Detection method of psychrophilic bacteria, primer pair, probe and application

Abstract

The invention belongs to the technical field of bacterium detection, and particularly relates to a psychrophilic bacterium detection method, a primer pair, a probe and application. The detection method comprises the following step: carrying out amplification detection by using one or more primer pairs and / or probes in a TY1F and TY1R primer pair and / or probe, a TY2F and TY2R primer pair and / or probe and a TY3F and TY3R primer pair and / or probe. According to the detection method, the psychrophilic bacteria in the sample can be rapidly detected, the species proportion of gram-negative bacteria, pseudomonas and pseudomonas fluorescens which is high in harm and produces alkaline metalloenzyme in the psychrophilic bacteria can be clearly determined, and the condition of the psychrophilic bacteria in the sample (such as raw milk) can be better controlled.

Description

Technical Field

[0001] This invention belongs to the field of bacterial detection technology, specifically relating to a method for detecting psychrophilic bacteria, primer pairs, probes, and their applications. Background Technology

[0002] Dairy products are an important source of protein and calcium for people. With rising living standards, the safety and hygiene of dairy products are receiving increasing attention. As a crucial raw material for dairy processing, raw milk is a major factor affecting the safety and hygiene of dairy products, especially the safety and hygiene issues caused by contamination with psychrophilic bacteria.

[0003] Psychrophiles are a general term for a group of bacteria that thrive best between 0°C and 20°C. This temperature range is significantly lower than the optimal growth temperature range of other bacteria, hence the name "psychrophiles." Psychrophiles mainly include two types of microorganisms: obligate psychrophiles and facultative psychrophiles (also known as cold-resistant bacteria). Obligate psychrophiles have a maximum growth temperature not exceeding 20°C, an optimal growth temperature of 15°C or lower, and can still grow and reproduce at 0°C. Facultative psychrophiles, on the other hand, can grow at temperatures above 20°C, have an optimal growth temperature above 15°C, and can also grow and reproduce at temperatures between 0°C and 5°C, exhibiting a wider temperature tolerance and a more widespread presence in the environment. Therefore, psychrophilic bacteria will multiply rapidly when raw milk is not cooled in time, the cooling temperature is not up to standard, or the storage time is too long. In addition, during the reproduction process, they will produce hot-resistant hydrolytic enzymes (such as proteolytic enzymes and lipolytic enzymes). These hot-resistant hydrolytic enzymes can withstand pasteurization (72℃ / 15s) and ultra-high temperature (UHT) (120℃-150℃ / 0.5-8.0s) sterilization processes. They are gradually activated during low-temperature storage and continue to decompose the fat and protein in dairy products during storage, resulting in bitterness, clumping, and stratification, which in turn causes milk spoilage.

[0004] Therefore, the detection of psychrophilic bacteria in raw milk is crucial for the quality control of dairy products, and there is an urgent need to establish a detection method for psychrophilic bacteria in raw milk. Summary of the Invention

[0005] This invention develops a PCR amplification method for detecting psychrophilic bacteria. This detection method has excellent specificity, high sensitivity, and short detection time, and is suitable for rapid detection of psychrophilic bacteria.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] This invention provides a method for detecting psychrophilic bacteria, the method comprising:

[0008] (1) Gram-negative bacteria were detected after specific amplification using TY1F and TY1R primer pairs;

[0009] (2) Detection of Pseudomonas spp. by one of the following methods: (a) Detection of Pseudomonas spp. by specific amplification using TY2F and TY2R primer pairs; (b) Detection of Gram-negative bacteria by specific amplification using TY1F and TY1R primer pairs; and then detection of Pseudomonas spp. by specific amplification using TY2F and TY2R primer pairs and TY1F and TY1R primer pairs; (c) Detection by specific amplification using a mixed primer pair consisting of TY2F and TY2R primer pairs and TY1F and TY1R primer pairs.

[0010] (3) Detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by one of the following methods: (a) Detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes after specific amplification using TY3F and TY3R primer pairs; (b) Detection of Gram-negative bacteria after specific amplification using TY1F and TY1R primer pairs; then detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by specific amplification using TY3F and TY3R primer pairs among the detected Gram-negative bacteria; (c) Detection of Pseudomonas spp. after specific amplification using TY2F and TY2R primer pairs; then detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by specific amplification using TY3F and TY3R primer pairs among the detected Pseudomonas spp.; (d) Detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by specific amplification using TY1F and TY1R primer pairs. Gram-negative bacteria were detected after amplification; then, Pseudomonas spp. were detected after specific amplification using TY2F and TY2R primer pairs among the detected Gram-negative bacteria; then, fluorescent Pseudomonas spp. producing alkaline metalloenzymes were detected after specific amplification using TY3F and TY3R primer pairs among the detected Pseudomonas spp.; (e) Specific amplification was performed using a mixed primer pair consisting of TY3F and TY3R primer pairs and TY1F and TY1R primer pairs; (f) Specific amplification was performed using a mixed primer pair consisting of TY3F and TY3R primer pairs and TY2F and TY2R primer pairs; (g) Specific amplification was performed using a mixed primer pair consisting of TY3F and TY3R primer pairs, TY1F and TY1R primer pairs, and TY2F and TY2R primer pairs;

[0011] The sequences of the TY1F and TY1R primer pairs, the TY2F and TY2R primer pairs, and the TY3F and TY3R primer pairs are shown below:

[0012] TY1F: 5'-CCAGGTGTGCATGGCTGCCG-3',

[0013] TY1R: 5'-GCCATGTCGCAGCCGTGACCTT-3';

[0014] TY2F: 5'-CGGCAACGTCACCTCAGGAAGCTCG-3',

[0015] TY2R: 5'-ACAGATACTAGGAAAATTAGACCAG-3';

[0016] TY3F: 5'-TAGAGACGGCCTGGTCCGCAACGACGGC-3',

[0017] TY3R: 5'-TCGGACGTACGGTGAATAAGCCCGG-3'.

[0018] It should be noted that the above detection methods can be selected according to different detection needs. If you only want to screen for Gram-negative bacterial strains in the sample, you can choose the detection method (1) above. If you want to screen for Pseudomonas strains in the sample, you can choose the detection method (2) above. For the detection method (2), you can use only the TY2F and TY2R primer pairs for screening. In order to improve accuracy, you can also combine the TY2F and TY2R primer pairs with the TY1F and TY1R primer pairs for screening. The combination of the TY2F and TY2R primer pairs with the TY1F and TY1R primer pairs for screening can be done in steps. Screening can also be performed using multiplex PCR amplification with mixed primer pairs. To screen for alkaline metalloenzyme-producing *Pseudomonas fluorescens* in a sample, the TY3F and TY3R primer pairs can be used directly. To improve accuracy, the TY3F and TY3R primer pairs can be combined with the TY1F and TY1R primer pairs, or the TY3F and TY3R primer pairs can be combined with the TY2F and TY2R primer pairs. To further improve accuracy, the TY3F and TY3R primer pairs, the TY2F and TY2R primer pairs, and the TY1F and TY1R primer pairs can be combined for screening. Of course, the above-mentioned combination screening is as described above.

