Lock-Structure Primers for Specific Tuberculosis HRCA Detection
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Solution Overview
Problem
Existing HRCA technology for Mycobacterium tuberculosis detection is prone to false-positive and false-negative results due to non-specific amplification and the influence of insertion sequence targets, leading to unreliable outcomes.
Innovation Solution
A primer set comprising Loop, Mag, Lock, Seal, and Branch primers, along with a recognition primer set, is designed to form a 'lock' structure that requires multiple characteristic sequences of Mycobacterium tuberculosis for accurate recognition and triggers HRCA reaction only when the target is present, enhancing specificity and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If conventional HRCA technology is used for Mycobacterium tuberculosis detection, then detection simplicity and cost are improved, but detection reliability deteriorates due to false-positive and false-negative results
Solution Approach 1:
The detection system is segmented into multiple functional components: recognition primers (Block1-Block3, Key1-Key3) for target identification, Lock primers (Lock1-Lock3) for forming a lock structure, Loop primer for circularization, and Branch primers (Branch1-Branch2) for amplification. This segmentation allows each component to perform a specific function, preventing non-specific amplification while maintaining detection simplicity.
Solution Approach 2:
The Lock structure is formed in advance through annealing of Lock1-Lock3 primers to the target DNA before the amplification step. This preliminary formation of the lock structure ensures that only specific target sequences can trigger the subsequent HRCA reaction, preventing false-positive results while maintaining the simplicity of the overall detection procedure.
2Productivity
If insertion sequence targets are used for HRCA detection, then detection speed is improved, but detection accuracy deteriorates due to non-specific amplification
Solution Approach 1:
Different regions of the primer set have different functions and specificities: Block primers (Block1-Block3) recognize specific insertion sequences, Key primers (Key1-Key3) bind to complementary sequences, and Lock primers (Lock1-Lock3) form a structured lock. This local differentiation of functionality ensures that each primer contributes to specific recognition, preventing non-specific amplification while maintaining fast detection speed.
Solution Approach 2:
The Lock structure acts as an intermediary between the recognition primers and the amplification process. It mediates the interaction by requiring specific recognition of multiple insertion sequences before allowing the HRCA reaction to proceed, thus ensuring detection accuracy while maintaining speed.
3Reliability
If multiple primers are used to form lock structure, then detection specificity is improved, but primer set complexity increases
Solution Approach 1:
Multiple primers (Lock1-Lock3) are merged into a single functional unit called the Lock structure. These primers work together synergistically to recognize and bind to the target sequence, forming a stable complex that triggers amplification only when the correct target is present. This merging increases specificity while the modular design keeps the complexity manageable.
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 method provides highly specific and sensitive detection of Mycobacterium tuberculosis, reducing detection time and costs while ensuring reliable results through a 'many-to-one' target gene recognition and accurate amplification.
Implementation Method 1
step 1), annealing the Loop primer, the Mag primer, the Lock1 primer, the Lock2 primer, and the Lock3 primer to synthesize a 'lock' structure
Implementation Method 2
step 6), adding the Branch1 primer and the Branch2 primer to polymerase to perform an HRCA reaction with a reaction system
Implementation Method 3
step 7), adding calcein to read a fluorescence signal of the reaction system
Data Source
AI summary
A primer set of Mycobacterium tuberculosis based on specific hyperbranched rolling circle amplification (HRCA) isothermal amplification includes: Lock and Key series primers for a recognition stage, and Loop and Link primers for an amplification stage. Using the primer set for an HRCA reaction enables rapid and sensitive detection with reliable and stable results. A method for detecting the Mycobacterium tuberculosis includes: (1) constructing a “lock” structure with recognition function; (2) performing magnetic selection and recognition; (3) conducting HRCA amplification; and (4) identifying a fluorescent signal to determine a positive or negative result determination. The method adopts the “lock” structure to design multiple conserved fragments of the Mycobacterium tuberculosis insertion sequence 6110 (IS6110) as target genes to jointly trigger the HRCA reaction, thereby enhancing the accuracy of the method.