Quantitative multiplexed identification of nucleic acid targets
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Publication Number
US-10538805-B2
Publication Date
2020-01-21
Expiration Date
Abstract
Methods and systems for detecting a target nucleic acid using the quantitative capabilities of real-time nucleic acid amplification systems and the multiplexing capabilities of hybridization systems, comprising: identifying a conservative sequence and a distinctive sequence within each target nucleic acid sequence; simultaneously amplifying the conservative region and the distinctive region; monitoring the amplification of the conservative region in real-time; identifying the distinctive region amplicon via multiplexed identification; and performing quantitative multiplexing analysis of the target by combining the real-time monitoring information with the multiplexed identification of the target nucleic acid.
Core Innovation
The invention provides a system for detecting in a sample at least one of a plurality of target nucleic acid molecules. Each target nucleic acid molecule has a first amplification region that is conserved among the plurality of target nucleic acid molecules and a second amplification region that is not conserved, with the first and second amplification regions located in different locations of the nucleic acid molecule. This enables separate handling of quantitative and identification aspects within the same target nucleic acid molecule.
The system uses a first primer pair to amplify the first amplification region of each target nucleic acid molecule to produce a first amplification product. A real-time nucleic acid amplification instrument detects the first amplification product in real time, providing quantitative measures for the conserved region. In parallel, a second primer pair mutually exclusive from the first primer pair amplifies the second amplification region to produce a second amplification product while the first amplification product is being produced.
The first and second amplification products are separate products, and the system includes a detection device for detecting the second amplification product. The approach combines real-time detection of the conserved quantitative region with multiplexed hybridization of the distinctive region to determine which target(s) are present, thereby providing multiplexed quantitative identification using the conserved TQR and the distinctive TDR across targets.
Claims Coverage
The claim coverage indicates one independent claim defining a core system architecture, with dependent features adding nucleic acid probes, substrate implementation for hybridization, and a quantitative probe melting temperature constraint.
Conserved and non-conserved amplification regions at different locations
Each target nucleic acid molecule has a first amplification region conserved among the plurality of target nucleic acid molecules and a second amplification region not conserved, with the first and second amplification regions located in different locations of the nucleic acid molecule.
Separate primer pairs for real-time conserved quantification and distinctive detection
A first primer pair amplifies the first amplification region to produce a first amplification product detected in real time by a real-time nucleic acid amplification instrument, while a second primer pair mutually exclusive from the first primer pair simultaneously amplifies the second amplification region to produce a second amplification product.
Separate first and second amplification products with a detection device for the second amplification product
The first amplification region and second amplification region are in different locations; the first and second amplification products are separate products; and a detection device detects the second amplification product.
First nucleic acid probe for hybridizing to the first region
A first nucleic acid probe hybridizes to the first region.
Second nucleic acid probe fixed to a substrate
A second nucleic acid probe is fixed to a substrate.
Microarray substrate configuration for the fixed second probe
The substrate is a microarray.
Probe melting temperature offset relative to primers
The first nucleic acid probe has a melting temperature at least 6°C higher than the melting temperature of either primer in the first primer pair.
Pathogen presence indicated by detection of the second amplification product
Detecting the second amplification product indicates the presence of a pathogen in the sample.
Overall, the claims cover a system that distinguishes conserved versus non-conserved amplification regions located at different positions on each target nucleic acid molecule, amplifies both regions using mutually exclusive primer pairs to yield separate amplification products, performs real-time detection of the conserved product, and detects the distinctive product to determine presence or identity. Dependent refinements add probe hybridization components, a substrate-immobilized probe implemented as a microarray, a probe-primer melting temperature constraint, and interpret the second amplification product as indicating pathogen presence.
Stated Advantages
Documented Applications
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