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

Xuetong Li; Yuan Su; Huashuo Chu; Shuxia Lyu; Jingjing Tian; Wentao Xu

doi:10.1016/j.foodchem.2019.125955

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

Food Chem. 2020 Apr 25:310:125955. doi: 10.1016/j.foodchem.2019.125955. Epub 2019 Dec 9.

Authors

Xuetong Li¹, Yuan Su², Huashuo Chu², Shuxia Lyu³, Jingjing Tian², Wentao Xu⁴

Affiliations

¹ Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; College of Biological Science and Technology, Agricultural University of Shenyang, Shenyang 110866, China. Electronic address: xuwentao@cau.edu.cn.
² Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
³ College of Biological Science and Technology, Agricultural University of Shenyang, Shenyang 110866, China.
⁴ Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; Key Laboratory of Safety Assessment of Genetically Modified Organism (Food Safety), Ministry of Agriculture, Beijing 100083, China. Electronic address: xuwentao@cau.edu.cn.

PMID: 31841941
DOI: 10.1016/j.foodchem.2019.125955

Abstract

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.

Keywords: Phosphate; Pyrophosphatase; Strand replacement primer; Vibrio parahaemolyticus; Visualization.

MeSH terms

DNA Primers / genetics*
DNA-Directed DNA Polymerase / genetics
Food Analysis / instrumentation*
Food Analysis / methods
Food Microbiology / methods*
Pyrophosphatases / genetics
Sensitivity and Specificity
Vibrio parahaemolyticus / genetics*

Substances

DNA Primers
DNA-Directed DNA Polymerase
Pyrophosphatases