实时荧光单引物等温扩增技术检测海产品中副溶血性弧菌的研究.docx

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Title: Real-time Fluorescent Probe Isothermal Amplification Technique for the Detection of Vibrio parahaemolyticus in Seafood
Abstract:
Vibrio parahaemolyticus is a pathogenic bacterium commonly found in seafood that can cause gastroenteritis in humans. Therefore, the development of rapid, sensitive, and specific detection methods is crucial for ensuring seafood safety. In this study, we investigated the use of real-time fluorescent probe isothermal amplification technique for the detection of V. parahaemolyticus in seafood. This isothermal amplification technique offers several advantages over traditional PCR methods, including faster amplification time, simplified procedures, and higher sensitivity. The results of this study demonstrated the feasibility and potential of using real-time fluorescent probe isothermal amplification technique as a rapid and reliable method for detecting V. parahaemolyticus in seafood.
Keywords: Vibrio parahaemolyticus, real-time fluorescent probe, isothermal amplification, seafood safety.
Introduction:
Seafood is a valuable source of nutrition, but it is also prone to contamination by various pathogens, including Vibrio parahaemolyticus. V. parahaemolyticus is one of the major causes of seafood-associated bacterial infections worldwide, leading to gastroenteritis in humans. Therefore, timely and accurate detection of V. parahaemolyticus in seafood is of great importance for ensuring food safety and preventing outbreaks of foodborne illnesses. Conventional detection methods such as culture-based methods and PCR-based methods have limitations in terms of sensitivity, specificity, and time-consuming procedures. Hence, it is necessary to develop alternative methods that can provide rapid and reliable detection of V. parahaemolyticus in seafood.
Materials and Methods:
Real-time fluorescent probe isothermal amplification technique was employed in this study for the detection of V. parahaemolyticus in seafood samples. The technique involves an isothermal amplification process that can generate a large amount of target DNA in a short period of time under constant temperature conditions. A real-time fluorescent probe was designed to specifically bind to the amplified target DNA, enabling real-time monitoring of the amplification process. The specificity and sensitivity of the assay were evaluated using purified V. parahaemolyticus DNA as a positive control and other related bacterial species as negative controls. Additionally, seafood samples were artificially contaminated with V. parahaemolyticus to simulate real-world conditions and assess the performance of the assay in detecting the bacterium in complex matrices.
Results and Discussion:
The real-time fluorescent probe isothermal amplification technique successfully detected V. parahaemolyticus in both purified DNA samples and artificially-contaminated seafood samples. The amplification process was monitored in real-time through the increase in fluorescent signal, which correlated with the amount of target DNA present. The technique demonstrated high specificity, as it only amplified the target DNA from V. parahaemolyticus and did not produce any false-positive results with other closely related bacterial species. The detection limit of the assay was determined to be X CFU/g (or mL) of seafood, highlighting its sensitivity in identifying low levels of V. parahaemolyticus contamination.
Conclusion:
The real-time fluorescent probe isothermal amplification technique proved to be a rapid, sensitive, and specific method for detecting V. parahaemolyticus in seafood samples. Its advantages over traditional PCR methods make it a promising alternative for routine testing in seafood processing facilities and food safety laboratories. Further optimization and validation of the technique are warranted to ensure its reliability in different seafood matrices and to establish its practicality in large-scale testing. Overall, the application of real-time fluorescent probe isothermal amplification technique in seafood safety can contribute to the prevention of foodborne illnesses and enhance consumer confidence in seafood products.