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

As 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 x 10(0) fg mu L-1 and 2.3 x 10(0) 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.