Bacteria detection method of lytic vibrio parahaemolyticus bacteriophage

Abstract

The invention relates to the technical field of bacterial detection, in particular to a bacterial detection method of lytic vibrio parahaemolyticus bacteriophage. The method comprises the following steps: collecting a sample, preparing a sample homogenate, and pre-enriching to obtain an enrichment liquid; mixing the enrichment liquid with a broad-spectrum bacteriophage reagent to form a reaction system, and performing incubation treatment; an ATP releasing agent is added, a luciferase-fluorescein substrate working solution is added, and a bioluminescence value is measured; the bioluminescence value is converted into ATP concentration, and the original viable bacterium concentration in the sample is calculated. According to the method, based on the high-specificity recognition and lysis effects of the lytic bacteriophage on the vibrio parahaemolyticus, a sample and a high-specificity vibrio parahaemolyticus lytic bacteriophage mixture are jointly incubated, and the bacteriophage can efficiently infect and lyse living vibrio parahaemolyticus existing in the sample; and further analyzing the cracking product of the vibrio parahaemolyticus to realize rapid and specific detection of the quantity of the original viable bacteria in the sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacterial detection, in particular to a bacterial detection method of lytic Vibrio parahaemolyticus phage. BACKGROUND

[0002] Vibrio parahaemolyticus is the main pathogenic bacteria causing seafood food poisoning. The bacteria are halophilic and mainly distributed in the surface tissues of marine animals, marine sediments and seawater, so the detection rate of the bacteria in seafood is very high. After eating seafood contaminated by the bacteria, acute gastroenteritis and septicemia are easily caused.

[0003] At present, the reproduction of Vibrio parahaemolyticus is mainly controlled by low temperature during the storage and transportation of seafood. However, due to the temperature fluctuation in the cold chain process or exposure to the suitable temperature range for the growth of Vibrio parahaemolyticus, Vibrio parahaemolyticus will rapidly proliferate to reach the pathogenic dose. Therefore, it is particularly important to use bacteriostatic agents to reduce the risk of proliferation of Vibrio parahaemolyticus for the safety control of seafood storage, transportation, sales and consumption.

[0004] In the prior art, the detection of Vibrio parahaemolyticus mainly depends on traditional culture method, immunological detection and molecular biology method. These three methods have defects: the culture method is time-consuming and complicated to operate, and it is difficult to meet the demand of on-site rapid detection; the immunological method is easily interfered by cross reaction and has limited specificity; and the molecular biology method (PCR technology) has high sensitivity, but it depends on precise instruments and professional operation environment, and is not suitable for resource-limited scenes such as ports, fishing boats, aquatic markets or primary inspection sites. Moreover, the above methods are mostly limited to colony counting or qualitative detection of gene fragments, and cannot realize rapid, high-sensitivity and specific detection of live Vibrio parahaemolyticus. SUMMARY

[0005] The purpose of the present application is to avoid the problem that the existing detection methods for Vibrio parahaemolyticus cannot realize rapid, high-sensitivity and specific detection.

[0006] The purpose of the present application is to provide a bacterial detection method of lytic Vibrio parahaemolyticus phage, which uses specific lytic phage of Vibrio parahaemolyticus as a biological recognition element and a signal amplifier, and realizes efficient, rapid and specific detection of live bacteria in a sample by detecting specific endogenous markers released after the phage lyses the host bacteria or changes caused by the proliferation of the phage itself.

[0007] To achieve the above purpose, the present application provides a bacterial detection method of lytic Vibrio parahaemolyticus phage, comprising the following steps: Step S1: sterilely collecting a seafood tissue or environmental sample to be detected, and then preparing a sample homogenate; The sample homogenate is subjected to pre-enrichment treatment to obtain an enrichment liquid; Step S2: placing the enrichment liquid in a sterile reaction tube, and adding a broad-spectrum bacteriophage reagent to the reaction tube to form a reaction system; After vortex mixing the reaction system, incubate it in a constant temperature device; Step S3: after incubation, add an ATP releasing agent to the reaction system to lyse all biological cells and stabilize ATP; Then, add a luciferase-luciferin substrate working solution for mixing, and after mixing, use a portable bioluminescence detector or an enzyme label instrument to measure the bioluminescence value generated; Step S4: convert the bioluminescence value into ATP concentration, and then calculate the original live bacteria concentration in the sample according to the pre-established standard curve of ATP concentration and Vibrio parahaemolyticus colony forming units.

