# Analysis of PathogenicVibrio Contamination in Marine Products along China Based on Fluorescence Quantitative PCR

Authors: Tong Wang; Shuo Wan; Jian-Lian Huang; Zhi-Hai Sui; Cui-Juan Gao; Yun-Guo Liu
Journal: Journal of Food Quality
 (2022)

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## Abstract

At present, aquatic product pollution has become the main root of frequent food safety problems and causes economic losses.Vibrio is one of the main pathogens causing foodborne diseases. In this study, in order to uncover the pollution status of pathogenic Vibrio in the marine products of China, a total of 646 aquatic products were collected and analyzed from 10 coastal cities in China. Five kinds of pathogenic Vibrio were separated from these samples and monitored to explore the relationship between pollution and the pathogen. Real-time fluorescence quantitative PCR was adopted to detect foodborne Vibrio quantitatively in marine aquatic products. Aquatic pathogenic Vibrio was collected in different regions, different types of aquatic products, and different sampling places, and the difference in detection rate was statistically significant through statistical analysis. This study made a frame for the pollution degree of pathogenic Vibrio in marine products in China and established the dominant flora of pathogenic Vibrio in different types of aquatic products, which provides a theoretical basis for food safety supervision departments to take targeted prevention and control measures.


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## Body

## 1. Introduction

Aquatic products are rich in variety and contain various nutrients required for a healthy daily diet. However, with the rapid development of modern industry and the wide use of pesticides, the aquatic animal breeding and fishing environment is deteriorating, polluting aquatic organisms, affecting the quality of aquatic products, and threatening the health of consumers [1]. The main chemical pollutants include excessive heavy metals, pesticide residues, fishery drug residues, and other organic pollutants. Careless consumption of these aquatic products will bring many health risks [2]. At present, aquatic product pollution has become the root of frequent food safety problems and causes immense economic losses [1]. Therefore, it is vital to develop rapid and reliable pathogen detection methods to reduce the frequency of outbreaks [3]. Vibrio is a Gram-negative rod-shaped or curved bacteria widely existing in seawater, seawater sediments, fish, shellfish, and other seafood, which includes the most common pathogenic genus in seawater and edible marine animals [4]. Eating raw or undercooked seafood can trigger foodborne diseases [5]. Vibrio infections can also occur as a result of skin damage (such as cuts, open wounds, or abrasions) exposed to the aquatic environment and marine animals. Due to the increasing outbreak of foodborne diseases, pathogenic Vibrio has become important in epidemiology [6].Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio fluvialis, and Vibrio anguillarum are common pathogens. V. parahaemolyticus is a halophilic pathogen that can be found in marine, estuarine, and aquaculture environments all over the world, and is detected constantly in aquatic products such as shellfish, crustaceans, fish, and cephalopods [7, 8]. Gastroenteritis is usually caused by eating contaminated shellfish and other raw or undercooked aquatic products containing V. parahaemolyticus [9]. V. vulnificus is another halophilic pathogen found in coastal areas and estuaries. It is pathogenic to marine animals and poses a threat to fish farming. Nevertheless, direct human infection would lead to sepsis [4]. V. alginolyticus is considered one of the most harmful Vibrio, which is pathogenic to humans and aquatic animals [10]. When exposed to polluted seawater, human soft tissues, ears and superficial wounds are vulnerable to V. alginolyticus [11]. Clinical symptoms include chronic diarrhea, otitis media and wound infection [12]. V. fluvialis is also considered a foodborne pathogen [13]. Studies have shown that V. fluvialis is associated with infant enterocolitis [14]. V. anguillarum is a severe aquatic pathogenic marine bacterium [15], causing vibriosis and sepsis in infected fish, crustaceans and bivalves [16], which caused significant economic losses [17].Under the current situation, rapid and reliable microbial detection technology is not only an indispensable tool for government departments confronting disease outbreaks but also an effective means of disease prevention and control [18]. Therefore, it depends on accurate and specific innovative analytical methods to identify pathogenic microorganisms from clinical and food samples. Traditional bacterial identification methods are time-consuming and laborious [19]. It will take several days to confirm the presence of Vibrio pathogens isolated from aquatic products [3], and the diagnostic sensitivity and specificity are inadequate [20]. At the same time, traditional PCR cannot quantify the target organism directly [3, 5]. Quantitative testing is necessary to determine the intensity of infection in the sample and the prevalence of organisms in the environment. Therefore, in order to control the harmfulness caused by pathogenic Vibrio, a rapid, reliable and sensitive detection technique is needed to track pathogenic Vibrio in aquatic products. Real-time fluorescence quantitative PCR has the characteristics of rapidity, high sensitivity and high specificity, low cost and low detection limit, and does not need PCR post-processing [21]. In addition, real-time fluorescence quantitative PCR can be used to identify Vibrio directly, which is highly efficient for detecting and quantifying pathogenic microorganisms [5].Real-time fluorescence quantitative PCR is a usual method to add fluorescent genes into the PCR reaction system and use the accumulation of fluorescent signals to trace the whole PCR process in real-time [22]. It quantifies the template’s initial concentration of DNA or cDNA in the sample through CT value and standard curve. It can be understood as adding fluorescent substances to the PCR reaction system. The fluorescence intensity emitted by fluorochrome is directly proportional to DNA yield [23]. The intensity of the fluorescence signal in the PCR reaction system was monitored continuously [24], then the amount of specific products in pathogenic bacteria was obtained to achieve the purpose of quantitative monitoring. Real-time fluorescence quantitative PCR has a high coincidence rate and sensitivity and can monitor the amplification of each cycle [25], which is more time saving and accurate than the traditional monitoring method [26].Aquatic products are an indispensable portion of nutrients in people’s life. However, water pollution in oceans, rivers, lakes and other places has been increasingly serious in recent years. If aquatic products are cultivated from such water resources, there will also be many bacteria in these products, which will further pose a severe risk to people’s health, especially the harm caused by pathogenicVibrio. Quantitative PCR technology is a detection method by using SYBR Green real-time PCR to track pathogenic Vibrio in marine products quantitatively. Compared with the traditional bacterial detection method, this is safer and more convenient, which has important practical significance for monitoring pathogenic Vibrio in aquatic products.In our previous study, 5 kinds of pathogenicVibrio in marine products of 10 coastal cities were monitored and analyzed in 2019 to explore their possible pollution causes in order to understand the pollution status of pathogenic Vibrio in marine products along China, accumulate basic data for risk assessment, and provide a guide for strengthening the monitoring and prevention of foodborne diseases in China. This study aimed to quantitatively detect foodborne pathogenic Vibrio in aquatic products by real-time fluorescence quantitative PCR so as to provide a powerful reference for controlling the pollution of pathogenic Vibrio in aquatic products and reducing the foodborne diseases caused by pathogenic Vibrio.