[0019] It should also be noted that psychrophilic bacteria are not a specific species but rather a combination of multiple species, which presents a significant challenge for detection. One of the detection methods mentioned above can accurately identify three types of psychrophilic bacteria in one go, solving the current problem that psychrophilic bacteria are not a specific species and therefore difficult to detect.

[0020] It should also be noted that, as described above, after PCR amplification, the detection results can be obtained by analyzing the PCR amplification products. For example, gel electrophoresis can be used to analyze the PCR amplification products to obtain the detection status of psychrophilic bacteria.

[0021] It should also be noted that the above detection methods are selected according to the specific detection purpose, and the order can be changed (i.e., there is no order relationship).

[0022] Furthermore, in the above-mentioned method for detecting psychrophilic bacteria, while using primer pairs for amplification, corresponding specific fluorescent probes are added for co-amplification, and then the psychrophilic bacteria are detected based on the CT value; wherein, the specific fluorescent probe sequence for Gram-negative bacteria is: 5'-AGGAGAGTTGATGAGCCGTA-3', the specific fluorescent probe sequence for Pseudomonas is: 5'-TCCGAGTAGCAGACGAATTCATTT-3', and the specific fluorescent probe sequence for alkaline metalloenzyme-producing fluorescent Pseudomonas is: 5'-AGCCGGCCCTTAAGCTTCAGCGGGCTTAGG-3'.

[0023] It should be noted that, in the above detection methods, in addition to using gel electrophoresis to analyze the PCR amplification products to obtain the detection status of psychrophilic bacteria, fluorescent probes can also be added during amplification (i.e., real-time fluorescent PCR amplification). Then, the detection status of psychrophilic bacteria can be determined based on the CT value. The detection status includes qualitative and quantitative detection. When performing quantitative detection, a standard curve can be obtained first, and then the amount of psychrophilic bacteria can be calculated based on the standard curve and the CT value.

[0024] It should also be noted that real-time fluorescent PCR amplification detection with added fluorescent probes takes less time than conventional PCR amplification. In this invention, the real-time PCR amplification detection method with added fluorescent probes can be preferred for the detection of psychrophilic bacteria.

[0025] Furthermore, the fluorescent reporter group at the 5' end of the specific fluorescent probe for the aforementioned Gram-negative bacteria can be FAM, and the fluorescent quenching group at the 3' end can be MGB; the fluorescent reporter group at the 5' end of the specific fluorescent probe for the genus Pseudomonas can be CY5, and the fluorescent quenching group at the 3' end can be BHQ3; the fluorescent reporter group at the 5' end of the specific fluorescent probe for alkaline metalloenzyme-producing fluorescent Pseudomonas can be ROX, and the fluorescent quenching group at the 3' end can be BHQ2.

[0026] It should be noted that those skilled in the art should know that when using fluorescent probes for real-time fluorescent PCR amplification, the fluorescent probes have a fluorescent reporter group and a fluorescent quenching group at both ends, respectively. The fluorescent reporter group and the fluorescent quenching group include all fluorescent reporter groups and fluorescent quenching groups in the art, and the fluorescent probes of the present invention preferably have the aforementioned fluorescent reporter group and fluorescent quenching group.

[0027] Furthermore, the aforementioned psychrophilic bacteria originated from cow's milk.

[0028] It should be noted that the psychrophilic bacteria in the detection method of the present invention can come from any item that can exist, especially cow's milk. That is, the above-mentioned detection method of psychrophilic bacteria can detect the presence of psychrophilic bacteria in cow's milk. Of course, it should be understood that when detecting the presence of psychrophilic bacteria in cow's milk, it is necessary to isolate the psychrophilic bacteria and then extract DNA for amplification. The isolation method and the DNA extraction method are well known in the art.

[0029] Another aspect of the present invention provides a primer pair for PCR amplification of psychrophilic bacteria, characterized in that the primer pair is selected from one or more combinations of the following primer pairs:

[0030] (1) Primer pairs used for PCR amplification of Gram-negative bacteria, the primer pair sequences are shown below:

[0031] TY1F: 5'-CCAGGTGTGCATGGCTGCCG-3',

[0032] TY1R: 5'-GCCATGTCGCAGCCGTGACCTT-3';

[0033] (2) Primer pairs used for PCR amplification of Pseudomonas spp., the primer pair sequences are shown below:

[0034] TY2F: 5'-CGGCAACGTCACCTCAGGAAGCTCG-3',

[0035] TY2R: 5'-ACAGATACTAGGAAAATTAGACCAG-3';

[0036] Primer pairs for PCR amplification of alkaline metalloenzymes produced by *Pseudomonas fluorescens* are shown below:

[0037] TY3F: 5'-TAGAGACGGCCTGGTCCGCAACGACGGC-3',

[0038] TY3R: 5'-TCGGACGTACGGTGAATAAGCCCGG-3'.

[0039] Another aspect of the present invention provides a primer pair and probe for PCR amplification of psychrophilic bacteria, characterized in that the primer pair and probe are selected from one or more combinations of the following primer pairs:

[0040] (1) Primer pairs and probes used for PCR amplification of Gram-negative bacteria, the primer pair and probe sequences are shown below:

[0041] TY1F: 5'-CCAGGTGTGCATGGCTGCCG-3',

[0042] TY1R: 5'-GCCATGTCGCAGCCGTGACCTT-3';

[0043] TY1Probe: 5'-AGGAGAGTTGATGAGCCGTA-3'

[0044] (2) Primer pairs and probes used for PCR amplification of Pseudomonas spp. are shown below:

[0045] TY2F: 5'-CGGCAACGTCACCTCAGGAAGCTCG-3',

[0046] TY2R: 5'-ACAGATACTAGGAAAATTAGACCAG-3';

[0047] TY2Probe:5'-TCCGAGTAGCAGACGAATTCATTT-3';

[0048] (3) Primer pairs and probes for PCR amplification of alkaline metalloenzymes produced by *Pseudomonas fluorescens*, the primer pair and probe sequences are shown below:

[0049] TY3F: 5'-TAGAGACGGCCTGGTCCGCAACGACGGC-3',

[0050] TY3R: 5'-TCGGACGTACGGTGAATAAGCCCGG-3',

[0051] TY3Probe: 5'-AGCCGGCCCTTAAGCTTCAGCGGGCTTAGG-3'.

