Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Multiplexing in polymerase chain reaction (PCR) allows simultaneous detection of multiple targets.
• Infrared-mediated PCR (IR-qPCR) offers advantages but faces challenges in distinguishing multiple fluorescence signals.
• Existing methods struggle with background noise and signal overlap in complex multiplex assays.

Purpose of the Study:

• To develop a novel frequency-modulated fluorescence encoding method for enhanced multiplexing in IR-qPCR.
• To improve the ability to distinguish and quantify multiple fluorophores and background signals.
• To demonstrate the utility of this method for sensitive DNA amplification and gene detection.

Main Methods:

• Utilized frequency modulation of laser excitation lines (488 nm and 561 nm) at distinct frequencies (73 Hz and 137 Hz).
• Employed a single photomultiplier tube for color-blind fluorescence detection.
• Applied frequency analysis for signal demodulation and separation of distinct fluorophore signals and background noise.

Main Results:

• Successfully resolved fluorescence contributions from dsDNA intercalating dye (EvaGreen) and reference dye (ROX).
• Achieved accurate quantification of DNA amplification from 10^4 to 10^7 starting copies with 96% efficiency.
• Demonstrated simultaneous amplification and detection of two genes from human genomic DNA using TaqMan probes.

Conclusions:

• Frequency-modulated fluorescence encoding significantly enhances multiplexing capabilities in IR-qPCR.
• The method effectively isolates signals of interest from background noise in the frequency domain.
• This approach is expected to simplify instrumentation for multiplexed microfluidic IR-qPCR systems.