IMLL Probes for Tm Mapping in Real-Time PCR
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current Tm mapping methods for identifying pathogenic microorganisms are limited by the need for high-accuracy real-time PCR instruments with measurement errors within ±0.1° C., restricting their widespread adoption due to the availability of only a few commercial instruments that meet this requirement, leading to delays in antimicrobial therapy and risks associated with incorrect antimicrobial agent selection.
Innovation Solution
The use of Imperfect-Match Linear Long (IMLL) probes, which are designed to bind to imperfect-match sequences and provide wide differences in melting temperatures, allowing for accurate identification of microorganisms using a broader range of commercial real-time PCR instruments with measurement errors up to ±0.5° C., enabling identification at least at the genus level and often at the species level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional Tm mapping method using PCR amplified products is used, then microorganism identification is achieved, but high measurement accuracy (±0.1° C.) is required which limits instrument availability
Solution Approach 1:
The invention changes the measurement parameter from Tm values of PCR amplified products to Tm values of IMLL probes. This parameter change allows the use of a broader range of commercial real-time PCR instruments with measurement errors up to ±0.5° C., while still achieving accurate microorganism identification at the genus and species levels.
Solution Approach 2:
The invention uses IMLL probes that bind to specific regions of PCR amplified products, creating a copied signal that reflects the Tm characteristics of the target microorganism DNA. This copying approach enables measurement on a broader instrument base while maintaining identification accuracy.
2Productivity
If conventional Tm mapping method is used, then microorganism identification is possible, but identification time takes 2 to 3 days from sample collection
Solution Approach 1:
The invention performs PCR amplification and probe hybridization in parallel during the same incubation period, rather than sequentially. The IMLL probes are added to the PCR reaction mixture before amplification, allowing both amplification and Tm measurement to occur simultaneously, thus reducing total identification time to about 4 hours.
Solution Approach 2:
The invention merges the PCR amplification step with the Tm measurement step into a single unified process. By adding IMLL probes to the PCR reaction mixture and measuring Tm values of the probes during/after amplification, the method combines two previously separate operations into one, achieving rapid identification in about 4 hours from sample collection.
3Measurement precision
If conventional Tm mapping method is used, then microorganism identification accuracy is achieved, but only a few commercial instruments fulfill the requirement
Solution Approach 1:
The invention changes the measurement parameter from Tm values of PCR amplified products (requiring ±0.1° C. accuracy) to Tm values of IMLL probes (tolerating up to ±0.5° C. error). This parameter change makes the method compatible with the majority of commercial real-time PCR instruments while maintaining the ability to accurately identify microorganisms at genus and species levels.
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 IMLL probes expand the scope of instruments capable of performing Tm mapping, enabling rapid identification of microorganisms in almost all real-time PCR instruments, reducing the time to identification from days to hours and improving the accuracy of antimicrobial therapy by allowing identification at least at the genus level and often at the species level.
Implementation Method 1
IMLL probes, which are designed to bind to imperfect-match sequences
Implementation Method 2
provide wide differences in melting temperatures, allowing for accurate identification of microorganisms
Data Source
AI summary
The improved Tm mapping method using imperfect-match linear long quenching probes can accurately distinguish among and identify microorganisms at least at the genus level and often at the species level even in a real-time PCR instrument having measurement errors of Tm values between PCR tubes within ±0.5° C. Therefore, the Tm mapping method can be performed in almost all real-time PCR instruments and can identify unspecified infection-causing pathogenic microorganisms in about 4 hours after sample collection.