Universal Plasmid Controls for Sequencing Assay Reliability
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Solution Overview
Problem
Current diagnostic sequencing assays require additional space, material, and increase the risk of cross-reactivity due to the need for separate positive, negative, and extraction controls, which complicates the detection of specific nucleic acids in clinical samples.
Innovation Solution
A method involving the use of universal plasmids that serve as both positive and negative controls, and extraction controls, allowing for the detection of a target nucleic acid sequence by sequencing in multiple vials with specific primers, eliminating the need for additional control reactions and reducing cross-reactivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If separate positive control, negative control, and extraction control reactions are conducted, then the reliability of nucleic acid detection is improved, but the device complexity and material requirements increase
Solution Approach 1:
The patent combines multiple control functions (positive control, negative control, and extraction control) into a single integrated control reaction. The control plasmid contains multiple control sequences that can simultaneously serve as positive control (amplifiable by target primers), negative control (non-amplifiable by target primers), and extraction control (amplifiable by control primers) within the same reaction well, thereby reducing the number of separate reactions needed
Solution Approach 2:
The control plasmid is designed with multi-functionality to perform multiple control roles simultaneously. It includes a first control sequence amplifiable by target-specific primers (positive control function), a second control sequence non-amplifiable by target primers (negative control function), and sequences amplifiable by control primers (extraction control function), making a single plasmid serve multiple purposes
2Reliability
If separate control reactions are conducted, then the reliability of detection is improved, but the quantity of substance and material requirements increase
Solution Approach 1:
The patent merges multiple control reactions into one by incorporating multiple control sequences into a single control plasmid. This allows all control functions to be tested in one reaction well using a shared reaction mixture, thereby reducing the total volume of reagents and materials required compared to running separate control reactions
3Measurement precision
If multiple primers are used for different control sequences, then the measurement precision is improved, but the risk of cross-reactivity increases
Solution Approach 1:
The patent applies local quality by designing specific binding regions within the control plasmid that have high sequence similarity to the target sequence only in the primer binding sites. The control plasmid contains a first control sequence with a first binding site that is highly similar to the target binding site (providing local quality for specific amplification), while the rest of the plasmid sequence differs from the target sequence, thereby maintaining measurement precision while minimizing cross-reactivity
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 simplifies the sequencing process, reduces material and space requirements, and ensures accurate detection of target nucleic acids by integrating control sequences within the assay, thereby improving the efficiency and specificity of nucleic acid detection in clinical samples.
Implementation Method 1
specific primers are used in the PCR reaction, which hybridize to sequences upstream and downstream of the sequence to be amplified
Implementation Method 2
an amplification reaction is usually carried out by a PCR reaction; thus, specific primers are used in the PCR reaction, which hybridize to sequences upstream and downstream of the sequence to be amplified
Implementation Method 3
the amplified nucleic acid is then sequenced in order to identify the target sequence
Data Source
Figure 1A~1C
AI summary
The present invention relates to a positive and negative control and an extraction control, respectively, for sequencing assays. The present application discloses plasmids, kits, their uses and a method of detecting a specific nucleic acid, wherein the controls according to the present invention are used.