Attenuating Probe Nested PCR for SARS-CoV-2 Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional PCR techniques face challenges with false negative signals in low-copy number samples and contamination risks in nested PCR procedures, leading to inconsistent diagnostic results, particularly in early detection of pathogens like COVID-19.
Innovation Solution
A single-tube nested PCR method using a probe with an attenuating site and a polymerase with 3'-5' exonuclease activity, which includes a fluorescent dye-quencher pair and a molecular moiety at the 3' end to prevent non-specific amplification and contamination, enhancing sensitivity and specificity for nucleic acid detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional TaqMan probe with 5'-3' exonuclease activity is used, then signal generation occurs through probe cleavage, but strong binding slows down primer extension and reduces PCR rate and yield
Solution Approach 1:
The patent changes the exonuclease activity direction from 5'-3' to 3'-5', and modifies the probe structure by placing the quencher at the 3' end instead of 5' end, enabling signal generation without strong probe binding that would slow primer extension
Solution Approach 2:
The patent introduces a molecular moiety at the 3' end of the probe that acts as an intermediary - it is cleaved by 3'-5' exonuclease activity to generate signal, preventing the probe from strongly binding and blocking primer extension while still enabling detection
2Reliability
If nested PCR is performed in separate tubes, then sensitivity and specificity are improved, but contamination risk increases due to multiple handling steps
Solution Approach 1:
The patent combines two separate nested PCR reactions into a single tube by using a probe with molecular moiety that can be cleaved by 3'-5' exonuclease activity, eliminating the need for intermediate product transfer and reducing contamination risk while maintaining detection reliability
Solution Approach 2:
The patent extracts the amplification and detection steps into a single continuous reaction by using a probe design where the molecular moiety is cleaved during the PCR process itself, eliminating the need for separate handling steps
3Measurement precision
If PCR cycles are increased to detect low-copy number sequences, then detection sensitivity improves, but false positive products increase from primer binding to incorrect regions
Solution Approach 1:
The molecular moiety at the 3' end of the probe acts as an intermediary that must be cleaved by 3'-5' exonuclease activity to generate signal, providing an additional specificity checkpoint that prevents false positives from non-specific primer binding while maintaining sensitivity for low-copy number detection
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 improves the sensitivity and specificity of nucleic acid detection, reduces contamination risks, and enables faster, more reliable quantification of target sequences, such as SARS-CoV-2, while minimizing false positives and negatives.
Implementation Method 1
a polymerase with 3'-5' exonuclease activity
Implementation Method 2
a fluorescent dye-quencher pair
Data Source
AI summary
An embodiment relates to a method comprising assembling a reaction mixture comprising: a target molecule comprising a nucleic acid sequence of interest; a set of oligonucleotides comprising: a 1st SW (selective wobble) primer comprising a 1st SW site; a 2nd SW primer comprising a 2nd SW site; at least a third primer; a probe comprising: (i) an attenuating site, (ii) a first label in a non 3′ site and (iii) a second label at the 3′ end; a polymerase with 3′-5′ exonuclease activity; conducting an amplification reaction of the target molecule comprising the nucleic acid sequence of interest using the reaction mixture; detecting or amplifying the target molecule comprising the nucleic acid sequence of interest or variants thereof present in the target molecule, wherein the SW sites are configured to enable non-disrupted nested amplification and quantification of the target molecule comprising the nucleic acid sequence of interest. The nucleic acid sequence of interest comprises a SARS-CoV-2 sequence.