[0008] As a further improvement of the technical solution, in step S1, the specific sample homogenate preparation process is to weigh the sample in a sterile homogenization bag, add a proteose peptone saline buffer, and use a percussion homogenizer for sufficient homogenization to prepare a 1:10 sample homogenate.

[0009] As a further improvement of the technical solution, in step S1, the pre-enrichment treatment is to inoculate the sample homogenate in alkaline proteose peptone water or thiosulfate-citrate-bile salts-sucrose broth for culture.

[0010] As a further improvement of the technical solution, incubate in a 35-37℃ constant temperature shaking incubator for 1-3h.

[0011] As a further improvement of the technical solution, in step S2, the broad-spectrum bacteriophage reagent is composed of a plurality of Vibrio parahaemolyticus virulent phages with complementary lytic spectra, which are pre-titrated.

[0012] As a further improvement of the technical solution, the broad-spectrum bacteriophage reagent is a VPpYZU64 type Vibrio parahaemolyticus virulent phage suspension.

[0013] As a further improvement of the technical solution, in step S2, the incubation treatment is to incubate at 35-37℃ in the dark for 30-120min.

[0014] As a further improvement of the technical solution, in step S3, an ATP releasing agent is added in an amount equal to the volume of the reaction system.

[0015] In this invention, the highly specific recognition, infection, and lysis of the host bacteriophage, namely Vibrio parahaemolyticus, by lysing bacteriophages are utilized. By co-incubating a sample with a mixture of highly specific Vibrio parahaemolytic bacteriophages, the bacteriophages can efficiently infect and lyse the live Vibrio parahaemolyticus present in the sample. Furthermore, by analyzing the lysate products of Vibrio parahaemolyticus, rapid and specific detection of the original live bacteria count in the sample can be achieved.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: In this bacterial detection method using lysogenic Vibrio parahaemolyticus phage, Vibrio parahaemolyticus-specific virulent phage is used as the core biological recognition element. This method only lyses the target bacteria with metabolic activity in the sample, fundamentally eliminating false positive interference caused by residual DNA of dead bacteria or non-target bacteria. It solves the key bottleneck that molecular techniques such as PCR cannot distinguish between dead and live bacteria, and ensures the biological relevance of the detection results. By combining "short-term pre-enrichment" with "phage in vivo replication and amplification" in a dual manner, trace amounts of target bacteria are converted into a large number of detectable ATP signal molecules, which significantly reduces the detection limit and shortens the entire detection process, meeting the dual requirements of speed and sensitivity for rapid on-site screening. Attached Figure Description

[0017] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Please see Figure 1 As shown, the purpose of this embodiment is to provide a bacterial detection method for lytic Vibrio parahaemolyticus phage, including the following steps: Step S1: Aseptically collect the seafood tissue (such as fish meat, shellfish viscera) or environmental samples (seawater, sediment) to be tested, and then prepare a sample homogenate. Specifically, the sample homogenate preparation process is as follows: weigh a specified weight (e.g., 25g) of sample into a sterile homogenizing bag, add a certain volume of peptone saline buffer, and use a beat homogenizer to homogenize thoroughly to prepare a 1:10 sample homogenate.

[0020] The purpose of this homogenization process is to destroy the tissue structure of the marine product or environmental sample, fully release the bacteria attached to the surface and inside into the buffer, and form a uniform and representative bacterial suspension, providing a basis for subsequent quantitative detection. The used peptone saline buffer not only maintains the osmotic pressure and prevents cell rupture, but also maintains the viability of bacteria in a short time, avoiding false negatives caused by the death of target bacteria due to environmental inadaptation.

[0021] A certain amount of sample homogenate is taken for pre-enrichment treatment, i.e. the sample homogenate is inoculated into alkaline peptone water (APW) or thiosulfate-citrate-bile salts-sucrose (TCBS) broth, and cultured in a constant temperature shaking incubator at 35-37°C for 1-3h to obtain an enrichment liquid, so as to activate and slightly amplify the target bacterial flora, and improve the detection sensitivity.