[0052] It should be noted that the primer pairs mentioned above for PCR amplification of psychrophilic bacteria all exhibit good specificity and sensitivity for their corresponding strains. For example, the real-time fluorescence PCR amplification detection method based on primer pairs for PCR amplification of Gram-negative bacteria shows a CT value of around 17 for Gram-negative bacteria, indicating high amplification efficiency and a detection sensitivity of up to 2 CFU / mL. Similarly, the real-time fluorescence PCR amplification detection method based on primer pairs for PCR amplification of *Pseudomonas* shows a CT value of around 20 for *Pseudomonas*, indicating high amplification efficiency and a detection sensitivity of up to 75 CFU / mL. Furthermore, the real-time fluorescence PCR amplification detection method based on primer pairs for PCR amplification of *Fluorescent Pseudomonas* producing basic metalloenzymes shows a CT value of around 20 for *Fluorescent Pseudomonas* producing basic metalloenzymes, with a detection sensitivity of up to 2 CFU / mL.

[0053] Furthermore, the fluorescent reporter group at the 5' end of the TY1Probe can be FAM, and the fluorescent quencher group at the 3' end can be MGB; the fluorescent reporter group at the 5' end of the TY2Probe can be CY5, and the fluorescent quencher group at the 3' end can be BHQ3; the fluorescent reporter group at the 5' end of the TY3Probe can be ROX, and the fluorescent quencher group at the 3' end can be BHQ2.

[0054] In another aspect, the present invention provides a detection product for detecting psychrophilic bacteria, comprising the primer pair for PCR amplification of psychrophilic bacteria as described in the present invention, or the primer pair and probe for PCR amplification of psychrophilic bacteria as described in the present invention.

[0055] It should be noted that the primer pairs for PCR amplification of psychrophilic bacteria in this invention, or the primer pairs and probes for PCR amplification of psychrophilic bacteria in this invention, can be combined with other auxiliary detection reagents to prepare a detection product for detecting psychrophilic bacteria. The auxiliary detection reagents are PCR amplification reagents known in the art, such as ddH2O reagent, DNA extraction reagent, positive control, and PCR amplification buffer. Furthermore, the detection product can be in any form that enables detection, such as a reagent or kit. Generally, for better transport and storage, it is preferred to prepare it in the form of a detection kit, which is in a form known in the art, such as including instructions for use and containers for holding the reagents.

[0056] In another aspect, this invention provides the application of primer pairs for PCR amplification of psychrophilic bacteria, or primer pairs and probes for PCR amplification of psychrophilic bacteria, in the detection of psychrophilic bacteria, wherein the application is selected from one of the following:

[0057] (a) Psychrophilic bacteria are Gram-negative bacteria. The application of primer pairs and / or probes used for PCR amplification of Gram-negative bacteria in the detection of Gram-negative bacteria.

[0058] (b) The psychrophilic bacteria are Pseudomonas spp., and the application of primer pairs and / or probes for PCR amplification of Pseudomonas spp. in the detection of Pseudomonas spp.; or the application of primer pairs and / or probes for PCR amplification of Gram-negative bacteria and primer pairs and / or probes for PCR amplification of Pseudomonas spp. in the detection of Pseudomonas spp.

[0059] (c) The use of primer pairs and / or probes for PCR amplification of alkaline metalloenzymes in *Pseudomonas fluorescens*, a psychrophilic bacterium producing alkaline metalloenzymes, in detecting alkaline metalloenzymes in *Pseudomonas fluorescens*; or the use of primer pairs and / or probes for PCR amplification of Gram-negative bacteria and for detecting alkaline metalloenzymes in *Pseudomonas fluorescens*; or the use of primer pairs and / or probes for PCR amplification of *Pseudomonas* spp. and for detecting alkaline metalloenzymes in *Pseudomonas fluorescens*; or the use of primer pairs and / or probes for PCR amplification of Gram-negative bacteria, primer pairs and / or probes for PCR amplification of *Pseudomonas* spp. and for detecting alkaline metalloenzymes in *Pseudomonas fluorescens*.

[0060] It should be noted that, as described above, the primer pairs for PCR amplification of psychrophilic bacteria in this invention have good specificity and sensitivity for their corresponding strains. Based on this, they can be applied to the detection of psychrophilic bacteria, enabling qualitative and quantitative analysis of psychrophilic bacteria with high accuracy and low detection limit.

[0061] The beneficial effects of this invention include at least the following:

[0062] (1) The method for detecting psychrophilic bacteria provided by the present invention can quickly detect psychrophilic bacteria in a sample and clearly identify the species ratio of Gram-negative bacteria, Pseudomonas, and fluorescent Pseudomonas that produce alkaline metalloenzymes, which are more harmful. This allows for better control of the status of psychrophilic bacteria in a sample (such as raw milk). Furthermore, the detection method does not require special and expensive equipment and has a short detection time.

[0063] (2) The primer pair for PCR amplification of Gram-negative bacteria provided by the present invention has excellent specificity for Gram-negative bacteria. The CT value of the real-time fluorescent PCR amplification detection method based on the primer pair is about 17, indicating that the amplification efficiency is high and the detection sensitivity can reach 2 CFU / mL.

[0064] (3) The primer pair for PCR amplification of Pseudomonas spp. provided by the present invention has excellent specificity for Pseudomonas spp. The CT value of Pseudomonas spp. detected by the real-time fluorescent PCR amplification detection method based on the primer pair is about 20, indicating that the amplification efficiency is high and the detection sensitivity can reach 75 CFU / mL.

[0065] (4) The primer pair provided by the present invention for PCR amplification of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes has excellent specificity for fluorescent Pseudomonas aeruginosa producing basic metalloenzymes. The real-time fluorescent PCR amplification detection method based on the primer pair detects fluorescent Pseudomonas aeruginosa producing basic metalloenzymes with a CT value of about 20 and a detection sensitivity of up to 2 CFU / mL. Attached Figure Description

[0066] Figure 1 This section details the primer screening results for Gram-negative bacteria.

