biochemical tests to identify *P. aeruginosa* is prone to misdiagnosis due to unstable biochemical reactions and poor repeatability of results.

[0006] In practical applications, it has been found that with the continuous discovery of variant and drug-resistant strains of *Pseudomonas aeruginosa* during clinical treatment, existing molecular detection methods based on virulence factors as detection targets are inaccurate due to the ease of target deletion and limited number of targets. Furthermore, with the rapid development of whole-genome sequencing technology and the continuous improvement of gene banks, existing targets do not fully cover *P. aeruginosa* strains, resulting in poor specificity of *P. aeruginosa* molecular targets. The limited number and poor specificity of targets pose significant challenges to the rapid and accurate identification of *P. aeruginosa*. Therefore, finding novel specific target molecules for the rapid and accurate detection of *P. aeruginosa* is of great significance. A literature search of existing technologies has not yet found any reports of novel *P. aeruginosa*-specific molecular targets and corresponding PCR and qPCR methods similar to those of this invention. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a molecular target and detection method for the identification of Pseudomonas aeruginosa. The detection method of this invention has the advantages of simple operation, easy result interpretation, short detection time, high specificity, low cost and good stability for the detection of Pseudomonas aeruginosa.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is: a molecular target for the identification of Pseudomonas aeruginosa, wherein the sequence of the molecular target is shown in any one of SEQ ID NO.1 to SEQ ID NO.4.

[0009] The inventors of this application analyzed the whole genome data of 1000 *Pseudomonas aeruginosa* strains and 1017 non-*Pseudomonas aeruginosa* strains from the NCBI database using pan-genome analysis. Through extensive screening and design validation, they ultimately obtained the aforementioned molecular target for *P. aeruginosa* identification. Extensive experimental validation has shown that the molecular target of this invention, used for *P. aeruginosa* identification, exhibits excellent specificity and greater coverage, achieving a detection rate of up to 100%, far exceeding the positive detection rate of the toxA gene for *P. aeruginosa* in existing technologies.

[0010] The present invention also provides a primer set for identifying Pseudomonas aeruginosa, the primer set being used to detect the molecular targets described in any one of SEQ ID NO. 1 to SEQ ID NO. 4; wherein the sequences of the primer set for detecting the molecular target described in SEQ ID NO. 1 are shown in SEQ ID NO. 5 and SEQ ID NO. 6; the sequences of the primer set for detecting the molecular target described in SEQ ID NO. 2 are shown in SEQ ID NO. 7 and SEQ ID NO. 8; the sequences of the primer set for detecting the molecular target described in SEQ ID NO. 3 are shown in SEQ ID NO. 9 and SEQ ID NO. 10; and the sequences of the primer set for detecting the molecular target described in SEQ ID NO. 4 are shown in SEQ ID NO. 11 and SEQ ID NO. 12.

[0011] The present invention also provides primer sets for identifying Pseudomonas aeruginosa, said primer sets being used to detect the molecular targets described in any one of SEQ ID NO. 1 to SEQ ID NO. 4; wherein, the sequences of the primer sets for detecting the molecular target described in SEQ ID NO. 1 are shown in SEQ ID NO. 13 and SEQ ID NO. 14; the sequences of the primer sets for detecting the molecular target described in SEQ ID NO. 2 are shown in SEQ ID NO. 15 and SEQ ID NO. 16; the sequences of the primer sets for detecting the molecular target described in SEQ ID NO. 3 are shown in SEQ ID NO. 17 and SEQ ID NO. 18; and the sequences of the primer sets for detecting the molecular target described in SEQ ID NO. 4 are shown in SEQ ID NO. 19 and SEQ ID NO. 20.

[0012] Based on the molecular targets for Pseudomonas aeruginosa identification obtained through screening, this invention designed a series of primer sets. After extensive screening and verification, it was found that the final primer sets have excellent specificity for the identification of Pseudomonas aeruginosa, with a coverage rate of 100%.

[0013] The present invention also provides a method for identifying Pseudomonas aeruginosa, comprising the following steps:

[0014] (1) Use at least one of the primer sets above to perform PCR amplification on the DNA of the sample to be tested;

[0015] (2) The amplification products were detected by gel electrophoresis;

[0016] (3) Observe whether the amplification products meet expectations.

[0017] The amplification products of the method for identifying Pseudomonas aeruginosa of the present invention have good specificity. The presence of Pseudomonas aeruginosa can be determined by observing whether the amplification products are in the expected positions.

