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.
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.
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana, USA.
Zhiyong
Z
Peng
Steven A
SA
Soper
Maneesh R
MR
Pingle
Francis
F
Barany
Lloyd M
LM
Davis
D000475:Alkenes / Q000737:chemistry*; D001419:Bacteria / Q000302:isolation & purification*; D002232:Carbocyanines / Q000737:chemistry; D003500:Cyclization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005506:Food Contamination; D008025:Ligases / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics
D000475:Alkenes; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D011108:Polymers; D012336:RNA, Ribosomal, 16S; C085321:cyanine dye 5; D008025:Ligases