## 2. Materials and Methods

### 2.1. Sample Collection

A total of 646 samples of marine aquatic products were collected in 10 cities from June to August 2019, that is Dalian, Liaoning; Yantai and Qingdao, Shandong; Lianyungang, Jiangsu; Nantong, Jiangsu;, Ningbo, Zhejiang; Wenzhou, Zhejiang; Xiamen, Fujian; Zhanjiang, Guangdong; and Beihai, Guangxi (Table1). Among the 646 samples, 136 were collected from restaurants, 120 from barbecue shops, 117 from farmers’ markets, 132 from supermarkets and 143 from online sales (Table 2). All aquatic products are stored at 4°C within 24 hours after being caught. The pollution of 5 kinds of foodborne pathogenic Vibrio (V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, and V. anguillarum) was screened and monitored, repeating each process three times.Table 1
Sample collection location.
Aquatic product categoryFishShrimpShellfishCrabsSnailsAlgaeTotalDalian, Liaoning12121010131168Yantai, Shandong1112109101264Qingdao, Shandong109129101262Lianyungang, Jiangsu101310812962Nantong, Jiangsu1111131113968Ningbo, Zhejiang12101012111166Wenzhou, Zhejiang13121112101371Xiamen, Fujian12101010101264Zhanjiang, Guangdong101010991058Beihai, Guangxi111212910963Total11211110899108108646Table 2
Sample collection site.
Type of sampling siteNumber of samples (copies)Restaurant134Barbecue shop120Farm product market117Supermarket132Network sales143Total646