[0018] As a preferred embodiment of the method for identifying Pseudomonas aeruginosa according to the present invention, the PCR amplification system is as follows: 2.5 μL of 10×PCR reaction buffer, 25 mmol / L, 2 μL of MgCl2, 1 μL of 2.5 mmol / L dNTP, 100 ng of template DNA, 1 μL each of 10 μmol / L primers, 1 U of Tag enzyme, and sterile double-distilled water to a final volume of 25 μL; the PCR amplification program is as follows: 98℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 60℃ annealing for 30 s; 72℃ extension for 45 s; 30 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 10 min.

[0019] The present invention also provides a method for quantitative detection of Pseudomonas aeruginosa, wherein the method uses at least one set of primers from the above-mentioned primer set to perform qPCR amplification of the DNA of the sample to be tested and analyzes the amplification results.

[0020] The amplification products of the method for quantitative detection of Pseudomonas aeruginosa of this invention have good specificity, and the concentration of Pseudomonas aeruginosa in the system is quantitatively detected by judging the intensity of the fluorescence signal in the system. The qPCR method established by this invention has ideal linearity, and the detection limit for pure bacteria can be as low as 10. 2 The detection method established in this invention has good sensitivity, comparable to or better than existing methods reported in the literature by 1-2 orders of magnitude, even at CFU / mL concentrations. Furthermore, the method established in this invention has strong anti-interference capabilities, even at coliform concentrations of 10... 8 At CFU / mL, the detection concentration is 10. 4 Pseudomonas aeruginosa does not cause interference.

[0021] As a preferred embodiment of the method for quantitative detection of Pseudomonas aeruginosa according to the present invention, the qPCR amplification system is as follows: 10 μL of 2×TBGreenPremix reaction solution, 100 ng of template DNA, 1 μL of each of 10 μmol / L primers, and sterile double-distilled water to make up to 20 μL; the qPCR amplification program is as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, and a total of 40 cycles of denaturation and annealing.

[0022] The present invention also provides the novel molecular target for the identification of Pseudomonas aeruginosa or the application of the primer set in the identification of Pseudomonas aeruginosa.

[0023] The present invention also provides the novel molecular target for the identification of Pseudomonas aeruginosa or the application of the primer set in the quantitative detection of Pseudomonas aeruginosa.

[0024] The present invention also provides a kit for identifying Pseudomonas aeruginosa, the kit comprising the primer set described above.

[0025] The beneficial effects of the present invention: The present invention provides a set of new molecular targets for the identification of Pseudomonas aeruginosa and their detection methods. (1) The detection method of the present invention can detect more Pseudomonas aeruginosa, with a larger coverage of Pseudomonas aeruginosa, and the coverage of Pseudomonas aeruginosa is 100%, which enhances its practicality; (2) The detection rate of the new molecular targets for identifying Pseudomonas aeruginosa of the present invention is 100% for 95 strains of Pseudomonas aeruginosa, while the positive detection rate of the toxA gene for strains reported in the literature is 82.1%, indicating that the new molecular targets for Pseudomonas aeruginosa of the present invention have good specificity; (3) The detection method of the present invention has the advantages of simple operation, easy result judgment, short detection time, strong specificity, low cost and good stability for the detection of Pseudomonas aeruginosa. The entire detection process of the method established by the present invention takes about 15 hours, while the traditional method takes about 3 to 5 days to complete. The detection method of the present invention greatly saves detection time and improves detection efficiency. Attached Figure Description

[0026] Figure 1 To evaluate the specificity of the PCR identification method for Pseudomonas aeruginosa using electrophoresis results;

[0027] Figure 2 Results of establishing a qPCR quantitative detection method for pure culture of Pseudomonas aeruginosa;

[0028] Figure 3 The results of establishing a qPCR quantitative detection method for Pseudomonas aeruginosa in artificially contaminated samples;

[0029] Figure 4 The results show the establishment of the qPCR quantitative detection method for Pseudomonas aeruginosa in actual samples in Example 7. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0031] Example 1: Design of screening for specific novel molecular targets for the identification of Pseudomonas aeruginosa

[0032] Based on the genomic data of 1000 *Pseudomonas aeruginosa* strains and 1017 non-*Pseudomonas aeruginosa* strains from the NCBI website, pan-genome analysis was performed. Through extensive screening and design validation, molecular targets for the identification of *Pseudomonas aeruginosa* were finally obtained. The nucleotide sequences of the molecular targets are shown in SEQ ID NO.1-SEQ ID NO.4.

