mecA Gene Detection Primer Pairs for High Sensitivity PCR
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Solution Overview
Problem
Current methods for detecting methicillin resistance genes, such as PCR, often lack sufficient sensitivity, leading to inadequate detection of low concentrations of MRSA, which can result in false negatives and hinder timely diagnosis and treatment.
Innovation Solution
Development of specific primer pairs for PCR that target the mecA gene, designed to amplify DNA fragments of 150bp to 700bp in size, focusing on highly conserved sequences and optimizing primer combinations to enhance detection sensitivity, allowing for the detection of trace amounts of the mecA gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods are used for detecting mecA gene, then the detection process is simple and quick, but the detection sensitivity is insufficient leading to false negatives
Solution Approach 1:
The patent applies parameter changes by optimizing PCR conditions including annealing temperature (55-65°C), extension time (30-60 seconds), and primer concentrations (0.1-1.0 μM) to enhance detection sensitivity. The primers are designed with specific melting temperatures and binding affinities that improve the ability to detect low concentrations of mecA gene, reducing false negatives while maintaining quick detection capability.
2Loss of time
If drug sensitivity tests are used to detect methicillin resistance, then the detection can be performed on patient blood, but the test requires culturing staphylococci for at least one day
Solution Approach 1:
The patent replaces the biological culturing process with a molecular biology-based PCR detection system. Instead of requiring staphylococci to be cultured and grown for at least one day, the method directly detects mecA gene DNA sequences from patient blood samples using nucleic acid amplification. This substitution eliminates the time-consuming culturing step while maintaining detection accuracy, enabling rapid results within hours rather than days.
3Loss of time
If immunoassays are used for methicillin resistance testing, then the test can be performed without culturing, but it still requires at least one day before results are obtained
Solution Approach 1:
The patent substitutes immunoassay-based detection with direct nucleic acid amplification and detection. The method uses PCR to amplify mecA gene sequences followed by rapid detection through fluorescence or other signal methods, eliminating the need for bacterial culturing and reducing detection time to several hours. The operational simplicity is maintained through standardized PCR protocols and automated detection systems.
4Measurement precision
If PCR detection is used, then the test can be performed directly on patient blood without culturing, but the detection sensitivity is not sufficient for clinical use
Solution Approach 1:
The patent applies parameter changes by optimizing multiple aspects of the PCR system including primer sequences (with specific melting temperatures and binding sites), annealing temperatures (55-65°C), extension times (30-60 seconds), and reagent concentrations. These optimizations enhance the amplification efficiency and detection sensitivity, enabling reliable detection of mecA gene at low concentrations in clinical samples while maintaining the advantage of direct blood sample analysis without culturing.
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 new primer pairs significantly improve the sensitivity and accuracy of mecA gene detection, enabling the identification of trace amounts of the gene in medical and food fields, facilitating rapid and reliable diagnosis.
Implementation Method 1
nucleic acid amplification tests, such as PCR, are very sensitive and do not require a culturing process and can be tested directly from the patient's blood
Data Source
AI summary
Provided is a primer pair of primers for methicillin-resistant gene detection for the purpose of achieving highly sensitive methicillin-resistant gene detection. Said primer pair comprises a combination of SEQ ID NO: 3 and SEQ ID NO: 7, a combination of SEQ ID NO: 2 and SEQ ID NO: 9, a combination of SEQ ID NO: 1 and SEQ ID NO: 8, a combination of SEQ ID NO: 1 and SEQ ID NO: 9, a combination of SEQ ID NO: 4 and SEQ ID NO: 11, a combination of SEQ ID NO: 5 and SEQ ID NO: 12, a combination of SEQ ID NO: 6 and SEQ ID NO: 10, or a combination of SEQ ID NO: 6 and SEQ ID NO: 12.