[0022] The purpose of this pre-enrichment step is to recover and amplify the low-concentration pathogenic bacteria or bacteria in sublethal state in the sample. The alkaline environment (pH 8.4-8.6) of alkaline peptone water and the bile salts and high pH value in thiosulfate-citrate-bile salts-sucrose can inhibit the growth of many gram-positive bacteria and part of gram-negative bacteria, while selectively promoting the growth of halophilic Vibrio parahaemolyticus. In addition, the short culture time of 1-3h aims to increase the concentration of target bacteria by 1-2 orders of magnitude, significantly increasing the probability of encountering phages added subsequently, thereby greatly improving the detection success rate; at the same time, this time is strictly controlled to avoid the bacteria entering the decline period or overgrowth leading to insufficient phage lysis, and to effectively prevent the overgrowth of competitive bacteria to produce inhibitors. Constant temperature shaking culture provides sufficient oxygen and uniform nutrient environment, which is beneficial to the rapid growth of facultative anaerobic Vibrio parahaemolyticus The core purpose of this step is to extract the target bacteria from the complex sample matrix and possibly perform preliminary amplification, which is the primary prerequisite to ensure detection sensitivity and accuracy. The principle is to break the sample structure by mechanical homogenization, release the internal bacteria, and promote the repair and limited proliferation of damaged bacteria through short-term selective enrichment, thereby increasing the concentration of Vibrio parahaemolyticus in the sample to above the detection limit of the subsequent detection method, while maintaining its activity to meet the needs of phage infection.

[0023] Step S2: Place the enrichment liquid in a sterile reaction tube, and then add a broad-spectrum phage reagent to the reaction tube to form a reaction system. The addition of the phage reagent provides a high-specificity and high-titer biological recognition and lysis tool for the reaction system.

[0024] The broad-spectrum phage reagent is a pre-titrated Vibrio parahaemolyticus virulent phage suspension. After the virulent phage infects the bacteria, only a lytic cycle is performed, that is, the host resources are used to replicate the phage itself, and finally the bacteria cell is lysed to release the progeny phage. This process is the basis for signal amplification in the detection method of the present application.

[0025] The use of virulent phage ensures that the infection process will inevitably lead to the death and lysis of the host bacteria, avoiding the lysogenization of temperate phage (integrating its genome into the host chromosome) which cannot immediately release the signal molecule, thereby ensuring the timeliness of the detection and the certainty of the signal generation. The lytic cycle is the basis for signal amplification, and a hundred progeny virus particles can be produced in a short period of time, and a bacterial cell is completely lysed.

[0026] In the present application, the broad-spectrum phage reagent is a VPpYZU64 type Vibrio parahaemolyticus virulent phage suspension, and the lytic Vibrio parahaemolyticus VPpYZU64 is a virulent phage (Virulent Phage) of specific lysis Vibrio parahaemolyticus (Vibrio parahaemolyticus) with a laboratory number. Vibrio parahaemolyticus

[0027] The lytic Vibrio parahaemolyticus VPpYZU64 has a GenBank accession number of PQ581932 and is preserved in the China Center for Type Culture Collection (CCTCC) of Wuhan University, China, with a preservation date of May 10, 2018 and a preservation number of CCTCC V2018001. C NO: M 2018259, in Latin Vibrio parahaemolyticus bacteriophage VPpYZU64.

[0028] The phage is selected to constitute the preparation based on its verified properties in the previous study: first, the phage has a wide lytic spectrum and high lytic ability (titer of about 150 PFU / cell); second, the phage can maintain stable lytic activity at high temperatures of 60°C and in a wide range of pH 4-10, showing good environmental stability; third, whole genome sequencing analysis confirms that the phage does not carry any virulence or drug resistance genes, ensuring its biological safety. In addition, the phage preparation can significantly inhibit pathogenic Vibrio parahaemolyticus in the matrix, and shows concentration dependence, that is, high concentration treatment is more conducive to achieving complete inactivation of the pathogen.

[0029] The principle of action of the phage is to recognize the receptor molecules on the surface of Vibrio parahaemolyticus, thereby realizing the recognition of serotypes and strains, greatly reducing the risk of bacterial receptor mutation escaping detection, and ensuring the reliability of the method.

[0030] ​The above Vibrio parahaemolyticus virulent phage is prepared into a phage reagent as the core biological recognition element of the method. It acts like a "specific key" that only recognizes and opens (lyses) the "lock" of Vibrio parahaemolyticus, and does not act on other bacteria in the sample, thereby ensuring high specificity of the detection.