[0033] Example 2: PCR method for identifying Pseudomonas aeruginosa

[0034] This embodiment provides four rapid detection methods. Primers can be designed based on any one of the four specific novel molecular targets of Pseudomonas aeruginosa to form a rapid detection method, which includes the following steps:

[0035] (1) Primer design: Based on the sequences SEQ ID NO.1 to SEQ ID NO.4 described in Example 1, a specific PCR amplification primer set was designed. The primer set sequences are shown in Table 1 below.

[0036] (2) DNA template preparation: The bacterial strains to be tested were enriched in LB liquid medium, and their bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as the templates to be tested.

[0037] (3) PCR amplification of the DNA sample to be tested was performed using the primer set described above; a. PCR detection system: 2.5 μL 10×PCR reaction buffer, 25 mmol / L MgCl2, 2 μL 2.5 mmol / L dNTP, 100 ng template DNA, 1 μL each of 10 μmol / L primers, 1 U Tag enzyme, and sterile double-distilled water to a final volume of 25 μL. b. PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 45 s; 30 cycles of denaturation, annealing, and extension were performed; and finally, 72℃ extension for 10 min.

[0038] (4) Perform gel electrophoresis on PCR amplification products;

[0039] (5) Observe whether there is a single amplification band at the position of the product size corresponding to each primer set. If it is present, it indicates that the sample contains Pseudomonas aeruginosa; if no corresponding single amplification band appears, the sample does not contain the corresponding Pseudomonas aeruginosa.

[0040] Table 1 Primer set for specific PCR detection

[0041]

[0042] Example 3: Specificity evaluation of the PCR detection method for Pseudomonas aeruginosa

[0043] Evaluation method: Ninety-five strains of *Pseudomonas aeruginosa* and 39 strains of other species were used as non-target bacteria, and PCR detection was performed according to the method in Example 2. Specifically, S1 DNA template preparation involved extracting genomic DNA from each bacterium; S2 PCR amplification used a primer set. A blank control was set up, with the template being an aqueous solution containing no genomic DNA.

[0044] Evaluation Results: The bacterial strains used and the test results are shown in Table 2 below. In the table, "+" indicates positive and "-" indicates negative in the test result column. The electrophoresis results of the PCR products are shown below. Figure 1 As shown in the figure; numbers 1-5 represent *Pseudomonas aeruginosa*; numbers 6-44 represent non-target strains; M stands for 2000 Maker. Figure 1 As shown in Table 2, the detection results of the PCR primer set showed that only Pseudomonas aeruginosa showed specific amplification bands, while non-target bacterial strains did not show specific bands, indicating that the identification method of the present invention has high specificity.

[0045] Table 2 Results of the specificity evaluation test for Pseudomonas aeruginosa identification

[0046]

[0047]

[0048] Example 4: Method for quantitative detection of Pseudomonas aeruginosa

[0049] This embodiment of a qPCR method for quantitative detection of Pseudomonas aeruginosa includes the following steps:

[0050] (1) Primer design: Based on the sequences SEQ ID NO.1-SEQ ID NO.4 described in Example 1, a specific qPCR amplification primer set was designed. The primer set sequence is shown in Table 3 below.

[0051] (2) DNA template preparation: Pseudomonas aeruginosa was enriched in LB liquid medium, and its bacterial genomic DNA was extracted using a commercially available bacterial genomic DNA extraction kit as the template to be tested.

[0052] (3) PCR amplification: The DNA of the test sample was amplified by PCR using primer set 5 as described above. The qPCR was performed on a 96 fluorescence quantitative amplification instrument. a. qPCR detection system: 10 μL of 2×TBGreenPremix reaction solution, 100 ng of template DNA, 1 μL of each of 10 μmol / L primers, and sterile double-distilled water to a final volume of 20 μL; b. qPCR amplification program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing for 30 s; a total of 40 cycles of denaturation and annealing were performed.

[0053] (4) Using software 96SW1.1 Analyze whether the amplification results meet expectations. If a fluorescence signal is generated in the blank control (where no fluorescence signal is present), it indicates the presence of *Pseudomonas aeruginosa* in the sample; if no fluorescence signal is generated, the sample does not contain *Pseudomonas aeruginosa*.