### 2.2. SYBR Green Real-Time PCR Detection Method

SYBR Green is a binding dye that can greatly enhance its fluorescence after binding in the small groove of double-stranded DNA. While free SYBR dye molecules will not emit any fluorescent signals, SYBR dye molecules specifically incorporate DNA double strands to emit fluorescent signals. The increase of PCR products is completely synchronized with the increase of fluorescent signals after SYBR fluorescent dye is added to the PCR reaction system. Then the whole PCR process was monitored by fluorescence signal accumulation, and the unknown concentration template was calculated quantitatively by using the standard curve. In this experiment, specific detection primers and prepared DNA templates are designed referring to the relevant literature (Table3). The PCR reaction procedure was set as follows: denaturation at 95°C for 5 s, annealing at 60°C for 20 s, extension at 72°C for 20 s, 40 cycles, fluorescence signals were collected at the annealing extension stage of each cycle, quantitative fluorescence detection was carried out, and the corresponding detection rate was obtained by analysis.Table 3
Oligonucleotide primer used in this study.
StrainPrime and sequence (5′ to 3′)Amplicon size (bp)ReferenceV. alginolyticusForward: ATT GAG AAC CCG ACAGAA GCG AAG340[19]Reverse: CCTAATGCGGTGATCAGTGTTACTV. parahaemolyticusForward: GAAAGTTGAACATCATCAGCACGA271[6]Reverse: GGTCAGAATCAAACGCCGV. vulnificusForward: TTCCAACTTCAAACCGAACTATGA205[27]Reverse: ATTCCAGTCGATGCGAATACGTTGV. anguillarumForward: TATCACTGTTGAAGAAGGTCAAGCACTG195[17]Reverse: CGCTTCAAGTGCAGGAAGCAGV. fluvialisForward: CGGGCTGGCCATGGACTAAACCATC280This studyReverse: GAACCGTCATACGCGGGTTCAGAGA


### 2.3. Statistical Analysis

SPSS 20.0 statistical software was used for data processing and analysis, and the chi-square test was applied to distinguish the difference in test results. The difference was statistically significant (P<0.05).


## 2.1. Sample Collection

A total of 646 samples of marine aquatic products were collected in 10 cities from June to August 2019, that is Dalian, Liaoning; Yantai and Qingdao, Shandong; Lianyungang, Jiangsu; Nantong, Jiangsu;, Ningbo, Zhejiang; Wenzhou, Zhejiang; Xiamen, Fujian; Zhanjiang, Guangdong; and Beihai, Guangxi (Table1). Among the 646 samples, 136 were collected from restaurants, 120 from barbecue shops, 117 from farmers’ markets, 132 from supermarkets and 143 from online sales (Table 2). All aquatic products are stored at 4°C within 24 hours after being caught. The pollution of 5 kinds of foodborne pathogenic Vibrio (V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, and V. anguillarum) was screened and monitored, repeating each process three times.Table 1
Sample collection location.
Aquatic product categoryFishShrimpShellfishCrabsSnailsAlgaeTotalDalian, Liaoning12121010131168Yantai, Shandong1112109101264Qingdao, Shandong109129101262Lianyungang, Jiangsu101310812962Nantong, Jiangsu1111131113968Ningbo, Zhejiang12101012111166Wenzhou, Zhejiang13121112101371Xiamen, Fujian12101010101264Zhanjiang, Guangdong101010991058Beihai, Guangxi111212910963Total11211110899108108646Table 2
Sample collection site.
Type of sampling siteNumber of samples (copies)Restaurant134Barbecue shop120Farm product market117Supermarket132Network sales143Total646


