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

Abstract

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 × 10 0 fg μL −1 and 2.3 × 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.

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

“…1 D presents the schematic representation of the RCA method. The RCA process involves the utilization of DNA/RNA polymerase (such as phi29 DNA polymerase or T7 RNA polymerase), a short linear single-stranded DNA or RNA primer, a circular template, and ligase ( Guo et al, 2022a , Liu et al, 2020 , Liu et al, 2021 , Prasad et al, 2023 , Xu et al, 2021 , Yuan et al, 2022 , Zhan et al, 2020 ). This combination of components leads to the generation of a lengthy single-strand product through DNA/RNA polymerase action, resulting in a single-strand RCA product (RCAP).…”

“…As a result, SRCA produces shorter, non-contiguous DNA segments, referred to as saltatory products. In recent studies, this method has been used to detect numerous pathogens in various types of food samples, including PIF, milk, pork, chicken meat, and oysters ( Huang et al, 2023b , Milton et al, 2021b , Milton et al, 2021a , Milton et al, 2021c , Prasad et al, 2023 , Yang et al, 2019 , Yuan et al, 2022 , Zhang et al, 2019a ).

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“…RCA technology shows great promise as a suitable candidate for on-site testing due to its convenient and easily accessible amplification requirements ( Guo et al, 2022a , Liu et al, 2020 , Liu et al, 2021 , Prasad et al, 2023 , Xu et al, 2021 , Yuan et al, 2022 , Zhan et al, 2020 ). However, certain drawbacks need to be addressed in the development of the RCA method, such as the background interference during signal detection, and nonspecific binding caused by the large molecular weight of RCA products, as well as the influence of complex food matrices ( Guo et al, 2022a , Liu et al, 2020 , Liu et al, 2021 , Prasad et al, 2023 , Xu et al, 2021 , Yuan et al, 2022 , Zhan et al, 2020 ). Moreover, the design and testing of padlock probes within the RCA assay are intricate and time-consuming.…”