[0054] Table 3 Primer set for specific qPCR detection

[0055]

[0056] Example 5: Specificity Evaluation of Pseudomonas aeruginosa Identification Using qPCR Method

[0057] Evaluation method: 63 strains of Pseudomonas aeruginosa and 32 strains of other species were used as non-target bacteria. Genomic DNA was extracted from each bacteria. One blank control was used. The primers used were designed based on primer set sequences. The template for the blank control was an aqueous solution without genome.

[0058] Evaluation Results: The bacterial strains used and the test results are shown in Table 4 below. In the table, "+" indicates positive and "-" indicates negative in the test result column. The qPCR fluorescence results are shown below. Figure 2 As shown in the figure; numbers 1-5 represent *Pseudomonas aeruginosa*; numbers 6-37 represent non-target strains. Figure 2 As shown in Table 4, the detection results of the qPCR primer set showed that only Pseudomonas aeruginosa showed fluorescent signals, while non-target bacterial strains and blanks did not produce fluorescent signals, indicating that the qPCR identification method of the present invention has high specificity.

[0059] Table 4. Results of the specificity evaluation test for the qPCR method for identifying Pseudomonas aeruginosa.

[0060]

[0061]

[0062]

[0063] Example 6: Sensitivity Evaluation of qPCR Quantitative Detection Method for Pure Culture of Pseudomonas aeruginosa

[0064] Evaluation method: Cultivate to a concentration of 10 8 The standard strain of Pseudomonas aeruginosa ATCC15442, with a concentration of CFU / mL, was serially diluted 10-fold with 0.85% sterile physiological saline to obtain a concentration of 10. 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 A pure culture of the strain at CFU / mL was used to extract DNA templates according to Example 4, which became the detection standard for *Pseudomonas aeruginosa* qPCR. qPCR reactions were performed according to Example 4, with each template tested in triplicate. A standard curve was plotted: the logarithm of the concentration of the pure culture of the standard was plotted on the x-axis, and the corresponding real-time Ct value of the qPCR was plotted on the y-axis. The resulting curve is the standard curve for *Pseudomonas aeruginosa*.

[0065] Evaluation results: Standard curve as follows Figure 3 As shown, Figure 3 The detection limit for primer set 5 (shown in .a) of pure bacteria is 10⁵. 3 The standard curve for *Pseudomonas aeruginosa* was calculated using CFU / mL as y = -3.0677x + 40.259, with a correlation coefficient R0. 2 =0.9901; Figure 3 The detection limit for primer set 6 (shown in .b) of pure bacteria is 10⁶. 2 The standard curve for *Pseudomonas aeruginosa* was calculated using CFU / mL as y = -2.3429x + 33.717, with a correlation coefficient R0. 2 =0.9915; Figure 3 The detection limit for primer set 7 shown in .c is 10⁻⁶ for pure bacteria. 2 The standard curve for *Pseudomonas aeruginosa* was y = -1.3954x + 35.83, with a correlation coefficient R0. 2 =0.9924; Figure 3 The detection limit for primer set 8 (shown in .d) of pure bacteria is 10⁸. 3 The standard curve for *Pseudomonas aeruginosa* was calculated using CFU / mL as y = -3.2347x + 40.592, with a correlation coefficient R0. 2 =0.9935.

[0066] Example 7: Sensitivity Evaluation of the qPCR Quantitative Detection Method for Pseudomonas aeruginosa in Artificially Spiked Samples

[0067] Evaluation Method: Sterile samples were prepared from fresh tomatoes by disinfecting them with alcohol and then removing the surface layer. After incubation on NA nutrient agar plates, the results showed no microorganisms present in the treated fresh vegetable samples, ensuring that any microorganisms in the fresh vegetable samples in subsequent experiments originated from artificial contamination. NA counting plate results showed that the initial concentration of *Pseudomonas aeruginosa* in the artificially contaminated samples was 1.33 × 10⁻⁶. 8 CFU / mL. The homogenate of the artificially contaminated sample was serially diluted 10-fold with 0.85% sterile physiological saline to prepare a Pseudomonas aeruginosa concentration of 10. 1 CFU / mL ~10 8 Artificially contaminated samples with CFU / mL were used. Bacterial genomic DNA extracted from homogenized solutions at each gradient was used as templates, and sterile distilled water was used as a blank control. qPCR reactions were performed according to Example 4, with each template tested in triplicate. Standard curves for the artificially spiked samples were established using the curve fitting method described in Example 5.