## 2.2. SYBR Green Real-Time PCR Detection Method

SYBR Green is a binding dye that can greatly enhance its fluorescence after binding in the small groove of double-stranded DNA. While free SYBR dye molecules will not emit any fluorescent signals, SYBR dye molecules specifically incorporate DNA double strands to emit fluorescent signals. The increase of PCR products is completely synchronized with the increase of fluorescent signals after SYBR fluorescent dye is added to the PCR reaction system. Then the whole PCR process was monitored by fluorescence signal accumulation, and the unknown concentration template was calculated quantitatively by using the standard curve. In this experiment, specific detection primers and prepared DNA templates are designed referring to the relevant literature (Table3). The PCR reaction procedure was set as follows: denaturation at 95°C for 5 s, annealing at 60°C for 20 s, extension at 72°C for 20 s, 40 cycles, fluorescence signals were collected at the annealing extension stage of each cycle, quantitative fluorescence detection was carried out, and the corresponding detection rate was obtained by analysis.Table 3
Oligonucleotide primer used in this study.
StrainPrime and sequence (5′ to 3′)Amplicon size (bp)ReferenceV. alginolyticusForward: ATT GAG AAC CCG ACAGAA GCG AAG340[19]Reverse: CCTAATGCGGTGATCAGTGTTACTV. parahaemolyticusForward: GAAAGTTGAACATCATCAGCACGA271[6]Reverse: GGTCAGAATCAAACGCCGV. vulnificusForward: TTCCAACTTCAAACCGAACTATGA205[27]Reverse: ATTCCAGTCGATGCGAATACGTTGV. anguillarumForward: TATCACTGTTGAAGAAGGTCAAGCACTG195[17]Reverse: CGCTTCAAGTGCAGGAAGCAGV. fluvialisForward: CGGGCTGGCCATGGACTAAACCATC280This studyReverse: GAACCGTCATACGCGGGTTCAGAGA


## 2.3. Statistical Analysis

SPSS 20.0 statistical software was used for data processing and analysis, and the chi-square test was applied to distinguish the difference in test results. The difference was statistically significant (P<0.05).


## 3. Results and Analysis

### 3.1. Detection Level of PathogenicVibrio in Aquatic Products of Varied Regions

646 samples of marine aquatic products were collected in 10 cities, includingDalian, Liaoning; Yantai and Qingdao, Shandong; Lianyungang, Jiangsu; Nantong, Jiangsu;, Ningbo, Zhejiang; Wenzhou, Zhejiang; Xiamen, Fujian; Zhanjiang, Guangdong; and Beihai, Guangxi. A total of 212 pathogenicVibrio were detected, with a detection rate of 32.82%. Among them, the detection rate of pathogenic Vibrio in aquatic products in Zhanjiang, Guangdong is the highest, 58.62%, followed by Beihai, Guangxi (52.38%) and Xiamen, Fujian (48.44%), and Dalian, Liaoning is the lowest, 17.65%. The difference in the detection rate of pathogenic Vibrio in aquatic products in 10 cities is statistically significant (P<0.01) through statistical analysis (Table 4).Table 4
Detection level of pathogenicVibrio in aquatic products in different regions.
RegionDetected quantity (copies)Number of samples (copies)Detection rate (%)Dalian, Liaoning126817.65Yantai, Shandong136420.31Qingdao, Shandong126219.35Lianyungang, Jiangsu166225.81Nantong, Jiangsu196827.94Ningbo, Zhejiang196628.79Wenzhou, Zhejiang237132.39Xiamen, Fujian316448.44Zhanjiang, Guangdong345858.62Beihai, Guangxi336352.38Total21264632.82χ2 value54.2P value<0.01


### 3.2. Detection of PathogenicVibrio in Different Categories of Aquatic Products

Among different kinds of aquatic products, the highest detection rate is crab (46.47%), followed by shrimp (45.95%), shellfish (34.26%), snails (31.48%), fish (27.68%), and the lowest detection rate is algae (12.04%). The difference in the detection rate of pathogenic bacteria among the six kinds of aquatic products is statistically significant (P<0.01) (Table 5).Table 5
Detection rate of pathogenicVibrio in various aquatic products.
Aquatic product categoryDetected quantity (copies)Number of samples (copies)Detection rate (%)Fish3111227.68Shrimp5111145.95Shellfish3710834.26Crabs469946.47Snails3410831.48Algae1310812.04Total21264632.82χ2 value42.9P value<0.01


### 3.3. Detection ofVibrio Contamination with Different Pathogenicities

As shown in Table6, Vibrio parahaemolyticus has the highest detection rate of 16.1%, followed by Vibrio alginolyticus (7.28%) and V. vulnificus (4.95%), while Vibrio anguillarum has the lowest detection rate of 1.7%. Through statistical analysis, there was a significant difference in the detection rate of five types of pathogenic Vibrio in aquatic products (P<0.01) (Table 6).Table 6
Detection of different types of pathogenicVibrio in aquatic products.
PathogenicVibrio typesDetected quantity (copies)Number of samples (copies)Detection rate (%)V. parahaemolyticus10464616.1V. vulnificus326464.95V. alginolyticus476467.28V. fluvialis186462.79V. anguillarum116461.7Total21264632.82χ2 value127.1P value<0.01


