Multiplexed Nucleic Acid Detection in Blood via Red Light Spectroscopy
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Solution Overview
Problem
Current fluorescently labelled real-time PCR methods are ineffective for detecting nucleic acid targets in samples containing significant amounts of blood due to high background interference and fluorescence attenuation, making direct multiplexed detection from whole blood samples impossible.
Innovation Solution
The use of high excitation power red light greater than 620nm, combined with a spectrophotometer calibrated to collect light at 650nm to 750nm, enables spectral deconvolution for simultaneous detection of multiple targets in a single fluorescence reading, employing a system with a reaction chamber and optical fiber array for efficient light delivery and emission collection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescently labelled real-time PCR methodology is used to detect nucleic acid targets, then detection sensitivity is improved, but the presence of blood causes high background interference and fluorescence attenuation making detection impossible
Solution Approach 1:
The patent changes the excitation wavelength parameter to greater than 620nm (red light region) where blood has lower absorption and quenching effects. This parameter change allows fluorescence detection to proceed with minimal interference from blood components while maintaining detection sensitivity for nucleic acid targets
Solution Approach 2:
The patent introduces a spectrophotometer as an intermediary detection device that can distinguish between blood-derived fluorescence and target-derived fluorescence through spectral analysis. This intermediary enables the system to filter out blood interference and detect the actual nucleic acid targets
2Loss of time
If direct PCR amplification from whole blood is performed, then time-consuming nucleic acid extraction is eliminated, but fluorescence inhibition from blood components prevents successful detection
Solution Approach 1:
The patent modifies the excitation wavelength to >620nm where blood components exhibit reduced quenching effects compared to conventional wavelengths. This allows direct PCR from whole blood to proceed with sufficient fluorescence signal for detection
Solution Approach 2:
The patent exploits the spectral properties of blood components by selecting an excitation wavelength (>620nm) where blood has lower absorption and quenching. This converts the previously harmful fluorescence inhibition into a manageable condition, enabling direct blood detection
3Adaptability or versatility
If multiplexed detection of multiple nucleic acid targets is attempted in the presence of blood, then diagnostic information is improved, but blood absorption peaks and red-shift prevent simultaneous detection
Solution Approach 1:
The patent transitions from single-wavelength detection to spectral-dimensional detection using a spectrophotometer. By analyzing fluorescence across multiple wavelengths (380-780nm range), the system can resolve overlapping signals from multiple targets and distinguish them from blood interference through spectral deconvolution
Solution Approach 2:
The spectrophotometer acts as an intermediary that performs spectral deconvolution to separate and identify multiple nucleic acid targets simultaneously. This intermediary device enables multiplexed detection by mathematically resolving overlapping fluorescence spectra from different targets and blood components
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly reduces fluorescence inhibition from blood, allowing for high-fidelity multiplexed detection of nucleic acid targets in whole blood samples, enabling differentiation between pathogens and internal controls, even in the presence of blood, with minimal signal loss.
Implementation Method 1
excitation of the reaction at a wavelength greater than 620nm... excitation is effected with a power of at least 2mW... detection of the emitted fluorescence signals
Implementation Method 2
spectrophotometer calibrated to collect light at 650nm to 750nm... spectral deconvolution for simultaneous detection of multiple targets
Implementation Method 3
employing a system with a reaction chamber and optical fiber array for efficient light delivery and emission collection
Data Source
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AI summary
A process and apparatus for the multiplexed detection of nucleic acid targets directly from samples containing blood by quantitative real-time PCR means. The process comprises the addition of blood directly into the PCR reaction, including the nucleic acid of the target species, and an optical interrogation method centred on illumination at wavelengths exceeding 620nm. Preferably the illumination is provided by red 635nm laser diodes delivering excitation power in excess of 2mW and delivered to the sample by optic fiber, further that spectrophotometry is utilised to make possible multiplexed detection of targets in the presence of high amounts of blood. Such a process and apparatus is capable of overcoming the >90% inhibition of fluorescence that the presence of blood would normally present, rendering QPCR possible.