Nested PCR Method for Bacterial 16S rRNA Quantification
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Solution Overview
Problem
Conventional PCR methods struggle to accurately determine the number and species of pathogenic bacteria in patient specimens, especially when the bacterial load is low, due to insufficient sensitivity and issues with contamination.
Innovation Solution
A method involving a two-step PCR process: a first PCR using universal primers for amplifying the bacterial 16S rRNA gene, followed by a nested PCR using internal primers to enhance sensitivity and specificity, along with calibration data to determine bacterial cell numbers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a bacterial universal primer is used in real-time PCR to detect pathogenic bacteria, then the sensitivity is improved, but contamination is easily detected making determination difficult
Solution Approach 1:
The patent divides the detection process into two distinct stages: first using a bacterial universal primer to amplify all bacterial DNA (including contaminants) with high sensitivity, then using a pathogen-specific primer to selectively amplify only the target pathogen DNA. This segmentation allows the system to benefit from high sensitivity in the first stage while achieving reliable pathogen-specific detection in the second stage, resolving the contradiction between sensitivity and reliability.
Solution Approach 2:
The patent introduces an intermediary step between universal PCR and specific PCR detection. The amplification product from the universal primer serves as a template for the subsequent specific primer amplification. This intermediary approach allows the system to first capture all bacterial DNA (ensuring sensitivity) and then selectively identify the pathogen (ensuring reliability), effectively mediating between the two conflicting requirements.
2Ease of manufacture
If conventional culturing method is used to determine bacterial number, then the procedure is simple, but it takes long time (2 to 3 days) for examination
Solution Approach 1:
The patent replaces the mechanical/biological culturing process with a molecular biology-based PCR system. Instead of relying on bacterial proliferation over days (culturing), the system uses enzymatic amplification of DNA sequences to detect and quantify bacteria within hours. This substitution maintains procedural simplicity while dramatically reducing examination time from 2-3 days to approximately 6 hours.
3Loss of time
If real-time PCR is used to determine bacterial number, then the examination time is reduced, but the sensitivity is sometimes not sufficient for accurately determining the number of pathogenic bacterial cells
Solution Approach 1:
The patent merges two PCR approaches into a single integrated system: the high-speed capability of real-time PCR and the high sensitivity of nested PCR. By combining these methods, the system achieves both rapid examination (maintaining the time advantage of real-time PCR) and accurate quantification of low-abundance pathogens (gaining the sensitivity advantage of nested PCR), thus resolving the contradiction between speed and accuracy.
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 enables rapid and accurate quantification of bacterial cells, even at low concentrations, while minimizing contamination errors, thereby improving the diagnosis and monitoring of infectious diseases.
Implementation Method 1
a first PCR step of carrying out a PCR method using a nucleic acid derived from a specimen as a template and a universal primer pair for amplifying a bacterial 16S rRNA gene
Implementation Method 2
a second PCR step of carrying out a nested PCR method using a primer pair(s) for amplifying an internal sequence(s) of the sequence of the first amplification product obtained by the first PCR step
Data Source
AI summary
A method for enabling rapid and accurate determination of the number of bacterial cells in a specimen using a PCR method includes the following steps: (1) a first PCR step of carrying out a PCR method using a nucleic acid derived from a specimen as a template and a universal primer pair for amplifying a bacterial 16S rRNA gene to obtain a first amplification product; (2) a second PCR step of carrying out a nested PCR method using a primer pair(s) for amplifying an internal sequence(s) of the sequence of the first amplification product obtained by the first PCR step to obtain a second amplification product; and (3) a bacterial number determination step of obtaining the number of bacterial cells in the specimen based on the amount of the second amplification product obtained in the second PCR step and using calibration data.