Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

Abstract

Background: Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry because of its strong pathogenicity, quick onset after infection and high mortality rate in aquatic animals. Fast, simple and specific methods are needed for on-site detection to effectively control outbreaks and prevent economic losses. The detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR)-based molecular approaches have been developed, but their application is limited due to the requirement of complicated thermal cycling machines and trained personnel. Results: A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted the virulence gene toxR, which is reported to have good coverage for V. alginolyticus pathogenic strains. To ensure the specificity of the method, the primer-probe set of the RPA system was carefully designed to recognize regions in the toxR gene that diverge in different Vibrio species but are conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening of amplification performance, primer-dimer formation and false positive signals. The RPA-LFD method was confirmed to have high specificity for V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species or other pathogenic bacteria and was able to detect as little as 1 colony forming unit (CFU) per reaction without DNA purification, or 170 fg of genomic DNA, or 6.25 × 10 3 CFU/25 g in spiked shrimp without any enrichment. The method finishes detection within 30 min at temperatures between 35°C and 45°C, and the visual signal on the dipstick can be directly read by the naked eye. In an application simulation, randomly spiked shrimp homogenate samples were 100% accurately detected.

A Recombinase Polymerase Amplification and Lateral Flow Strip Combined Method That Detects Salmonella enterica Serotype Typhimurium With No Worry of Primer-Dependent Artifacts

“…In addition to the primer pair, using a probe in the RPA reactions could increase the specificity and reduce the primerdependent artifacts (Dong et al, 2020;Wang et al, 2020). This measure has been proven to be effective in some cases (Kersting et al, 2014).…”

A Real-Time Recombinase Polymerase Amplification Method for Rapid Detection of Vibrio vulnificus in Seafood

“…Recombinase polymerase amplification (RPA), another isothermal amplification technology, has been widely applied for foodborne pathogen detections in recent years for its rapidness, simplicity, and convenience (Piepenburg et al, 2006;Du et al, 2018;Dong et al, 2020). The amplification products of RPA can be analyzed by gel electrophoresis, lateral flow chromatography, or fluorescence (Li et al, 2018).…”

Quality and Safety Assessment of Edible Seaweeds Alaria esculenta and Saccharina latissima Cultivated in Scotland

“…V. alginolyticus is one of the most common pathogenic Vibrio species and has been found to cause serious infections in both humans and animals [ 53 ]. In humans, soft tissues, the ear and superficial wounds are easily invaded by V. alginolyticus , while in animals, septicemia, melanosis, white spot syndrome and necrosis are among the most common diseases caused by this microorganism, resulting in increased mortality rates [ 54 ]. Regarding S. latissima , none of the aforementioned pathogenic bacteria was detected.…”