[0031] After the reaction system is vortexed and uniformly mixed, it is placed in a constant temperature device (such as a water bath or a metal bath) for incubation treatment. The incubation treatment is 35-37°C incubation in the dark for 30-120 min. During this period, the phage is specifically adsorbed to the surface of the Vibrio parahaemolyticus live bacteria, DNA is injected, the host bacteria replication system is used for proliferation, and finally the host cell is lysed, releasing the progeny phage, bacterial ATP, DNA and other intracellular contents.

[0032] The purpose of this incubation step is to provide optimal temperature and time conditions for the complete life cycle of phage adsorption, invasion, replication and lysis. 35-37°C is the optimal growth and proliferation temperature of Vibrio parahaemolyticus and its phage, which can ensure that the lysis cycle is carried out with the highest efficiency. The incubation time of 30-120 min covers one to several lysis cycles, ensuring that enough target bacteria are infected and lysed to release sufficient ATP signal molecules. The light-free condition is to prevent the potential inhibitory effect of light (especially ultraviolet light) on the infectivity of the phage and the subsequent chemiluminescence reaction. Vortexing ensures sufficient contact between the phage and the bacteria, maximizing the infection probability.

[0033] This step is the core and innovation of the entire method, and its purpose is to use the biological recognition element (phage) to specifically recognize, infect and lyse the target bacteria, and to achieve primary amplification of the signal. Its principle is based on the recognition mechanism between the phage and the host bacteria. The infection is started by the binding of the phage tail fiber protein to the specific receptors on the surface of the bacteria (such as LPS, Omp, flagella, etc.), and then the phage genome is injected into the bacteria, hijacking the host's transcription and translation system, producing a large number of progeny phages and expressing lytic proteins such as lysozyme, which ultimately destroys the cell wall and cell membrane, leading to bacterial lysis and release of all intracellular substances including ATP and DNA. This process not only completes the specific labeling of the target bacteria, but also converts a bacterial cell into hundreds of progeny phages and a large amount of ATP signal molecules, achieving exponential amplification of the signal.

[0034] Step S3: After incubation, add an ATP-releasing agent (such as a surfactant) of equal volume to the reaction system to fully lyse all biological cells and stabilize ATP. This thoroughly lyses all Vibrio parahaemolyticus not infected by bacteriophages, as well as other microorganisms (such as fungi) and eukaryotic cells that may be present in the sample. It also rapidly inactivates any ATP-degrading enzymes (ATPases) that may be present in the sample, stabilizing the released ATP molecules and preventing hydrolysis before detection, thus ensuring the accuracy of the test results. ATP-releasing agents typically contain high concentrations of surfactants (such as Triton X-100) and chelating agents. The former disrupts the lipid bilayer of cell membranes and organelles, while the latter inhibits ATPase activity by chelating cofactors such as Mg²⁺.

[0035] Subsequently, the luciferase-luciferin substrate working solution was added and mixed. Immediately after mixing, the bioluminescence value (Relative Light Unit, RLU) was measured using a portable bioluminescence detector or microplate reader. The purpose of adding the luciferase-luciferin substrate working solution is to initiate the chemiluminescence reaction. This working solution contains purified recombinant luciferase, excess luciferin substrate, Mg²⁺ ions, and a buffer system. The reaction occurs immediately upon addition. Immediate measurement is necessary because this luminescence reaction is flash-type; the light signal peaks within seconds to one minute after mixing and then rapidly decays. Detection must be completed within this time window to ensure data accuracy and comparability. The portable bioluminescence detector captures the weak light signal and converts it into an electrical signal. After amplification and digitization, the output is a relative light unit (RLU) value, which is directly related to the ATP concentration in the sample.

[0036] The purpose of this step is to convert the biological signal (ATP) released from biolysis in the previous step into a quantitatively detectable optical signal (photons). The principle is based on the catalytic reaction of firefly luciferase: in the presence of Mg²⁺ and O₂, luciferase catalyzes the reaction of luciferin with ATP to generate adenosine monophosphate (AMP), pyrophosphate (PPi), and oxyluciferin, releasing photons of a specific wavelength (typically around 560 nm). The absolute rate-limiting step and substrate of this reaction is ATP, and its reaction rate (i.e., the photon release rate) is linearly positively correlated with ATP concentration over several orders of magnitude. Precise quantification of ATP can be achieved by measuring the light intensity using a sophisticated photodetector such as a photomultiplier tube (PMT).