[0067] Figure 2 The results of primer screening for Pseudomonas aeruginosa;

[0068] Figure 3 Primer screening results for fluorescent Pseudomonas aeruginosa producing basic metalloenzymes;

[0069] Figure 4 Evaluation of the specificity of rapid detection of Gram-negative bacteria;

[0070] Figure 5 Evaluation of the specificity of rapid detection of Pseudomonas aeruginosa;

[0071] Figure 6 Evaluation of the specificity of rapid detection of alkaline metalloenzyme-producing bacteria;

[0072] Figure 7 The sensitivity evaluation of the quantitative real-time PCR detection method for Gram-negative bacteria;

[0073] Figure 8 The sensitivity evaluation of the quantitative real-time PCR detection method for Pseudomonas aeruginosa;

[0074] Figure 9 The sensitivity evaluation of the fluorescence quantitative PCR detection method for alkaline metalloenzyme-producing Pseudomonas aeruginosa. Detailed Implementation

[0075] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0076] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0077] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0078] Example 1: Isolation, Screening and Identification of Psychrophilic Bacteria

[0079] (1) Preliminary screening of psychrophilic bacteria

[0080] Spread 1 mL of raw milk sample onto a PCA counting plate and incubate at 4°C for 72 h. Then pick a single colony, purify the cultured strain, and store it in a freezer at -80°C.

[0081] (2) Activation and identification of strains

[0082] Glycerol tubes were inoculated into sterile liquid BHI brain heart extract culture medium and cultured at 4°C for 48 hours. After activation three times until no contaminants were detected under a microscope, the bacterial culture was inoculated into liquid culture medium at a 2% inoculation rate. The bacterial culture was then sent to Shanghai Sangon Biotech for sequencing.

[0083] (3) After sequencing the psychrophilic bacteria isolated from the samples, a BLAST analysis was performed in NCBI. The results showed that the psychrophilic bacteria were mainly Gram-negative bacteria. Among the Gram-negative bacteria, *Pseudomonas* species had the most significant impact on the quality of raw milk products. Within the *Pseudomonas* genus, protease-producing bacteria (including alkaline metalloenzymes) and lipase-producing bacteria (especially *Pseudomonas fluorescens*, which produces alkaline metalloenzymes) directly affected the quality of UHT milk during its shelf life. Classification of the psychrophilic bacteria, through whole-genome sequencing and BLAST analysis, revealed that the isolated psychrophilic bacteria included four Gram-negative bacteria and one Gram-positive bacteria. The four Gram-negative bacteria were named *Pseudomonas psychrophilus* TY1, *Pseudomonas psychrophilus* TY2, *Pseudomonas fluorescens* TY3, and *Pseudomonas fluorescens* TY4, respectively, and the one Gram-positive bacteria was named *Bacillus cereus* TY5. The 16S sequences of the above five strains were SEQ ID NO:1 (*Pseudomonas psychrophilus* TY1), SEQ ID NO:2 (*Pseudomonas psychrophilus* TY2), SEQ ID NO:3, and SEQ ID NO:4, respectively. SEQ ID NO:3 (Fluorescent Pseudomonas TY3), SEQ ID NO:4 (Fluorescent Pseudomonas TY4), and SEQ ID NO:5 (Bacillus cereus TY5); among them, those belonging to the genus Pseudomonas include Psychotropic Pseudomonas TY1, Psychotropic Pseudomonas TY2, Fluorescent Pseudomonas TY3, and Fluorescent Pseudomonas TY4; among the Pseudomonas genus, the fluorescent Pseudomonas that produce basic metalloenzymes are Psychotropic Pseudomonas TY2 and Fluorescent Pseudomonas TY3.

[0084] In summary, this invention aims to establish a rapid detection method for Gram-negative bacteria, Pseudomonas spp., and fluorescent Pseudomonas spp. producing basic metalloenzymes in milk, in order to accurately detect psychrophilic bacterial populations that significantly affect the quality of raw milk. Example 2: Design of Primers for Quantitative Real-Time PCR.

[0085] (1) Experimental strains

[0086] *Pseudomonas psychrophilus* CGMCC1.8930, *Pseudomonas psychrophilus* CGMCC1.3356, *Pseudomonas fluorescens* CGMCC1.1823, *Listeria monocytogenes* ATCC19115, *Escherichia coli* ATCC8739, *Bacillus cereus* CMCC(B)63303, *Bacillus subtilis* ATCC6633, and *Citrobacter freundii* ATCC43864 were purchased from the China Industrial Microbial Culture Collection Center (CICC) and the China General Microbial Culture Collection Center (CGMCC). *Pseudomonas psychrophilus* TY1, *Pseudomonas psychrophilus* TY2, *Pseudomonas fluorescens* TY3, *Pseudomonas fluorescens* TY4, and *Bacillus cereus* TY5 were isolated from raw cow's milk using the methods described above. The *Pseudomonas* species were classified according to their Gram-negative nature, *Pseudomonas* genus, and alkaline metalloenzyme-producing *Pseudomonas fluorescens*, as shown in Table 1 below.

[0087] Table 1 Classification of strains used in the embodiments of the present invention

[0088]

[0089] (2) Activation of the strain

[0090] Bacillus cereus CMCC(B)63303, Bacillus subtilis ATCC6633, Listeria monocytogenes ATCC19115, Escherichia coli ATCC8739, and Citrobacter freundii ATCC43864 were inoculated into LB broth medium and cultured at 37°C to the logarithmic development phase for later use. Pseudomonas psychrophilus CGMCC1.8930, Pseudomonas psychrophilus CGMCC1.3356, Pseudomonas fluorescens CGMCC1.1823, Pseudomonas psychrophilus TY1, Pseudomonas psychrophilus TY2, Pseudomonas fluorescens TY3, and Pseudomonas fluorescens TY4 were inoculated into BHI brain heart extract liquid medium and cultured at 21°C to the logarithmic development phase for later use.

[0091] (3) Primer and probe design

[0092] Based on the conserved universal sequences of Gram-negative bacteria, Pseudomonas spp., and fluorescent Pseudomonas fluorescens producing alkaline metalloenzymes, DNAMAN was used for alignment to identify highly conserved and variable regions. Universal primers were designed in the conserved regions using Primer Premier 6 and Oligo 6. Specific sequences were found based on the designed primers, and fluorescent labels were added to obtain probes. The specificity of the primers was verified by calculating the Tm value and using NCBI. Finally, the designed primer sequences were sent to Shanghai Sangon Biotech for synthesis.

[0093] Example 3: Establishment of a real-time PCR method for detecting psychrophilic bacteria

[0094] (1) DNA preparation of the strain

[0095] 1) DNA preparation of control bacteria

[0096] DNA was extracted from the *Pseudomonas psychrophilus* CGMCC1.8930, *Pseudomonas psychrophilus* CGMCC1.3356, *Pseudomonas fluorescens* CGMCC1.1823, *Listeria monocytogenes* ATCC19115, *Escherichia coli* ATCC8739, *Bacillus cereus* CMCC(B)63303, *Bacillus subtilis* ATCC6633, and *Citrobacter freundii* ATCC43864 cultured to the logarithmic phase in Example 2 above using the Ezup column-based bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). After extraction, the DNA was stored at -80℃ for later use as a positive control template for PCR reactions, and the DNA concentration was detected using a NanoDrop one ultra-micro UV spectrophotometer.

