Decoy Oligonucleotides Suppress Carryover Contamination in Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid amplification techniques, particularly isothermal methods like RPA, are vulnerable to carryover contamination, leading to false positive results due to the inability to effectively eliminate pre-existing amplicons, which complicates diagnostics and requires resource-intensive and complex methods to prevent.
Innovation Solution
A reagent kit comprising a decoy-oligonucleotide and a probe with a cleavage site, where the decoy-oligonucleotide has a 3'-OH group and mismatch bases, competes with the probe to suppress the amplification of carryover contaminants, reducing false positives by producing non-detectable amplicons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid amplification methods are used, then amplification sensitivity is improved, but susceptibility to carryover contamination increases
Solution Approach 1:
The invention converts the harmful effect of carryover contamination into a beneficial feature by designing the probe to specifically detect only authentic amplification products. The probe requires a specific structural feature (e.g., a nick or gap) that is only present in genuine amplicons generated from template nucleic acid, but absent in contaminant amplicons. This transforms the contamination problem into a detection advantage, where the same amplification sensitivity that produces vulnerable amplicons also creates the structural signature needed for specific probe binding.
Solution Approach 2:
The invention applies local quality by making the probe's binding requirements highly specific to particular structural features at localized positions in the amplicon. Rather than requiring uniform properties throughout the entire amplification process, the probe is designed to recognize only specific local structural characteristics (such as a nick created by a specific enzyme or a particular sequence arrangement) that distinguish authentic products from contaminants. This localized specificity allows high sensitivity amplification while rejecting carryover contamination.
2Reliability
If stringent workflow controls and sophisticated detection methods are implemented, then false positive results are reduced, but device complexity and resource requirements increase
Solution Approach 1:
The invention enables self-service by incorporating the contamination resistance directly into the molecular reagents themselves. The probe is designed with intrinsic properties that allow it to automatically distinguish authentic amplification products from contaminants without requiring external intervention. The probe's specific binding requirements (such as requiring a nick created by a template-dependent enzyme step) ensure that only genuine products are detected, eliminating the need for complex external verification procedures or sophisticated instrumentation.
Solution Approach 2:
The invention extracts the contamination vulnerability from the amplification system by separating the amplification function from the detection function. The amplification reagents (polymerase, primers) operate independently to generate both authentic and contaminant products, while the probe provides a separate, specific detection layer that selectively recognizes only authentic products. This extraction allows the amplification process to maintain high sensitivity while the detection process independently ensures specificity, without requiring complex integrated systems.
3Ease of manufacture
If isothermal amplification methods are used, then equipment requirements are reduced, but susceptibility to carryover contamination increases
Solution Approach 1:
The invention applies preliminary action by incorporating contamination-resistant probe design into the reagent preparation stage, before the amplification reaction begins. The probe is pre-designed with specific structural requirements and binding characteristics that will later enable it to distinguish authentic products from contaminants. This preliminary configuration of the detection reagent ensures that even when using simple isothermal amplification equipment, the system inherently resists carryover contamination through the probe's selective binding properties.
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
The use of the reagent kit significantly reduces carryover contamination, enhancing the specificity of nucleic acid amplification and detection, thereby improving diagnostic accuracy without the need for extensive laboratory equipment or stringent workflow controls.
Implementation Method 1
the probe comprises a cleavage site and a binding site hybridisable to a pre-determined nucleotide sequence
Implementation Method 2
The probe is converted to a detection-competent state by an endonuclease (e.g. endonuclease IV from E.coli, which cleaves double-stranded DNA at abasic sites) present during amplification
Data Source
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AI summary
The invention relates to reagent kits comprising a decoy-oligonucleotide and a probe, wherein the probe comprises a cleavage site, and a binding site hybridisable to a pre-determined nucleotide sequence and the decoy-oligonucleotide comprises a 3'-OH group, at least one mismatch base compared to the binding site of the probe. The invention further relates to methods for detecting the presence of a pre-determined nucleotide sequence in a sample, the method comprising the steps of adding said reagent kit additionally comprising at least one DNA primer; adding one or more enzyme(s) providing activities of DNA polymerase activity and strand-displacement activity to the sample to be analysed for the presence of the pre-determined nucleotide sequence; amplifying the pre-determined nucleotide sequence; and detecting whether amplification products are present in the sample, wherein presence of the amplification product in the sample is indicative of the presence of the pre-determined nucleotide sequence in the sample.