Development of real‐time fluorescence saltatory rolling circle amplification for rapid detection of Vibrio parahaemolyticus in seafood

Abstract

AbstractAs a marine food‐borne pathogen, Vibrio parahaemolyticus (V. parahaemolyticus) can cause human gastrointestinal disorders and pose a serious threat to food safety and public health worldwide. Using a portable scanner, herein, a real‐time fluorescence saltatory rolling circle amplification (RF‐SRCA) has been established for the rapid detection of V. parahaemolyticus in seafood targeting toxR gene. The RF‐SRCA results could be effectively determined within 20–60 min via real‐time fluorescence curve. Significant specificity of RF‐SRCA was exhibited against 12 V. parahaemolyticus strains and 29 non‐V. parahaemolyticus strains. The sensitivity and detection limit of V. parahaemolyticus in artificially contaminated raw oyster by RF‐SRCA were 3.2 × 100 fg μL−1 and 2.3 × 100 CFU g−1, respectively. Compared with SRCA by electrophoresis, RF‐SRCA has higher sensitivity, lower detection limit, and avoids complicated electrophoresis. Compared with visual SRCA, the RF‐SRCA takes shorter time, provides more accurate results and can eliminate the risk of cross‐contamination. Moreover, 236 seafood samples were investigated for V. parahaemolyticus contamination, and the results showed 100% sensitivity, 95.88% specificity and 98.31% accuracy compared with the ISO method. Therefore, RF‐SRCA possesses great potential as an accurate, specific, sensitive, rapid and convenient molecular diagnostic method for V. parahaemolyticus detection from various seafoods.