A Visual Protocol for Sensitive Vibrio parahaemolyticus Detection Based on Hybridization chain reaction cascaded DNAzyme

Abstract

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the most harmful pathogenic bacteria derived from seafood and could cause great damage to aquaculture and human health even at low concentrations. Rapid and accurate detection of V. parahaemolyticus in clinic samples is essential but still remains a challenge. Herein, we developed a highly sensitive, simple and visual method for V. parahaemolyticus detection. First, V. parahaemolyticus specific aptamer, its block stands named initiator (I), and magnetic beads (MB) composed the capture probe. Then in the present of V. parahaemolyticus, the aptamer on the probe specifically recognize and bind to V. parahaemolyticus, lead to the separation of I strand. Next, the I strand sequentially initiates hybridization chain reaction (HCR) for signal amplification, releases the G-quadruplex DNAzyme which was blocked in hairpin H2 and recovers its peroxidase-mimicking activity. Thus, the results are visualized through the color variation of 2,2’-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS2−), which displays green for V. parahaemolyticus contained samples while colorless for negative samples. With the signal amplification of HCR, this proposal could achieve a detection limit down to 0.82 CFU/mL with a wide linear detection range from 100 to 106 CFU/mL. Besides, it also exhibited excellent specificity against common non-target bacteria in aquaculture and performed well in the detection of V. parahaemolyticus-spiked pacific white shrimp. Furthermore, this method realized accurate detection in shrimp seedlings from six of different breeding bases in Wenchang, which indicates tremendous potential for early disease diagnosis and prevention.