期刊
BIOSENSORS & BIOELECTRONICS
卷 81, 期 -, 页码 317-323出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2016.03.006
关键词
P. aeruginosa; Toxin genes; Multiple loop-mediated isothermal amplification (mLAMP); Lateral flow nucleic acid biosensor (LFNAB); Visual detection
类别
资金
- Ministry of Science and Technology of Beijing [XX2014B069]
- National GMO Cultivation Major Project of New Varieties [2014ZX0801202B]
This study describes a simple and sensitive approach for visual and point-of-care detection of P. aeruginosa and its toxin genes based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow nucleic acid biosensor (LFNAB). Differentiation of the internal standard gene ecfX and toxin genes (ExoS and ExoU) in P. aeruginosa was determined using FITC-, hex-and digoxin-modified primers in the mLAMP process. In the presence of biotin-and FITC- (hex-, digoxin-) modified primers and Bst DNA polymerase large fragments, the mLAMP produced numerous biotin- and F1TC- (hex-, digoxin-) attached duplex DNA products. The products were detected by LFNAB through dual immunoreactions (anti-biotin antibodies on the gold nanoparticle (Au-NP) and biotin on the duplex, anti-FITC (hex, digoxin) antibodies on the LFNAB test line and FITC (hex, digoxin) on the duplex). The accumulation of Au-NPs produced a characteristic red band, enabling visual detection of P. aeruginosa and its toxin genes without instrumentation. After systematic optimization of LFNAB preparation and detecting conditions, the current approach was capable of detecting concentrations as low as 20 CFU/mL P. aeruginosa or its toxin genes within 50 min without complicated instrument, which is more sensitive than PCR. Therefore, this approach provides a simple, pollution free, sensitive, and low-cost point-of-care test for the detection of P. aeruginosa and its toxin genes. (C) 2016 Elsevier B.V. All rights reserved.
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