[0097] 2) DNA preparation of the psychrophilic bacteria of the test strain

[0098] DNA was extracted from *Pseudomonas psychrophilus* TY1, TY2, TY3, TY4, and *Bacillus cereus* TY5 using a high-temperature lysis method, as follows: 1 mL of each of the following bacterial cultures cultured to the logarithmic growth phase was placed in a sterile 1.5 mL Eppendorf centrifuge tube. The tubes were centrifuged at 8000 rpm for 5 min at 4°C. The supernatant was aspirated and added to another sterile 1.5 mL Eppendorf centrifuge tube. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded. 200 μL of deionized water was added to resuspend the bacterial cells. The tubes were heated in a boiling water bath for 10 min and immediately placed in liquid nitrogen. After cooling, the tubes were centrifuged at 8000 rpm for 10 min. The supernatant was used as a template for the PCR reaction.

[0099] (2) Establishment of the real-time PCR method

[0100] The DNA of the control bacteria mentioned above was used as a positive control template, and the DNA of Pseudomonas psychrophilus TY1, Pseudomonas psychrophilus TY2, Pseudomonas fluorescens TY3, Pseudomonas fluorescens TY4 and Bacillus cereus TY5 extracted by high-temperature lysis method was used as the test sample template. ddH2O was used as a negative control. A real-time PCR amplification method was established based on the primers and probes mentioned above. The real-time PCR amplification system and real-time PCR amplification program are shown in Table 2.

[0101] Table 2. Quantitative PCR amplification system and amplification procedure

[0102]

[0103]

[0104] (3) Primer screening

[0105] DNA was extracted from the positive controls *Pseudomonas psychrophilus* CGMCC 1.8930, *Pseudomonas psychrophilus* CGMCC 1.3356, *Pseudomonas fluorescens* CGMCC 1.1823, *Listeria monocytogenes* ATCC19115, *Escherichia coli* ATCC8739, *Bacillus cereus* CMCC(B)63303, *Bacillus subtilis* ATCC6633, *Citrobacter freundii* ATCC43864, and *Pseudomonas psychrophilus* TY1, TY2, TY3, TY4, and TY5, and stored at -80℃ for later use.

[0106] 1) Screening of primers for Gram-negative bacteria-specific amplification

[0107] Six pairs of primers and probes were designed for Gram-negative bacteria, numbered as primer 1 and probe 1, primer 2 and probe 2, primer 3 and probe 3, primer 4 and probe 4, primer 5 and probe 5, and primer 6 and probe 6. The primer and probe sequences are shown in Table 3 below.

[0108] Table 3 Primer and probe sequences for Gram-negative bacteria

[0109]

[0110]

[0111] Then, DNA extracted from the bacterial cultures of Gram-positive bacteria such as Bacillus cereus CMCC(B)63303, Bacillus subtilis ATCC6633, Bacillus cereus TY5, and Listeria monocytogenes ATCC19115, and Gram-negative bacteria such as Pseudomonas psychrophilus CGMCC 1.8930, Pseudomonas psychrophilus CGMCC 1.3356, Pseudomonas fluorescens CGMCC 1.1823, Escherichia coli ATCC8739, Citrobacter freundii ATCC43864, Pseudomonas psychrophilus TY1, Pseudomonas psychrophilus TY2, Pseudomonas fluorescens TY3, and Pseudomonas fluorescens TY4 were mixed and used as templates. Then, amplification was performed using primers 1 and probes 1 through 6 and probes 6 as described in Table 3 according to the above-mentioned quantitative PCR amplification method (see Table 2).

[0112] Amplification results as follows Figure 1 As shown, primer 1 and probe 1 (corresponding to Figure 1 The CT value of primer 1 (i.e., primers TY1F and TY1R in this invention and probe TY1Probe) is around 18, and it exhibits a standard S-shaped curve, which meets the expected screening and can be used for later experiments; while primer 2 and probe 2 (corresponding to Figure 1 The CT value of primer 2) is around 26, which is relatively high. Using this value in later experiments might result in low sensitivity and a high detection limit, which is not suitable for practical applications. Primers 3 and 3 to 6 and 6 (corresponding to...) Figure 1 Neither the middle primer (3-6) nor the negative control showed a line, which did not meet the expected screening criteria.

[0113] 2) Screening of Pseudomonas spp.-specific amplification primers

[0114] Six pairs of primers and probes were designed for Pseudomonas spp., numbered as primer 1 and probe 1, primer 2 and probe 2, primer 3 and probe 3, primer 4 and probe 4, primer 5 and probe 5, and primer 6 and probe 6. The primer and probe sequences are shown in Table 4 below.

[0115] Table 4 Primer and probe sequences for Pseudomonas spp.

[0116]

[0117] Then, DNA extracted from the bacterial cultures of Gram-positive bacteria such as Bacillus cereus CMCC(B)63303, Bacillus subtilis ATCC6633, Bacillus cereus TY5, Escherichia coli ATCC8739, Citrobacter freundii ATCC43864, Listeria monocytogenes ATCC19115, and Pseudomonas psychrophilic CGMCC 1.8930, Pseudomonas psychrophilic CGMCC 1.3356, Pseudomonas fluorescens CGMCC1.1823, Pseudomonas psychrophilic TY1, Pseudomonas psychrophilic TY2, Pseudomonas fluorescens TY3, and Pseudomonas fluorescens TY4 was mixed and used as a template. Then, amplification was performed using primers 1 and probes 1 through 6 in Table 4 according to the above-described quantitative PCR amplification method (see Table 2).

[0118] Amplification results as follows Figure 2 As shown, primer 1 and probe 1 (corresponding to Figure 2 The CT value of primer 1 (i.e., primers TY2F and TY2R in this invention and probe TY2Probe) is around 17, and it exhibits a standard S-shaped curve, which meets the expected screening and can be used for later experiments; while primers 2 and probes 2-6 (corresponding to...) Figure 2 The CT values ​​of primers 2-6 were around 34, which is relatively high. If used in later experiments, this may lead to low sensitivity and a high detection limit, which is not in line with practical application. The negative controls did not show any lines.

[0119] 3) Screening of primers for specific amplification of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes

[0120] Six pairs of primers and probes were designed for alkaline metalloenzyme-producing Pseudomonas fluorescens, numbered as primer 1 and probe 1, primer 2 and probe 2, primer 3 and probe 3, primer 4 and probe 4, primer 5 and probe 5, and primer 6 and probe 6. The primer and probe sequences are shown in Table 5 below.