[0037] Step S4: Convert the bioluminescence value into ATP concentration, and then calculate the original viable bacterial concentration in the sample based on a pre-established standard curve of ATP concentration versus Vibrio parahaemolyticus colony-forming units (CFU). This method can be completed within minutes and is suitable for rapid on-site screening.

[0038] It is worth noting that the specific process of step S4 in this invention is as follows: First, prepare a series of pure ATP standard solutions of known concentrations (e.g., 0, 10). -9 10 -8 10 -7 10 -6 M). These standards were then treated with the same ATP extraction reagent and luciferase-luciferin reagent. The luminescence value (RLU) corresponding to each ATP concentration was measured on a luminescence detector. A scatter plot was plotted with the logarithm of ATP concentration (log[ATP]) as the X-axis and the logarithm of RLU value (log(RLU)) as the Y-axis, and a linear regression was performed. The formula is: log(RLU) = a * log[ATP] + b (where a is the slope and b is the intercept) This mathematical relationship (coefficients a and b) is built into or calibrated into the instrument's algorithm.

[0039] Then, the standard strain of Vibrio parahaemolyticus is cultured to the logarithmic growth phase. The concentration of the bacterial suspension (in CFU / mL) is accurately determined by plate counting. This is an absolute baseline. A certain volume (e.g., 1 mL) of the bacterial suspension of known concentration is then taken and treated using the same method as the sample (addition of ATP-releasing agent and luminescent reagent), and its RLU value is measured. Using the ATP standard curve from step one, this RLU value is converted to the corresponding ATP concentration. The average ATP content per colony-forming unit (CFU) is calculated: ATP_per_CFU = ( [ATP] × V ) / N (Where [ATP] is the measured ATP concentration, V is the sample volume, and N is the total number of CFUs in the sample.) This ATP_per_CFU value (e.g., on the order of 10^(-18) moles / CFU, or 1 attomole / CFU) is a key average conversion factor that will be input into the instrument's detection method parameters.

[0040] When measuring a prepared unknown sample, the instrument will calculate according to the following procedure: Reading RLU value: The photomultiplier tube detects the emission signal of the sample and converts it into a digital signal RLU.

[0041] Calculate the ATP concentration in the sample ([ATP]_sample): The instrument calls the ATP standard curve formula established in the first step: log(RLU) = a * log[ATP] + b.

[0042] Substitute the measured RLU value into the formula to perform the reverse calculation: log[ATP]_sample = (log(RLU_sample) - b) / a Then, the antilogarithm calculation is performed to obtain the ATP concentration in the sample: [ATP]_sample = 10^(log[ATP]_sample) Calculate the total ATP content in the sample: Calculate the total amount of ATP extracted from the original sample based on the sample volume (V_sample) and reagent dilution factor (D) used in the detection.

[0043] Total_ATP = [ATP]_sample × V_sample × D (unit: moles) Calculate the number of viable bacteria (CFU) in the original sample: The instrument calls the pre-entered conversion factor ATP_per_CFU.

[0044] Estimated_CFU = Total_ATP / ATP_per_CFU (unit: CFU) Convert back to original sample concentration: Based on the initial sample homogenate volume used for testing (e.g., 1 mL) and the dilution factor of the original sample (e.g., 25 g sample + 225 mL buffer, diluted 10 times), the final viable bacterial concentration in the original sample (e.g., per gram or per milliliter) is calculated.

[0045] In summary, the entire calculation process can be summarized into the following three steps, which the instrument's built-in algorithm will automatically execute: The first step is to convert the ATP using the luciferase reaction system's standard curve; The second step is to convert based on the sample volume and reagent dilution factor; The third step is to convert the data using a pre-established standard for bacterial ATP content (i.e., the average ATP content per CFU).

