Anti-Interference Detection of Vibrio parahaemolyticus from Aquatic Food Based on Target-Cyclized RCA with Dynamic Adapter Followed by LAMP

Abstract

Vibrio parahaemolyticus (V. parahaemolyticus) is considered the most concerning pathogen for seafood. Like other pathogens in food samples, its gene detection suffers from a problem of background interference when isothermal detection methods are used. The sensitivity and specificity greatly decrease due to large amounts of background genome. Here we describe a novel isothermal detection technology based on target-cyclized rolling circle amplification combined with loop-mediated isothermal amplification (tRCA-lamp). By avoiding unexpected ligation, a short dynamic adapter is employed to increase the sensitivity of target cyclization in the presence of the background genome. At the amplification step, highly specific detection is obtained by linear RCA and simplified LAMP (only two primers are used). Furthermore, visual detection is easily realized with hydroxynaphthol blue (HNB). In the oyster samples, the tRCA-lamp approach can detect V. parahaemolyticus with a detection limit of 22 cfu/g with none necessary to enrich the bacteria and remove the host DNA. This method gets rid of the complicated primer design process and can be extended to the detection of other pathogens in food samples.

Rapid detection methods for foodborne pathogens based on nucleic acid amplification: Recent advances, remaining challenges, and possible opportunities

“…1 B presents the schematic representation of the LAMP amplification. The LAMP primer set includes internal primers (FIP and BIP) as well as external primers (F3 and B3) ( Du et al, 2022 , Jia et al, 2023 , Jiang et al, 2020 , Li et al, 2021 , Priya et al, 2020 , Wang et al, 2020a , Xie et al, 2022a , Xiong et al, 2020 , Zendrini et al, 2021 , Zhang et al, 2021 , Zhang et al, 2022 ). FIP comprises the F1c and F2 regions, while BIP comprises the B1c and B2 regions.…”

“…In recent years, a great number of studies have been devoted to exploring the capability of LAMP technology in detecting various pathogens in various types of foods, including milk, seafood, and meats ( Du et al, 2022 , Jia et al, 2023 , Jiang et al, 2020 , Li et al, 2021 , Priya et al, 2020 , Wang et al, 2020a , Xie et al, 2022a , Xiong et al, 2020 , Zendrini et al, 2021 , Zhang et al, 2021 , Zhang et al, 2022 ). Most of these studies used fluorescence to indicate the presence of pathogens in the amplified product and some of them applied necessary pre-treatment on their samples before DNA amplification ( Table S3 ).…”

“…Regarding the LAMP method, researchers have observed that it displays substantial resistance to reagent inhibition and is well-suited for complex samples. However, the commercialization of LAMP devices encounters challenges, including the preservation of LAMP reagent and primer stability, the resolution of contamination concerns, the management of aerosol pollution during LAMP reactions, and the mitigation of substantial noise background signals in scenarios involving multiple target sites ( Du et al, 2022 , Jia et al, 2023 , Jiang et al, 2020 , Li et al, 2021 , Priya et al, 2020 , Wang et al, 2020a , Xie et al, 2022a , Xiong et al, 2020 , Zendrini et al, 2021 , Zhang et al, 2021 , Zhang et al, 2022 ). To address the challenge of noise background signals, Ding et al (2019) proposed the implementation of the “Dual-Priming Isothermal Amplification (DAMP)” approach.…”

Establishment and application of a rapid visualization method for detecting Vibrio parahaemolyticus nucleic acid

“…Infection with V. parahaemolyticus not only gives rise to gastrointestinal diseases characterized by symptoms such as watery diarrhoea, nausea, vomiting, and abdominal cramps, but can also lead to wound infections. In severe cases, these infections can result in sepsis, shock, and even mortality [3] . Reports of V. parahaemolyticus gastroenteritis outbreaks have been documented in multiple countries worldwide, thus highlighting its status as a prevalent and significant public health concern [4] , [5] , [6] .…”

CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

“…As a zoonotic pathogen, V. parahaemolyticus causes gastrointestinal illness characterized by watery diarrhea, nausea, vomiting, abdominal cramps, and headache. In severe cases, it may result in septicemia and fatal diseases [ 3 ]. Consumption of raw, parboiled, or contaminated seafood is one of the most common causes of food poisoning in humans [ 4 ].…”