### 3.4. Detection of PathogenicVibrio at Different Sampling Sites

Among different sampling sites, the highest detection rate is the farm product market (67.52%), followed by network sales (39.86%), barbecue shops (30.83%), supermarkets (17.42%), and the lowest detection rate is restaurants (11.94%); Through statistical analysis, there was a significant difference in the detection rate of pathogenicVibrio in aquatic product samples collected at each sampling site (P<0.01) (Table 7).Table 7
Detection levels of pathogenicVibrio in different sampling sites.
Type of sampling siteDetected quantity (copies)Number of samples (copies)Detection rate (%)Restaurant1613411.94Barbecue shop3712030.83Farm product market7911767.52Supermarket2313217.42Network sales5714339.86Total21264632.82χ2 value109.4P value<0.01


## 3.1. Detection Level of PathogenicVibrio in Aquatic Products of Varied Regions

646 samples of marine aquatic products were collected in 10 cities, includingDalian, Liaoning; Yantai and Qingdao, Shandong; Lianyungang, Jiangsu; Nantong, Jiangsu;, Ningbo, Zhejiang; Wenzhou, Zhejiang; Xiamen, Fujian; Zhanjiang, Guangdong; and Beihai, Guangxi. A total of 212 pathogenicVibrio were detected, with a detection rate of 32.82%. Among them, the detection rate of pathogenic Vibrio in aquatic products in Zhanjiang, Guangdong is the highest, 58.62%, followed by Beihai, Guangxi (52.38%) and Xiamen, Fujian (48.44%), and Dalian, Liaoning is the lowest, 17.65%. The difference in the detection rate of pathogenic Vibrio in aquatic products in 10 cities is statistically significant (P<0.01) through statistical analysis (Table 4).Table 4
Detection level of pathogenicVibrio in aquatic products in different regions.
RegionDetected quantity (copies)Number of samples (copies)Detection rate (%)Dalian, Liaoning126817.65Yantai, Shandong136420.31Qingdao, Shandong126219.35Lianyungang, Jiangsu166225.81Nantong, Jiangsu196827.94Ningbo, Zhejiang196628.79Wenzhou, Zhejiang237132.39Xiamen, Fujian316448.44Zhanjiang, Guangdong345858.62Beihai, Guangxi336352.38Total21264632.82χ2 value54.2P value<0.01


## 3.2. Detection of PathogenicVibrio in Different Categories of Aquatic Products

Among different kinds of aquatic products, the highest detection rate is crab (46.47%), followed by shrimp (45.95%), shellfish (34.26%), snails (31.48%), fish (27.68%), and the lowest detection rate is algae (12.04%). The difference in the detection rate of pathogenic bacteria among the six kinds of aquatic products is statistically significant (P<0.01) (Table 5).Table 5
Detection rate of pathogenicVibrio in various aquatic products.
Aquatic product categoryDetected quantity (copies)Number of samples (copies)Detection rate (%)Fish3111227.68Shrimp5111145.95Shellfish3710834.26Crabs469946.47Snails3410831.48Algae1310812.04Total21264632.82χ2 value42.9P value<0.01


## 3.3. Detection ofVibrio Contamination with Different Pathogenicities

As shown in Table6, Vibrio parahaemolyticus has the highest detection rate of 16.1%, followed by Vibrio alginolyticus (7.28%) and V. vulnificus (4.95%), while Vibrio anguillarum has the lowest detection rate of 1.7%. Through statistical analysis, there was a significant difference in the detection rate of five types of pathogenic Vibrio in aquatic products (P<0.01) (Table 6).Table 6
Detection of different types of pathogenicVibrio in aquatic products.
PathogenicVibrio typesDetected quantity (copies)Number of samples (copies)Detection rate (%)V. parahaemolyticus10464616.1V. vulnificus326464.95V. alginolyticus476467.28V. fluvialis186462.79V. anguillarum116461.7Total21264632.82χ2 value127.1P value<0.01