[0121] Table 5 Primer and probe sequences of alkaline metalloenzyme-producing fluorescent Pseudomonas.

[0122]

[0123] Then, DNA extracted from the bacterial cultures of Gram-positive bacteria such as Bacillus cereus CMCC(B)63303, Bacillus subtilis ATCC6633, Bacillus cereus TY5, Escherichia coli ATCC8739, Citrobacter freundii ATCC43864, CGMCC 1.8930, Pseudomonas psychrophilus CGMCC1.3356, Pseudomonas psychrophilus TY1, Pseudomonas fluorescens TY4, Listeria monocytogenes ATCC19115, and Pseudomonas fluorescens CGMCC 1.1823, Pseudomonas psychrophilus TY2, and Pseudomonas fluorescens TY3 (which produce basic metalloenzymes) was mixed and used as a template. Then, amplification was performed using primers 1 and probes 1 through 6 in Table 5 according to the above-described quantitative PCR amplification method (see Table 2).

[0124] Amplification results as follows Figure 3 As shown, primer 1 and probe 1 (corresponding to Figure 3 The CT value of primer 1 (i.e., primers TY3F and TY3R and probe TY3Probe in this invention) is around 20, and it exhibits a standard S-shaped curve, which meets the expected screening and can be used for later experiments; while primer 2 and probe 2 (corresponding to Figure 3 Primer 2), primer 3 and probe 3 (corresponding to primer 2), primer 3 and probe 3 (cor Figure 3 Primer 3) and primer 4 and probe 4 (corresponding to Figure 3 The CT value of primer 4 is around 32-36, which is relatively high. Using this value in later experiments might result in low sensitivity and a high detection limit, which is not suitable for practical applications. Primer 5 and probe 5 (corresponding to...) Figure 3 Primer 5) and primer 6 and probe 6 (corresponding to Figure 3 Neither the middle primer 6 nor the negative control showed a line, which did not meet the expected screening criteria.

[0125] Example 4: Specificity evaluation of the real-time quantitative PCR detection method for psychrophilic bacteria

[0126] DNA was extracted from the positive control bacteria, including *Pseudomonas psychrophilus* CGMCC1.8930, *Pseudomonas psychrophilus* CGMCC1.3356, *Pseudomonas fluorescens* CGMCC1.1823, *Listeria monocytogenes* ATCC19115, *Escherichia coli* ATCC8739, *Bacillus cereus* CMCC(B)63303, *Bacillus subtilis* ATCC6633, *Citrobacter freundii* ATCC43864, and *Pseudomonas psychrophilus* TY1, *Pseudomonas psychrophilus* TY2, *Pseudomonas fluorescens* TY3, *Pseudomonas fluorescens* TY4, and *Bacillus cereus* TY5, respectively (extraction was performed according to the method described in Example 3 above), and then mixed to obtain a DNA mixture, which was stored at -80°C for later use.

[0127] (1) Evaluation of primer specificity for amplifying Gram-negative bacteria

[0128] Using the extracted DNA mixture as a template, and primer 1 and probe 1 from Table 3 as amplification primers and probes, amplification was performed according to the real-time PCR amplification method constructed in Example 3 above. The amplification results are as follows: Figure 4 As shown, Gram-positive bacteria such as Bacillus cereus CMCC(B)63303, Bacillus subtilis ATCC6633, Bacillus cereus TY5, and Listeria monocytogenes ATCC19115, as well as the negative control ddH2O, did not show a line, indicating good specificity. However, Gram-negative bacteria such as Pseudomonas psychrophilus CGMCC 1.8930, Pseudomonas psychrophilus CGMCC 1.3356, Pseudomonas fluorescens CGMCC 1.1823, Escherichia coli ATCC8739, Citrobacter freundii ATCC43864, Pseudomonas psychrophilus TY1, Pseudomonas psychrophilus TY2, Pseudomonas fluorescens TY3, and Pseudomonas fluorescens TY4 all showed standard S-shaped curves with CT values ​​around 17, indicating that the rapid detection method specifically targeting Gram-negative bacteria met expectations.

[0129] (2) Evaluation of primer specificity for amplifying Pseudomonas spp.

[0130] Using the extracted DNA mixture as a template, and primer 1 and probe 1 from Table 4 as amplification primers and probes, amplification was performed according to the real-time PCR amplification method constructed in Example 3 above. The amplification results are as follows: Figure 5 As shown, no lines were observed in Gram-positive bacteria such as Bacillus cereus CMCC(B)63303, Bacillus subtilis ATCC6633, Bacillus cereus TY5, Escherichia coli ATCC8739, Citrobacter freundii ATCC43864, and Listeria monocytogenes ATCC19115, as well as the negative control ddH2O, indicating good specificity. Meanwhile, Pseudomonas psychrophilic (CGMCC 1.8930, CGMCC 1.3356, CGMCC 1.1823, TY1, TY2, TY3, and TY4) all exhibited standard S-shaped curves with CT values ​​around 20, indicating that the rapid detection method for Pseudomonas specificity met expectations.

[0131] (3) Evaluation of primer specificity for amplifying alkaline metalloenzyme-producing fluorescent Pseudomonas aeruginosa.

[0132] Using the extracted DNA mixture as a template, and primer 1 and probe 1 from Table 5 as amplification primers and probes, amplification was performed according to the real-time PCR amplification method constructed in Example 3 above. The amplification results are as follows: Figure 6As shown, no lines were observed in Gram-positive bacteria such as Bacillus cereus CMCC(B)63303, Bacillus subtilis ATCC6633, Bacillus cereus TY5, Escherichia coli ATCC8739, Citrobacter freundii ATCC43864, CGMCC 1.8930, Pseudomonas psychrophilus CGMCC 1.3356, Pseudomonas psychrophilus TY1, Pseudomonas fluorescens TY4, and Listeria monocytogenes ATCC19115, as well as the negative control ddH2O, indicating good specificity. However, the Pseudomonas fluorescens CGMCC 1.1823, Pseudomonas psychrophilus TY2, and Pseudomonas fluorescens TY3, which are alkaline metalloenzyme-producing strains, all showed standard S-shaped curves with CT values ​​around 20, indicating that the rapid detection method for alkaline metalloenzyme-producing strains met expectations.

[0133] In summary, the fluorescence quantitative PCR amplification method established based on primers 1 and probe 1 in Table 3, Table 4, and Table 5 can rapidly detect psychrophilic bacteria in samples and clearly identify the species ratios of Gram-negative bacteria, Pseudomonas, and alkaline metalloenzymes among psychrophilic bacteria, thus enabling better control of the microbial status in raw milk.

