Systems and methods for ultra-specific and ultra-sensitive nucleic acid detection
Inventors
Assignees
Publication Number
US-12359265-B2
Publication Date
2025-07-15
Expiration Date
2041-10-27
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Methods according to aspects of the disclosure, compositions and kits therefore, include at least one, two, or three sets of amplification primers and hydrolysis probes with at least two separate corresponding readouts per set. According to aspects of the present disclosure, the at least two hydrolysis probes and associated pair of primers of each set are directed to opposite strands of an amplification product of the set. According to aspects of the present disclosure, one of the two hydrolysis probes in each set is directed to a first strand of the amplification product and therefore has a sequence complementary to the first strand of the amplification product and the second of the two hydrolysis probes in the set is directed to the second strand of the amplification product and therefore has a sequence complementary to the second strand of the amplification product.
Core Innovation
The invention relates to highly specific and sensitive systems and methods for detection of a target nucleic acid, particularly methods that provide ultra-specific and ultra-sensitive detection of nucleic acids such as SARS-CoV-2. The methods utilize at least one, two, or three sets of amplification primers and hydrolysis probes, each set including two hydrolysis probes directed to opposite strands of a double-stranded amplification product, and each probe labeled with distinct fluorophores. Signals produced by the hydrolysis probes during quantitative polymerase chain reaction (qPCR), including RT-qPCR, indicate presence of the target nucleic acid.
The problem being solved is the need for systems capable of ultra-specific detection of very low copy numbers of nucleic acids amid a background of other nucleic acids, with marked reduction of false positives and false negatives. Current molecular detection systems, including those recommended by WHO and CDC, may underperform in specificity and detection limit, which is critical in contexts such as the Covid-19 pandemic where high-throughput and reliable testing is essential for surveillance and mitigation.
The present disclosure provides methods that include forming one, two, or three reaction mixtures each containing a test sample, nucleic acid amplification reagents, and a set of amplification primers and two hydrolysis probes labeled with two distinct fluorophores and quenchers. The hydrolysis probes are specific to opposite strands of an amplification product generated by the primers, and their signals allow for ultra-specific detection when Ct values fall below a predetermined threshold. Further, controls with separate primers and hydrolysis probes for non-target nucleic acids are included to validate assays. Multiplexing options and composition details including primer and probe sequences and fluorophore selection are disclosed to optimize specificity, sensitivity, and fluorophore signal differentiation.
Claims Coverage
The patent claims include one independent method claim and multiple dependent claims detailing specific aspects of nucleic acid detection for SARS-CoV-2 using RT-qPCR with particular primer and probe sets, fluorophores, quenchers, and controls.
Highly specific RT-qPCR method for detecting SARS-CoV-2 nucleocapsid phosphoprotein nucleic acid
The method comprises forming a reaction mixture including the test sample, amplification reagents, and up to three sets of amplification primers and hydrolysis probes specific for SARS-CoV-2 nucleic acid sequences (SEQ ID NOs provided). Each set includes two hydrolysis probes labeled with distinct fluorophores and quenchers hybridizing to opposite strands of amplification products, reaction under amplification conditions producing detectable fluorophore signals, Ct value calculation, and determination of SARS-CoV-2 presence when all fluorophores yield positive Ct values below a threshold.
Use of distinguishable fluorophores for multiplex detection
Each of the six fluorophores corresponding to the hydrolysis probes in the three sets are detectably different, allowing simultaneous or multiplex detection and discrimination of signals.
Inclusion of a control in the detection method
A control is included, which may be a separate reaction mixture or included in the same mixture, containing at least one control hydrolysis probe and a pair of control primers specific for a non-target nucleic acid, with a distinct fluorophore and quencher, to validate the assay.
Formation of a separate control reaction mixture
The control can be implemented by forming a separate reaction mixture including the test sample, amplification reagents, a control hydrolysis probe, and control primers specific for a non-target nucleic acid, where the control hydrolysis probe carries a control fluorophore.
The claims cover a method for ultra-specific detection of SARS-CoV-2 nucleocapsid nucleic acid by RT-qPCR using multiple primer/probe sets with dual hydrolysis probes directed to opposite strands, distinct fluorophores for multiplexing, and controls to improve specificity and reliability.
Stated Advantages
Ultra-specific detection of target nucleic acids with marked elimination of false positives and false negatives.
Ultra-sensitive detection capable of detecting very low copy numbers, including as low as 1 copy per microliter.
Highly reliable and precise detection demonstrated by consistent amplification efficiency and correlation across multiple sets of primers and probes.
Distinguishable fluorophore labeling allows multiplexing with clear signal discrimination.
Assays show no cross-reactivity to human nucleic acids or other common coronavirus nucleic acids, improving specificity.
Documented Applications
Detection of nucleic acids for medical diagnosis, including detection of SARS-CoV-2 viral RNA in biological samples from mammalian subjects, such as humans.
Environmental monitoring for viral contamination in biological and non-biological environmental samples, such as sewage and surface samples.
High-throughput testing to assess viral shedding and spreading, useful for surveillance and mitigation during pandemics such as COVID-19.
Interested in licensing this patent?