Dual Probe Assay for HCV Detection Inclusivity
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Solution Overview
Problem
Current molecular diagnostic assays face challenges in detecting and quantifying nucleic acids with sequence variations and mutations, leading to false-negative or underquantified results due to variable viral genomes, especially in pathogens like HIV and HCV, where conserved target regions may not be easily identified and oligonucleotides for amplification and detection can interfere.
Innovation Solution
A method involving DNA polymerase, nucleotide monomers, primers, and at least two non-overlapping detectable probes specific for different sequence portions of an amplicon is used for amplification and detection, allowing for the simultaneous amplification and detection of nucleic acids with sequence variations and mutations, reducing the risk of interference and enhancing inclusivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a single probe is used for detection, then the assay is simpler, but the inclusivity is reduced and false-negative results increase due to sequence variations
Solution Approach 1:
The patent divides the detection system into multiple independent probes (at least two non-overlapping detectable probes) that target different sequence portions of the same amplicon. Each probe can detect different sequence variations, and their signals are combined to achieve comprehensive detection coverage across multiple genotypes and mutants, thereby improving inclusivity while maintaining manageable assay complexity through modular design.
2Reliability
If two independent target regions are amplified, then inclusivity increases, but interference between oligonucleotides occurs
Solution Approach 1:
The patent merges the detection of multiple target sequences into a single amplicon by designing at least two non-overlapping detectable probes that bind to different portions of the same amplified region. This approach allows simultaneous detection of multiple sequence variations within one amplicon, achieving high inclusivity without the interference problems associated with co-amplifying two separate target regions.
3Measurement precision
If probes target variable sequence regions, then sensitivity to mutants increases, but false-negative results increase due to mutations
Solution Approach 1:
The patent applies local quality by designing probes with different specificities: some probes target highly conserved regions to ensure reliable amplification and detection across all variants, while other probes target more variable regions to detect specific mutants. The combination of probes with different binding characteristics ensures both high sensitivity for detecting sequence variations and high reliability by maintaining at least one reliable detection signal.
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 increases the inclusivity of molecular assays, reducing the risk of false-negative results and improving the detection and quantification of viral subgroups and mutants, enabling the detection of a variety of genotypes and subtypes with high sensitivity and accuracy.
Implementation Method 1
Detection of the obtained amplicon is brought about by detecting hybridization of the probes mentioned above to said different sequence portions of the amplicon
Data Source
AI summary
The present invention relates to a method for amplifying and detecting a target nucleic acid of HCV in a sample, wherein an amplification of the nucleic acids in said sample is carried out. This amplification involves a polymerase, primers for generating an amplicon and at least two detectable probes specific for different sequence portions of said amplicon. Detection of the obtained amplicon is brought about by detecting hybridization of the probes mentioned above to said different sequence portions of the amplicon.The invention further provides reaction mixtures and kits for amplifying and detecting a target nucleic acid of HCV involving the use of at least two detectable probes specific for different sequence portions of an amplicon.