Ligase Detection Reaction Generation of Reverse Molecular Beacons for Near Real-Time Analysis of Bacterial Pathogens Using Single-Pair Fluorescence Resonance Energy Transfer and a Cyclic Olefin Copolymer Microfluidic Chip

• Published in: Analytical chemistry (Washington) 2010-12, Vol.82 (23), p.9727-9735
• Main Authors: Peng, Zhiyong, Soper, Steven A, Pingle, Maneesh R, Barany, Francis, Davis, Lloyd M
• Format: Article
• Language: English
• Subjects: Alkenes - chemistry; Analytical chemistry; Bacteria - isolation & purification; Carbocyanines - chemistry; Chemistry; Copolymers; Cyclization; Enzyme kinetics; Exact sciences and technology; Fluorescence; Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes - chemistry; Food Contamination; General, instrumentation; Genomics; Ligases - metabolism; Microfluidic Analytical Techniques - methods; Molecules; Oligonucleotide Probes - chemistry; Pathogens; Polymerase chain reaction; Polymers - chemistry; Real time; RNA, Ribosomal, 16S - chemistry; RNA, Ribosomal, 16S - genetics; Spectrometric and optical methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac101843n

Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET.