Helicase-Dependent Amplification for Rapid Cronobacter Detection
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Solution Overview
Problem
Conventional methods for detecting Cronobacter in environmental samples are time-consuming and require enrichment or prolonged incubation, limiting their ability to rapidly identify bacterial contamination.
Innovation Solution
The development of compositions and methods for rapid detection of Cronobacter using helicase-dependent amplification (HDA) that allow for the direct detection of target nucleic acids without enrichment, utilizing specific primers and probes to amplify and detect Cronobacter DNA in environmental samples within a short timeframe.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used, then Cronobacter can be detected, but the detection process is time-consuming and requires enrichment or prolonged incubation
Solution Approach 1:
The method performs preliminary cell lysis and nucleic acid extraction directly on the sample without requiring prior enrichment culture steps. By preparing the nucleic acid template in advance through direct lysis, the detection can proceed immediately to amplification, eliminating the time-consuming enrichment phase while maintaining detection sensitivity through the use of highly specific primers and probes
Solution Approach 2:
The invention changes the detection parameters by using helicase-dependent amplification instead of conventional PCR or culture methods. This amplification technique operates at isothermal conditions (constant temperature) rather than requiring thermal cycling, and uses helicase to unwind DNA at the replication fork, enabling rapid amplification within 1-2 hours while maintaining high specificity through temperature-optimized primer and probe hybridization
2Measurement precision
If enrichment is performed to increase target organism concentration, then detection sensitivity is improved, but the overall detection time increases
Solution Approach 1:
The method extracts and isolates the essential target component (nucleic acid) directly from the sample through cell lysis, removing the need for time-consuming enrichment steps. By taking out the nucleic acid template directly and using highly specific primers and probes that target unique Cronobacter sequences, the method achieves high detection sensitivity without requiring increased target organism concentration through enrichment
Solution Approach 2:
The invention uses helicase-dependent amplification to rapidly copy and amplify the target nucleic acid sequences in vitro. This copying process occurs isothermally and can be completed within 1-2 hours, providing sufficient amplification for sensitive detection without requiring prior biological enrichment of the bacteria themselves, thus maintaining high productivity
3Quantity of substance
If prolonged incubation is used to increase target molecule concentration, then detectable levels are achieved, but the detection time is extended
Solution Approach 1:
The method replaces the biological mechanical system of bacterial growth and multiplication (incubation) with an in vitro enzymatic amplification system. Helicase-dependent amplification uses helicase to unwind DNA and polymerase to synthesize new strands, rapidly increasing target molecule concentration through controlled enzymatic reactions rather than relying on prolonged bacterial incubation, achieving sufficient target concentration within 1-2 hours
Solution Approach 2:
The invention changes the physical parameters of the amplification process by operating at isothermal conditions (constant temperature around 37-42°C) rather than requiring prolonged incubation at various temperatures. This parameter change enables continuous rapid amplification without the time constraints of biological incubation, achieving high target molecule concentration in a fraction of the time
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
Enables rapid detection of Cronobacter in environmental samples within 1-2 hours, improving sensitivity and specificity compared to conventional methods, and can detect low concentrations of target bacteria, reducing the need for enrichment or prolonged incubation.
Implementation Method 1
amplifying the target cDNA by helicase-dependent amplification (HDA)
Data Source
AI summary
The present disclosure provides compositions, methods and kits for detection of Cronobacter from a sample, such as an environmental sample.