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 it can cause great damage to aquaculture and human health, even at low concentrations. The rapid and accurate detection of V. parahaemolyticus in clinical samples is essential but remains a challenge. Herein, we have developed a highly sensitive, simple and visual method for V. parahaemolyticus detection. The capture probe is composed of a V. parahaemolyticus-specific aptamer, with its blocking strands designated as the initiator (I), and magnetic beads (MB). In the presence of V. parahaemolyticus, the aptamer on the probe specifically recognizes and binds to V. parahaemolyticus, leading to the separation of the I strand. Next, the I strand sequentially initiates a hybridization chain reaction (HCR) for signal amplification, releasing the G-quadruplex, which was blocked in hairpin H2, and recovers its peroxidase-mimicking activity. Thus, the results can be visualized through the color variation of 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS²⁻), which displays green for V. parahaemolyticus-containing samples and colorless for negative samples. With the signal amplification of HCR, this method can achieve a detection limit of 0.82 CFU mL⁻¹ with a wide linear detection range from 10⁰ to 10⁶ CFU mL⁻¹. 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 the accurate detection of V. parahaemolyticus in shrimp seedlings from six different breeding bases in Wenchang, Hainan Province, which indicates tremendous potential for early disease diagnosis and prevention.