Abstract
Vibrio parahaemolyticus is a marine microorganism that can cause seafood-borne gastroenteritis in humans. The infection can be spread and has become a pandemic through the international trade of contaminated seafood. Strains carrying the tdh gene encoding the thermostable direct hemolysin (TDH) and/or the trh gene encoding the TDH-related hemolysin (TRH) are considered to be pathogenic with the former gene being the most frequently found in clinical strains. However, their distribution frequency in environmental isolates is below 1%. Thus, very sensitive methods are required for detection and quantitation of tdh+ strains in seafood. We previously reported a method to detect and quantify tdh+ V. parahaemolyticus in seafood. This method consists of three components: the most-probable-number (MPN), the immunomagnetic separation (IMS) targeting all established K antigens, and the loop-mediated isothermal amplification (LAMP) targeting the tdh gene. However, this method faces regional issues in tropical zones of the world. Technicians have difficulties in securing dependable reagents in high-temperature climates where we found MPN underestimation in samples having tdh+ strains as well as other microorganisms present at high concentrations. In the present study, we solved the underestimation problem associated with the salt polymyxin broth enrichment for the MPN component and with the immunomagnetic bead-target association for the IMS component. We also improved the supply and maintenance of the dependable reagents by introducing a dried reagent system to the LAMP component. The modified method is specific, sensitive, quick and easy and applicable regardless of the concentrations of tdh+ V. parahaemolyticus. Therefore, we conclude this modified method is useful in world tropical, sub-tropical, and temperate zones.
Highlights
Vibrio parahaemolyticus inhabits estuarine and marine environments (Joseph et al, 1982). This bacterium thrives in hightemperature environments and it is prevalent in tropical areas year around and is found at lower concentrations only in summer in temperate regions
The pairs of the samples varying in PickPen Operation time that were compared for the difference in capture efficiency (CE) (%) using the Student’s t-test are indicated; ∗pairs showing a significant difference (p = 0.05)
Time during the washing step of immunomagnetic beads (IMBs) helped to minimize the loss of IMB and improve the CE of IMB
Summary
Vibrio parahaemolyticus inhabits estuarine and marine environments (Joseph et al, 1982). Vibrio parahaemolyticus is the major cause of seafood-borne infections in the world (Raghunath, 2015). This bacterium can cause gastroenteritis in humans only when it propagates in the harvested seafood to the number exceeding the infectious dose when consumed by humans without proper cooking (Okuda et al, 1997a). A large number of V. parahaemolyticus cells distributed in the eutrophic coastal environments may accumulate in the digestive tract of molluscan bivalves because they filterfeed (Nishibuchi and Kaper, 1995; Manas et al, 2014). Most clinical isolates of V. parahaemolyticus carry the tdh and trh genes, either alone or in combination; distribution of these genes in environmental isolates is usually low (1–2%; Nishibuchi and Kaper, 1995; Yamazaki et al, 2010) some workers reported extremely frequent detection (up to 48%; Rodriguez-Castro et al, 2010; Gutierrez West Casandra et al, 2013)
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