[0134] Example 5: Sensitivity Evaluation of the Detection Method for Psychrophilic Bacteria by Real-Time PCR

[0135] (1) Sensitivity evaluation of primer 1 for amplifying Gram-negative bacteria

[0136] To better simulate the state of psychrophilic bacteria in raw milk, *Pseudomonas psychrophilus* CGMCC1.8930, *Pseudomonas psychrophilus* CGMCC1.3356, *Pseudomonas fluorescens* CGMCC1.1823, *Escherichia coli* ATCC8739, *Citrobacter freundii* ATCC43864, *Pseudomonas psychrophilus* TY1, *Pseudomonas psychrophilus* TY2, *Pseudomonas fluorescens* TY3, and *Pseudomonas fluorescens* TY4 were mixed and added to raw milk, and the viable count was determined to be 2.3 × 10⁻⁶. 5 CFU / mL.

[0137] DNA was isolated, activated, and extracted according to the methods described in Examples 2 and 3 above. Primer 1 and probe 1 from Table 3 were used as amplification primers and probes, and amplification was performed according to the real-time quantitative PCR method constructed in Example 3 above. The results are as follows: Figure 7 As shown, the results indicate that from the original solution to a 10-fold dilution... -5The amplification curves for all six different concentrations showed a line formation within 35 cycles (Ct value ≤ 35), and the S-shape was obvious. However, the amplification curves for lower concentrations and the negative control did not show a line formation within 40 cycles, indicating that the sensitivity of this method can detect 2 CFU / mL. This means that if the sample contains 2-3 psychrophilic bacteria per milliliter, this method can detect them.

[0138] (2) Sensitivity evaluation of primer 2 for amplifying Pseudomonas spp.

[0139] To better simulate the state of psychrophilic bacteria in raw milk, *Pseudomonas psychrophilus* CGMCC1.8930, *Pseudomonas psychrophilus* CGMCC1.3356, *Pseudomonas fluorescens* CGMCC1.1823, *Pseudomonas psychrophilus* TY1, *Pseudomonas psychrophilus* TY2, *Pseudomonas fluorescens* TY3, and *Pseudomonas fluorescens* TY4 were mixed and added to the raw milk, and the viable count was determined to be 7.5 × 10⁻⁶. 5 CFU / mL.

[0140] DNA was isolated, activated, and extracted according to the methods described in Examples 2 and 3 above. Primer 1 and probe 1 from Table 4 were used as amplification primers and probes, and amplification was performed according to the real-time quantitative PCR amplification method constructed in Example 3 above. The results are as follows: Figure 8 As shown, the results indicate that from the original solution to a 10-fold dilution... -5 The amplification curves for all six different concentrations showed a line formation within 35 cycles (Ct value ≤ 35), and exhibited a clear S-shape. However, the amplification curves for lower concentrations and the negative control showed no line formation within 40 cycles, and the melting curves showed no melting temperature, indicating that the method's sensitivity can detect up to 75 CFU / mL. This means that if the sample contains approximately 75 psychrophilic bacteria per milliliter, this method can detect them.

[0141] (3) Sensitivity evaluation of primer 3 for amplifying fluorescent Pseudomonas aeruginosa producing alkaline metalloenzymes

[0142] To better simulate the state of psychrophilic bacteria in raw milk, *Pseudomonas psychrophilus* CGMCC1.1823, *Pseudomonas psychrophilus* TY2, and *Pseudomonas fluorescens* TY3 were mixed and added to the raw milk, and the viable count was determined to be 1.8 × 10⁻⁶. 5 CFU / mL.

[0143] DNA was isolated, activated, and extracted according to the methods described in Examples 2 and 3 above. Primer 1 and probe 1 from Table 5 were used as amplification primers and probes, and amplification was performed according to the real-time quantitative PCR method constructed in Example 3 above. The results are as follows: Figure 9 As shown, the results indicate that from the original solution to a 10-fold dilution... -5The amplification curves for all six different concentrations showed a line formation within 35 cycles (Ct value ≤ 35), and exhibited a clear S-shape. However, the amplification curves for lower concentrations and the negative control showed no line formation within 40 cycles, and the melting curves showed no melting temperature, indicating that the method's sensitivity can detect up to 2 CFU / mL. This means that if the sample contains 2 psychrophilic bacteria per milliliter, this method can detect them.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. 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 spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting psychrophilic bacteria, characterized in that, The detection methods include: (1) Gram-negative bacteria were detected after specific amplification using TY1F and TY1R primer pairs; (2) Detection of Pseudomonas spp. by one of the following methods: (a) Detection of Pseudomonas spp. by specific amplification using TY2F and TY2R primer pairs; (b) Detection of Gram-negative bacteria by specific amplification using TY1F and TY1R primer pairs; and then detection of Pseudomonas spp. by specific amplification using TY2F and TY2R primer pairs and TY1F and TY1R primer pairs; (c) Detection by specific amplification using a mixed primer pair consisting of TY2F and TY2R primer pairs and TY1F and TY1R primer pairs. (3) Detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by one of the following methods: (a) Detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes after specific amplification using TY3F and TY3R primer pairs; (b) Detection of Gram-negative bacteria after specific amplification using TY1F and TY1R primer pairs; then detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by specific amplification using TY3F and TY3R primer pairs among the detected Gram-negative bacteria; (c) Detection of Pseudomonas spp. after specific amplification using TY2F and TY2R primer pairs; then detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by specific amplification using TY3F and TY3R primer pairs among the detected Pseudomonas spp.; (d) Detection of fluorescent Pseudomonas aeruginosa producing basic metalloenzymes by specific amplification using TY1F and TY1R primer pairs. Gram-negative bacteria were detected after amplification; then, Pseudomonas spp. were detected after specific amplification using TY2F and TY2R primer pairs among the detected Gram-negative bacteria; then, fluorescent Pseudomonas spp. producing alkaline metalloenzymes were detected after specific amplification using TY3F and TY3R primer pairs among the detected Pseudomonas spp.; (e) Specific amplification was performed using a mixed primer pair consisting of TY3F and TY3R primer pairs and TY1F and TY1R primer pairs; (f) Specific amplification was performed using a mixed primer pair consisting of TY3F and TY3R primer pairs and TY2F and TY2R primer pairs; (g) Specific amplification was performed using a mixed primer pair consisting of TY3F and TY3R primer pairs, TY1F and TY1R primer pairs, and TY2F and TY2R primer pairs; The sequences of the TY1F and TY1R primer pairs, the TY2F and TY2R primer pairs, and the TY3F and TY3R primer pairs are shown below: TY1F: 5'-CCAGGTGTGCATGGCTGCCG-3', TY1R: 5'-GCCATGTCGCAGCCGTGACCTT-3'; TY2F: 5'-CGGCAACGTCACCTCAGGAAGCTCG-3', TY2R: 5'-ACAGATACTAGGAAAATTAGACCAG-3'; TY3F: 5'-TAGAGACGGCCTGGTCCGCAACGACGGC-3', TY3R: 5'-TCGGACGTACGGTGAATAAGCCCGG-3'.