## 3.4. Detection of PathogenicVibrio at Different Sampling Sites

Among different sampling sites, the highest detection rate is the farm product market (67.52%), followed by network sales (39.86%), barbecue shops (30.83%), supermarkets (17.42%), and the lowest detection rate is restaurants (11.94%); Through statistical analysis, there was a significant difference in the detection rate of pathogenicVibrio in aquatic product samples collected at each sampling site (P<0.01) (Table 7).Table 7
Detection levels of pathogenicVibrio in different sampling sites.
Type of sampling siteDetected quantity (copies)Number of samples (copies)Detection rate (%)Restaurant1613411.94Barbecue shop3712030.83Farm product market7911767.52Supermarket2313217.42Network sales5714339.86Total21264632.82χ2 value109.4P value<0.01


## 4. Discussion

Taking in contaminated aquatic products would cause foodborne diseases.Vibrio is a serious foodborne pathogen worldwide [28]. Especially in the southeast coastal areas of China, Vibrio is the main pathogen causing food poisoning. This study reflects the general situation of foodborne pathogenic bacteria pollution of aquatic products in coastal cities in China. Pathogenic Vibrio is detected in aquatic products of different regions, different types of aquatic products, and different sampling places. Meanwhile, our research shows that the pollution rate of V. parahaemolyticus and V. alginolyticus in these aquatic products is high apparently, and they are considered the main pathogens causing foodborne diseases. When detecting Vibrio from fish and shellfish in Egypt, it is found that the detection rate of V. alginolyticus is the highest, which reflects actual local factors, such as the type and source of aquatic products, transportation and storage, treatment process of geographical source, and season [29]. Although the detection rates of V. vulnificus, V. fluvialis and V. anguillarum are low, studies have shown that they are highly related to extensive food poisoning and even lead to infection and death. Therefore, all these five kinds of pathogenic Vibrio should be paid attention to.From the analysis of different links, this study found that the detection rate of pathogenicVibrio was different in different types of aquatic products. The highest detection rate was found in crabs and shrimps, followed by shellfish, fish, snails and algae. The shells of shrimp and crabs may be conducive for these species to adhere, settle, and promote their survival in the aquatic environment [30]. These aquatic products are a regular diet in people’s daily life, and the clean and hygienic food source is closely related to people’s health and safety. Furthermore, this study demonstrates varied levels of pathogenic Vibrio when comparing the main pollution sources in marine products in China. For detail, in restaurants and supermarkets, the detection rate of pathogenic Vibrio in aquatic products is low, while in barbecue shops, farmers’ markets and online sales, the detection rate is significantly high. These results suggest that the main pollution source of pathogenic Vibrio in coastal marine products of China is cross pollution during transportation and sales. When detecting pathogenic Vibrio in aquatic products in different regions, it is found that Zhanjiang, Guangdong, has the highest detection rate, followed by Beihai, Guangxi, and Shenzhen, Guangdong. With the advanced transportation service and trades between inland and coastal areas, pathogenic Vibrio has also spread from coastal areas into inland areas, becoming an apparent food safety risk factor that cannot be ignored in inland areas. Another problem related to Vibrio species is their increasing prevalence of antibiotic resistance in the marine environment, which is an important public health issue of widespread concern in various countries [31]. Particularly Vibrio strains with multiple antibiotic resistance may pose a serious threat to human health and the seafood industry [32,33]. Therefore, in future perspectives, we will examine the antibiotic resistance of these vibrios.In Asian countries, including China, South Korea, and Japan, eating raw seafood is the main cause of gastrointestinal diseases. To sum up, the pollution of pathogenicVibrio in aquatic products of different regions is serious. Especially, there are multiple Vibrio with simultaneous cross contamination in the farm product market. It is urgent to strengthen the management of all links of aquatic products and reduce pathogenic Vibrio pollution and prevent foodborne diseases. In addition, we recommend not eating raw or undercooked seafood in order to avoid taking in food contaminated by Vibrio. It would be worth suggesting adequate cooking of the seafoods before consumption to prevent future foodborne outbreaks.This study mastered the pollution degree and source of pathogenicVibrio in marine products in China and the dominant flora of pathogenic Vibrio in different types of aquatic products. It provides a theoretical basis for monitoring, early warning and molecular traceability of foodborne diseases. Based on the results of this study, further efforts are needed to quickly identify pathogens and control Vibrio contamination in aquatic products. In addition, there is an urgent need for consultation and cooperation between researchers and the government in order to minimize the risk of pathogenic Vibrio infection and ensure the safety of aquatic products.



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