Anti-Interference Detection of Vibrio parahaemolyticus from Aquatic Food Based on Target-Cyclized RCA with Dynamic Adapter Followed by LAMP
Boying Zhang, Wenhua Sun, Lingling Ran, Chenru Wang, Jing Wang, Ran An, Xingguo Liang
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.