Mycobacterium tuberculosis Genotyping via SNP Mass Spectrometry
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genotyping methods for Mycobacterium tuberculosis (MTB) are limited by high DNA sample requirements, time consumption, insufficient sensitivity and specificity, and inability to genotype certain strains, necessitating an improved method for accurate strain differentiation.
Innovation Solution
A primer set and extension primers are developed for PCR-based genotyping of MTB, utilizing single-nucleotide polymorphism markers detected by mass spectrometry, which allows for the amplification and identification of specific DNA fragments, enabling more precise strain classification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional genotyping methods (spoligotyping, MIRU-VNTR) are used, then strain classification can be achieved, but large amounts of DNA sample are required and time consumption increases
Solution Approach 1:
The patent extracts and utilizes specific single-nucleotide polymorphism (SNP) markers from the MTB genome at positions 301-320 of the PE/PPE gene family. By focusing on these specific diagnostic SNPs rather than analyzing the entire genome or using conventional methods, the method reduces DNA sample requirements while maintaining strain classification capability. The primer sets are designed to amplify only these specific SNP regions, extracting the essential genotyping information with minimal DNA input.
Solution Approach 2:
The patent changes the genotyping approach from conventional methods (spoligotyping, MIRU-VNTR) to SNP-based detection using mass spectrometry. This parameter change enables rapid detection of multiple samples simultaneously, reducing both DNA sample requirements and time consumption. The method transforms the genotyping process into a high-throughput assay that can process multiple samples in parallel, dramatically improving productivity while reducing material requirements.
2Measurement precision
If conventional genotyping methods are used, then strain differentiation is possible, but sensitivity and specificity are insufficient
Solution Approach 1:
The patent applies local quality by focusing on specific diagnostic SNP markers at positions 301-320 of the PE/PPE gene family, which are known to be highly discriminatory for MTB strain typing. Instead of using generic genotyping methods, the method targets these specific local regions that provide high sensitivity and specificity for strain differentiation. The primer sets are designed to specifically amplify these diagnostic SNPs, ensuring accurate detection and classification.
Solution Approach 2:
The patent replaces conventional mechanical/chemical genotyping methods (PCR-based spoligotyping and MIRU-VNTR) with mass spectrometry-based SNP detection. This substitution enables more precise and reliable strain differentiation by directly detecting nucleotide variations through mass spectrometry, which provides higher sensitivity and specificity compared to conventional methods. The mass spectrometry technology allows for accurate identification of specific SNP alleles, improving overall genotyping reliability.
3Productivity
If conventional genotyping methods are used, then basic strain typing can be performed, but the methods are time-consuming and cannot rapidly detect multiple samples
Solution Approach 1:
The patent merges multiple genotyping operations into a single integrated assay. By combining primer set 1 (for SNP amplification) and primer set 2 (for extension) in a unified workflow, along with using mass spectrometry for simultaneous detection of multiple samples, the method achieves high-throughput processing. This merging of functions allows rapid detection of multiple samples in parallel, dramatically reducing the time required compared to conventional sequential methods.
Solution Approach 2:
The patent replaces time-consuming conventional genotyping methods with mass spectrometry-based SNP detection, which enables rapid simultaneous analysis of multiple samples. The mass spectrometry technology provides fast, accurate detection that can process multiple samples in parallel, reducing genotyping time from days to hours while maintaining high productivity.
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 method provides enhanced sensitivity and specificity for MTB genotyping, reducing DNA sample requirements and enabling rapid detection of multiple samples, improving strain differentiation and epidemiological analysis.
Implementation Method 1
amplifying and obtain at least one of first DNA fragment by using one or more primer sets selected from the group consisting of primer sets 1 to 25
Implementation Method 2
amplifying and obtain at least one of second DNA fragment by using the obtained first DNA fragment as template and using one or more extension primers selected from the group consisting of SEQ ID Nos. 51 to 75
Implementation Method 3
detecting the second DNA fragment by using mass spectrometry
Data Source
AI summary
The present application provides a method for genotyping M. tuberculosis, comprising obtaining amplifying and obtaining a first DNA fragment from a DNA sample by using one or more primer sets selected from the group consisting of primer sets 1 to 25 (SEQ ID Nos. 1 to 50); amplifying and obtaining a second DNA fragment from the obtained first DNA fragment by using one or more extension primers selected from the group consisting of SEQ ID Nos. 51 to 75; and detecting the second DNA fragment by using mass spectrometry, particularly by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).