Real-time PCR Melting Temperature Analysis for Bacterial Identification
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Solution Overview
Problem
Current methods for detecting and identifying pathogenic bacteria in septicemia are time-consuming, often taking over 18 hours, leading to inappropriate antimicrobial drug selection and potential emergence of multi-drug resistant bacteria, due to the need for numerous hybridization probes for each bacterial species.
Innovation Solution
A method utilizing real-time PCR with specific primers targeting common 16S rRNA gene regions and analyzing melting temperatures (Tm values) to identify pathogenic bacteria, allowing for rapid detection and identification of a wide range of bacterial species using a database of Tm values specific to each bacterium.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization probes specific to each bacterial species are used for detection, then identification accuracy is improved, but the number of probes required increases, making the system impractical for identifying a wide range of bacterial species
Solution Approach 1:
The patent uses a single universal primer set that can amplify 16S rRNA genes from all bacterial species, replacing the need for multiple species-specific probes. This universal primer approach allows one reagent to perform the function of many specific probes, making the system practical for identifying a wide range of bacteria
Solution Approach 2:
The patent shifts from using multiple specific probes to using melting temperature (Tm) analysis as a distinguishing parameter. By analyzing the Tm values of PCR amplification products, the system can identify different bacterial species using a single primer set, thereby reducing probe complexity while maintaining identification accuracy
2Measurement precision
If traditional microbiological detection methods are used, then comprehensive bacterial identification is achieved, but the detection time exceeds 18 hours, delaying appropriate antimicrobial drug selection
Solution Approach 1:
The patent replaces traditional culture-based mechanical detection methods with PCR-based molecular amplification and Tm analysis. This substitution reduces detection time from over 18 hours to a few hours while maintaining identification accuracy, enabling timely antimicrobial drug selection
Solution Approach 2:
The patent performs DNA extraction and PCR amplification of 16S rRNA genes as preliminary actions before final identification. By preparing the DNA template and amplifying target sequences in advance, the system enables rapid Tm analysis and species identification within hours rather than days
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 diagnosis of pathogenic bacteria within two hours, facilitating timely and appropriate antimicrobial drug selection and monitoring of therapeutic effects, while minimizing measurement errors through the use of SYBR Green I and a reference Tm value for accurate identification.
Implementation Method 1
subjecting the DNA as a template to gene amplification by PCR or the like using a specific primer set
Implementation Method 2
analyzing the combination of the melting temperatures (Tm values) specific to the microorganism or the difference between the Tm values
Implementation Method 3
minimum inhibition concentration (MIC) value, it is possible to select an appropriate antimicrobial drug
Data Source
Figure 1
AI summary
A system rapidly detects and identifies pathogenic bacteria responsible for infection (particularly septicemia), and selects an appropriate antimicrobial drug. A method according to the present invention for detecting and identifying pathogenic bacteria includes performing gene amplification such as real-time PCR, and analyzing the combination of the melting temperatures (Tm values) determined by gene amplification product melting curve analysis or the difference between the Tm values. Specifically, real-time PCR is performed using 4 to 16 primer sets including 1 to 7 primer sets for the 16S ribosomal RNA of bacteria, 1 to 6 primer sets for the 18S ribosomal RNA of fungi, and one primer set respectively for the spa gene and the mecA gene specific to MRSA, and the combination of the Tm values of the amplification product or the combination of the differences between the Tm values is compared with a database to identify pathogenic bacteria responsible for septicemia. Pathogenic bacteria responsible for infection (particularly septicemia) can be rapidly detected and identified using the method according to the invention so that a rapid septicemia diagnosis method and evidence-based medicine in septicemia treatment are implemented.