Abstract
As an important foodborne pathogen, Vibrio vulnificus gives a significant threat to food safety and public health. Rapid and accurate detection methods for V. vulnificus are required to control its spread. The conventional detection methods are time-consuming and labor-intensive, while the polymerase chain reaction (PCR)- and quantitative PCR (qPCR)-based methods are limited because of their dependence on laboratory equipment. Nucleic acid isothermal amplification technologies have been applied to develop simpler assays. In this study, a rapid detection method based on real-time recombinase polymerase amplification (RPA) targeting the extracellular metalloprotease (empV) gene of V. vulnificus has been established. The method finished the detection in 2–14 min at 39°C with good specificity. The limit of detection was 17 gene copies or 1 colony-forming unit (CFU) per reaction, or 1 CFU/10 g of spiked food with enrichment. In a clinical sample detection test, the results of real-time RPA were 100% consistent with bioassay and qPCR. Moreover, the method could resist the effect of food matrix and could tolerate crude templates. The real-time RPA method established in this study is rapid and simple and has the potential to be widely applied for V. vulnificus detection in food safety control.
Highlights
Foodborne infectious diseases are of significant public health concern
Reference strains of V. vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Salmonella Typhimurium, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus were kindly provided by the Wuhan Institute for Food and Cosmetic Control (Wuhan, China)
A total of 1 μl of the supernatant was used as template for real-time recombinase polymerase amplification (RPA) or quantitative polymerase chain reaction (PCR) detection
Summary
Researchers have focused on the development of rapid and reliable methods of pathogen detection for food safety. Vibrio vulnificus is a Gram-negative, halophilic bacterium found in coastal or estuarine environment worldwide and has been isolated from sediments, water, and a variety of seafood (Dalsgaard et al, 1996; Jones and Oliver, 2009). Consumption of raw or undercooked seafood contaminated with V. vulnificus can result in severe infection that causes life-threatening septicemia and acute gastroenteritis. Real-Time-RPA Detection for Vibrio vulnificus (Jones and Oliver, 2009). The mortality rate of V. vulnificus infection is up to 60%, making it a serious public health and food safety concern (Jones and Oliver, 2009). Rapid, specific, and reliable detection methods for V. vulnificus are required to facilitate better control of its spread
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