期刊
BIOSENSORS-BASEL
卷 11, 期 5, 页码 -出版社
MDPI
DOI: 10.3390/bios11050151
关键词
Vibrio cholerae; Vibrio vulnificus; recombinase polymerase amplification; lateral flow strip; on-site detection; multiplexing
资金
- National Natural Science Foundation of China [31470275]
- Key Natural Science Research Project of the Jiangsu Higher Education Institutions of China [20KJA416002]
- Fishery Science and Technology Innovation Program of China [Y2018-14]
- Nantong Municipal Science and Technology Plan of China [MS12020059]
- Lianyungang Science and Technology Project of China [SF2003]
- Science and Technology Project of Lianyungang High-tech Zone of China [HZ201901]
- Open-end Funds of Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening [HY202004]
- Priority Academic Program Development of Jiangsu Higher Education Institutions of China
The duplex detection biosensor based on RPA technology and LFS strips showed good specificity and high sensitivity for Vibrio cholerae and Vibrio vulnificus. It is simple and fast, suitable for on-site detection, and capable of simultaneous detection of these two important foodborne bacteria.
Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and economic consequences. Molecular detection methods targeting species-specific genes are effective tools in the fight against bacterial infections for food safety. In this study, a duplex detection biosensor based on isothermal recombinase polymerase amplification (RPA) and a three-segment lateral flow strip (LFS) has been established. The biosensor used lolB gene of Vibrio cholerae and empV gene of Vibrio vulnificus as the detection markers based on previous reports. A duplex RPA reaction for both targets were constructed, and two chemical labels, FITC and DIG, of the amplification products were carefully tested for effective and accurate visualization on the strip. The biosensor demonstrated good specificity and achieved a sensitivity of 10(1) copies per reaction or one colony forming unit (CFU)/10 g of spiked food for both bacteria. Validation with clinical samples showed results consistent with that of real-time polymerase chain reaction. The detection process was simple and fast with a 30-min reaction at 37 degrees C and visualization on the strip within 5 min. With little dependence on laboratory settings, this biosensor was suitable for on-site detection, and the duplex system enabled simultaneous detection of the two important foodborne bacteria. Moreover, the principle can be extended to healthcare and food safety applications for other pathogens.
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