2. The method for detecting psychrophilic bacteria according to claim 1, characterized in that, While using primer pairs for amplification, corresponding specific fluorescent probes were added for co-amplification, and then psychrophilic bacteria were detected based on CT values. The specific fluorescent probe sequence for Gram-negative bacteria was 5'-AGGAGAGTTGATGAGCCGTA-3', the specific fluorescent probe sequence for *Pseudomonas* spp. was 5'-TCCGAGTAGCAGACGAATTCATTT-3', and the specific fluorescent probe sequence for alkaline metalloenzyme-producing *Pseudomonas* spp. was: 5'-AGCCGGCCCTTAAGCTTCAGCGGGCTTAGG-3'.

3. The method for detecting psychrophilic bacteria according to claim 2, characterized in that, The specific fluorescent probes for Gram-negative bacteria have a fluorescent reporter group (FAM) at the 5' end and a fluorescent quencher group (MGB) at the 3' end; the specific fluorescent probes for Pseudomonas spp. have a fluorescent reporter group (CY5) at the 5' end and a fluorescent quencher group (BHQ3) at the 3' end; and the specific fluorescent probes for alkaline metalloenzyme-producing fluorescent Pseudomonas spp. have a fluorescent reporter group (ROX) at the 5' end and a fluorescent quencher group (BHQ2) at the 3' end.

4. The method for detecting psychrophilic bacteria according to any one of claims 1 to 3, characterized in that, Psychrophilic bacteria originate from cow's milk.

5. A primer pair for PCR amplification of psychrophilic bacteria, characterized in that, Primer pairs are selected from one or more combinations of the following primer pairs: (1) Primer pairs used for PCR amplification of Gram-negative bacteria, the primer pair sequences are shown below: TY1F: 5'-CCAGGTGTGCATGGCTGCCG-3', TY1R: 5'-GCCATGTCGCAGCCGTGACCTT-3'; (2) Primer pairs used for PCR amplification of Pseudomonas spp., the primer pair sequences are shown below: TY2F: 5'-CGGCAACGTCACCTCAGGAAGCTCG-3', TY2R: 5'-ACAGATACTAGGAAAATTAGACCAG-3'; Primer pairs for PCR amplification of alkaline metalloenzymes produced by *Pseudomonas fluorescens* are shown below: TY3F: 5'-TAGAGACGGCCTGGTCCGCAACGACGGC-3', TY3R: 5'-TCGGACGTACGGTGAATAAGCCCGG-3'.

6. A primer pair and probe for PCR amplification of psychrophilic bacteria, characterized in that, The primer pairs and probes are selected from one or more combinations of the following primer pairs: (1) Primer pairs and probes used for PCR amplification of Gram-negative bacteria, the primer pair and probe sequences are shown below: TY1F: 5'-CCAGGTGTGCATGGCTGCCG-3', TY1R: 5'-GCCATGTCGCAGCCGTGACCTT-3', TY1Probe:5'-AGGAGAGTTGATGAGCCGTA-3'; (2) Primer pairs and probes used for PCR amplification of Pseudomonas spp. are shown below: TY2F: 5'-CGGCAACGTCACCTCAGGAAGCTCG-3', TY2R: 5'-ACAGATACTAGGAAAATTAGACCAG-3', TY2Probe:5'-TCCGAGTAGCAGACGAATTCATTT-3'; (3) Primer pairs and probes for PCR amplification of alkaline metalloenzymes produced by *Pseudomonas fluorescens*, the primer pair and probe sequences are shown below: TY3F: 5'-TAGAGACGGCCTGGTCCGCAACGACGGC-3', TY3R: 5'-TCGGACGTACGGTGAATAAGCCCGG-3', TY3Probe: 5'-AGCCGGCCCTTAAGCTTCAGCGGGCTTAGG-3'.

7. The primer pair and probe for PCR amplification of psychrophilic bacteria according to claim 6, characterized in that, The 5' end of the TY1Probe has a fluorescent reporter group of FAM and the 3' end has a fluorescent quencher group of MGB; the 5' end of the TY2Probe has a fluorescent reporter group of CY5 and the 3' end has a fluorescent quencher group of BHQ3; the 5' end of the TY3Probe has a fluorescent reporter group of ROX and the 3' end has a fluorescent quencher group of BHQ2.

8. A detection product for detecting psychrophilic bacteria, characterized in that, Includes the primer pair for PCR amplification of psychrophilic bacteria as described in claim 5, or the primer pair and probe for PCR amplification of psychrophilic bacteria as described in claim 6 or 7.

9. The testing product according to claim 8, characterized in that, The product being tested is a testing reagent or testing kit.

10. The application of the primer pair for PCR amplification of psychrophilic bacteria as described in claim 5, or the primer pair and probe as described in claim 6 or 7, in the detection of psychrophilic bacteria, wherein the application is selected from one of the following: (a) Psychrophilic bacteria are Gram-negative bacteria. The application of primer pairs and / or probes used for PCR amplification of Gram-negative bacteria in the detection of Gram-negative bacteria. (b) The psychrophilic bacteria are Pseudomonas spp., and the application of primer pairs and / or probes for PCR amplification of Pseudomonas spp. in the detection of Pseudomonas spp.; or the application of primer pairs and / or probes for PCR amplification of Gram-negative bacteria and primer pairs and / or probes for PCR amplification of Pseudomonas spp. in the detection of Pseudomonas spp. (c) The use of primer pairs and / or probes for PCR amplification of alkaline metalloenzymes in *Pseudomonas fluorescens*, a psychrophilic bacterium producing alkaline metalloenzymes, in detecting alkaline metalloenzymes in *Pseudomonas fluorescens*; or the use of primer pairs and / or probes for PCR amplification of Gram-negative bacteria and for PCR amplification of alkaline metalloenzymes in *Pseudomonas fluorescens*; or the use of primer pairs and / or probes for PCR amplification of *Pseudomonas* spp. and for PCR amplification of alkaline metalloenzymes in *Pseudomonas fluorescens*; or the use of primer pairs and / or probes for PCR amplification of Gram-negative bacteria, primer pairs and / or probes for PCR amplification of *Pseudomonas* spp. and for PCR amplification of alkaline metalloenzymes in *Pseudomonas fluorescens*.

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