Rapid strand replacement primer thermostat visual sensor based on Bst DNA polymerase and pyrophosphatase for detecting Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a major hidden danger of food safety. To develop a rapid, sensitive and on-site detecting method of Vibrio parahaemolyticus (V. parahaemolyticus), a strand replacement primer thermostat phosphate (SRPP) visual sensor was proposed, based on Bst DNA polymerase and pyrophosphatase. The novel strand replacement primer (SRP) facilitates chain substitution and to open a self-folding hairpin by adding region at its 3' end. Under the action of the SRP, a pair of external primers and two inner primers, target DNA is specifically amplified at 63 °C relies mainly on the hairpin. Many pyrophosphates (PPi) are simultaneously generated as by-products, which can be converted into phosphates (Pi) by pyrophosphatase for phosphomolybdate blue visual detection within 5 min. The proposed biosensor can detect 1.29 × 103 copies of V. parahaemolyticus within 35 min.