Quantitative multiplexed identification of nucleic acid targets
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9284613-B2
Publication Date
2016-03-15
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
A method detects in a sample at least one of a plurality of target nucleic acid molecules by using two different amplification regions per target. The method identifies a first amplification region that is conserved among the plurality of target nucleic acid molecules and a second amplification region that is not conserved, where for each target nucleic acid molecule the first amplification region and the second amplification region are in different locations of the nucleic acid molecule.
The method generates an amplification reaction mixture comprising at least two different target nucleic acid molecules, a first primer pair designed to amplify the first amplification region, a second primer pair designed to amplify the second amplification region, and a first nucleic acid probe designed to hybridize to the first amplification region. The amplification reaction produces simultaneously a first amplification product of the first amplification region of each target nucleic acid molecule present in the sample and a second amplification product of the second amplification region of each target nucleic acid molecule present in the sample, where the first and second amplification products are separate products.
The method detects the production of the first amplification product in real time using the first nucleic acid probe hybridizing to the first amplification region, thereby providing real-time quantitative information for detection. The method also detects the second amplification product to indicate the presence of each target nucleic acid molecule present in the sample. The approach uses conserved and non-conserved regions to combine quantification from real-time detection with multiplex identification based on the distinct amplification region.
Claims Coverage
The independent claim covers a multiplex nucleic-acid detection method using two different amplification regions per target, one conserved and one not conserved in different locations, with real-time detection for the conserved-region product and detection for the non-conserved-region product to indicate presence. Dependent claims add alignment-based selection of conserved regions and design of primers and probe, a probe melting temperature limitation, microarray substrate, detection of at least two pathogens, and quantification of target nucleic acid molecules.
Two-region target discrimination with separate conserved and non-conserved amplification products
Identifying a first amplification region conserved among a plurality of target nucleic acid molecules and a second amplification region not conserved among the plurality, wherein for each target nucleic acid molecule the first and second amplification regions are in different locations, and producing separate first and second amplification products corresponding to the respective regions.
Real-time detection of conserved-region amplification product using a hybridizing probe
Generating an amplification reaction mixture with a first primer pair to amplify the conserved first amplification region and a first nucleic acid probe designed to hybridize to the first amplification region, producing a first amplification product, and detecting the production of the first amplification product in real time while the probe hybridizes to the first amplification region.
Presence indication from detection of the distinct-region amplification product
Generating and producing a second amplification product from the non-conserved second amplification region using a second primer pair, and detecting the second amplification product to indicate the presence of each target nucleic acid molecule present in the sample.
Alignment-based selection of conserved regions and design of primers and probe
Identifying the first amplification region for each plurality of target nucleic acid molecules by using sequence alignment, selecting the amplification region from segments of the nucleic acid sequence, and designing a first primer pair, a second primer pair, and a probe to hybridize to the first amplification region.
Probe melting temperature relative to primer melting temperatures
Limiting the probe such that its melting temperature is at least 6°C higher than the melting temperature of the first primer and the melting temperature of the second primer.
Multiplex detection using a microarray substrate
Performing the method using a microarray as the substrate.
Multiplex detection of multiple pathogens
The method according to the independent claim is capable of detecting at least two pathogens in the sample.
Quantification of target nucleic acid molecules
Further detecting and quantifying the amount of target nucleic acid molecules present in the sample.
Across the independent and dependent claims, the core coverage is multiplex detection using conserved and non-conserved amplification regions in different locations for each target, with real-time detection of the conserved-region product using a hybridizing probe and detection of the non-conserved-region product to indicate presence. Dependent limitations add pathogen and multiplex capability, quantification of target amount, alignment-based selection of amplification regions and design of primers and probe, a melting-temperature constraint relating the probe to primers, and performing the method with a microarray substrate.
Stated Advantages
Provides real-time detection of the conserved-region amplification product using a probe that hybridizes to the first amplification region.
Indicates the presence of each target nucleic acid molecule present in the sample by detecting the non-conserved-region amplification product.
Detects at least two pathogens in the sample.
Detects and quantifies the amount of target nucleic acid molecules present in the sample.
Documented Applications
Pathogen detection in a sample, including detection of at least two pathogens.
Interested in licensing this patent?