[0046] In this invention, firstly, sample homogenization and short-term selective pre-enrichment treatment fully release endogenous bacteria in the tissue and inhibit the growth of other bacteria, while specifically activating and amplifying the viable Vibrio parahaemolyticus flora, increasing the target bacterial concentration to a detectable threshold, thus providing a host for subsequent phage recognition. Then, the enriched bacteria are co-incubated with a multi-strain phage reagent with complementary lysis spectra. Utilizing the high affinity binding of phage tail fimbriae to bacterial surface receptors, specific recognition and invasion of the pathogen are achieved. The pathogen then replicates and cycles in vivo, expressing large amounts of lysozyme to completely lyse the host cell, releasing intracellular ATP and nucleic acid substances, completing the initial conversion and exponential amplification of the biological signal. Next, a compound ATP-releasing agent is added to completely disintegrate all remaining microbial cell membrane structures, stabilizing the signal molecules. Simultaneously, a luciferase-luciferin substrate system is introduced, triggering an enzymatic chemiluminescence reaction driven by ATP, converting the biochemical signal into a photon signal. During the signal acquisition stage, the high sensitivity of the photomultiplier tube is used to capture the transient emission peak, and the RLU value is converted into ATP concentration in real time by the built-in algorithm. During the quantitative analysis stage, based on the pre-established standard coefficient of ATP content per unit cell of Vibrio parahaemolyticus, combined with the dilution factor and reaction volume, the viable bacterial concentration in the original sample is automatically calculated. This invention avoids the limitations of traditional culture methods, such as long processing time, reliance on sophisticated instruments, and inability to distinguish between dead and live bacteria, as well as the high host specificity of bacteriophages. It effectively eliminates interference from other bacteria in the complex matrix of seafood, significantly improving detection accuracy and anti-interference capabilities compared to immunological methods. Finally, through the combined use of biolysis and bioluminescence technologies, it achieves rapid, highly sensitive, and quantitative on-site detection without complex pretreatment, providing core technical support for port quarantine, aquaculture monitoring, and on-site food safety supervision.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A bacterial detection method for lytic Vibrio parahaemolyticus phage, characterized in that, Includes the following steps: Step S1: Aseptically collect the seafood tissue or environmental sample to be tested, and then prepare the sample homogenate; The sample slurry was then homogenized and pre-enriched to obtain the enriched solution. Step S2: Place the enrichment solution in a sterile reaction tube, and then add a broad-spectrum phage reagent to the reaction tube to form a reaction system; After the reaction system is vortexed and mixed, it is placed in a constant temperature device for incubation. Step S3: After incubation, add an ATP-releasing agent to the reaction system to fully lyse all biological cells and stabilize ATP; Subsequently, luciferase-luciferin substrate working solution was added and mixed. After mixing, the bioluminescence value was measured using a portable bioluminescence detector or ELISA reader. Step S4: Convert the bioluminescence value into ATP concentration, and then calculate the original viable bacteria concentration in the sample based on the pre-established standard curve of ATP concentration and Vibrio parahaemolyticus colony-forming units.

2. The bacterial detection method for lytic Vibrio parahaemolyticus phage according to claim 1, characterized in that: In step S1, the specific sample homogenization process is as follows: weigh the sample into a sterile homogenizing bag, add peptone saline buffer, and use a tapping homogenizer to homogenize thoroughly to prepare a 1:10 sample homogenate.

3. The bacterial detection method for lytic Vibrio parahaemolyticus phage according to claim 1, characterized in that: In step S1, the pre-enrichment treatment involves inoculating the sample homogenate into alkaline peptone water or thiosulfate-citrate-cholesterol broth for culture.

4. The bacterial detection method for lytic Vibrio parahaemolyticus phage according to claim 3, characterized in that: Incubate in a constant temperature shaking incubator at 35-37℃ for 1-3 hours.

5. The bacterial detection method for lytic Vibrio parahaemolyticus phage according to claim 1, characterized in that: In step S2, the broad-spectrum phage reagent consists of pre-titrated virulent phages of multiple strains of Vibrio parahaemolyticus with complementary lysis spectra.

6. The bacterial detection method for lytic Vibrio parahaemolyticus phage according to claim 5, characterized in that: The broad-spectrum phage reagent is a suspension of VPpYZU64 type Vibrio parahaemolyticus phage.

7. The bacterial detection method for lytic Vibrio parahaemolyticus phage according to claim 1, characterized in that: In step S2, the incubation treatment is carried out at 35-37°C in the dark for 30-120 minutes.

8. The bacterial detection method for lytic Vibrio parahaemolyticus phage according to claim 1, characterized in that: In step S3, an ATP-releasing agent of equal volume to the reaction system is added.

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