[0068] Evaluation results: Standard curve as follows Figure 4 As shown, Figure 4 The detection limit for artificial contamination using primer set 5 (shown in .a) is 1.33 × 10⁻⁶. 4The standard curve for *Pseudomonas aeruginosa* was y = -1.0071x + 36.621, with a correlation coefficient R0. 2 It is 0.9944; for example Figure 4 The detection limit for artificial contamination using primer set 6 (shown in .b) is 1.33 × 10⁶. 3 The standard curve for *Pseudomonas aeruginosa* was y = -2.1233x + 35.354, with a correlation coefficient R0. 2 It is 0.9851; for example Figure 4 The detection limit for artificial contamination using primer set 7 (shown in .c) is 1.33 × 10⁻⁶. 3 The standard curve for *Pseudomonas aeruginosa* was y = -2.8239x + 39.775, with a correlation coefficient R0. 2 It is 0.9814; for example Figure 4 The detection limit for primer set 8 (shown in .d) against artificial contaminants is 1.33 × 10⁸. 4 The standard curve for *Pseudomonas aeruginosa* was y = -1.1851x + 27.745, with a correlation coefficient R0. 2 It is 0.9853.

[0069] Example 8: PCR and qPCR quantification results of Pseudomonas aeruginosa in actual samples

[0070] Experimental Methods: Twenty-nine samples of fresh lettuce, cucumber, lettuce, and tomatoes were collected from local vegetable markets in Guangzhou and transported to the laboratory in sterile homogenization bags. Under aseptic conditions in a clean bench, the vegetable samples were further divided and weighed, with each portion weighing 25g. Each portion was placed in 225ml of *Pseudomonas aeruginosa* enrichment broth and enriched for 18 hours. Genomic DNA was then extracted from each sample and detected using the qPCR method described in Example 4, using primer sets 5-8. Control methods, including traditional culture-mass spectrometry and the aforementioned PCR method, were also used.

[0071] The experimental results are shown in Table 5. As can be seen from Table 5, compared with the traditional gold standard method, the PCR and qPCR methods of this invention accurately detected *Pseudomonas aeruginosa* in all positive and negative samples among the 29 ready-to-eat vegetable samples tested. This embodiment demonstrates that the qualitative and quantitative detection method for *Pseudomonas aeruginosa* of this invention has high reliability and can effectively identify the target *Pseudomonas aeruginosa* in actual samples.

[0072] Table 5. Results of Pseudomonas aeruginosa detection in actual samples.

[0073]

[0074]

[0075] 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. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A molecular marker for the identification of Pseudomonas aeruginosa, characterized in that, The sequence of the molecular marker is shown in SEQ ID NO.

3.

2. A primer set for identifying Pseudomonas aeruginosa, characterized in that, The primer sequence for detecting the molecular marker shown in SEQ ID NO.3 is shown in any of the following primer combinations: Combination 1: SEQ ID NO.9 and SEQ ID NO.10, Combination 2: SEQ ID NO.17 and SEQ ID NO.

18.

3. A method for identifying Pseudomonas aeruginosa for non-disease diagnostic and therapeutic purposes, characterized in that, Includes the following steps: (1) Perform PCR amplification on the DNA of the sample to be tested using at least one set of primers from claim 2; (2) The amplification products were detected by gel electrophoresis; (3) Observe whether the amplification products meet expectations.

4. The method for identifying Pseudomonas aeruginosa for non-disease diagnostic and therapeutic purposes according to claim 3, characterized in that, The PCR amplification system consisted of: 2.5 μL of 10×PCR reaction buffer, 25 mmol / L MgCl2, 1 μL of 2.5 mmol / L dNTP, 100 ng of template DNA, 1 μL each of 10 μmol / L primers, 1 U of Tag enzyme, and sterile double-distilled water to a final volume of 25 μL. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 60℃ annealing for 30 s; 72℃ extension for 45 s; 30 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 10 min.

5. The application of the primer set according to claim 2 in the identification of Pseudomonas aeruginosa for non-disease diagnosis and treatment purposes.

6. A kit for identifying Pseudomonas aeruginosa, characterized in that, The kit includes the primer set as described in claim 2.

Images