Phage-Based Mycobacteria Detection Assay
Solution Overview
Problem
Current methods for detecting Mycobacterial infections, such as those caused by Mycobacterium avium subspecies paratuberculosis (MAP), are unreliable, slow, and of low reproducibility, particularly in human samples, which hinders diagnosis and treatment of conditions like Crohn's disease.
Innovation Solution
A semiquantitative phage-based assay that involves lysing cells to release viable mycobacterial cells, infecting them with a bacteriophage, and quantifying plaques formed on a substrate to detect and differentiate between diseased and asymptomatic subjects, with the use of broad host range mycobacteriophages like D29 and DNA analysis using PCR or whole genome sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current blood culture method is used to detect MAP, then detection is possible, but the method requires 3 to 6 months of growth and has low reproducibility
Solution Approach 1:
The patent introduces bacteriophage as an intermediary agent to detect mycobacteria. Instead of directly culturing slow-growing mycobacteria, the method uses bacteriophage that specifically infects mycobacteria. The phage acts as a mediator that accelerates detection by targeting the bacteria directly, bypassing the need for long-term mycobacterial culture while maintaining specificity through phage-host interaction.
Solution Approach 2:
The patent replaces the mechanical/biological system of direct mycobacterial culture with a phage-based detection system. Instead of relying on the slow mechanical process of mycobacterial growth and division, the method uses phage infection and plaque formation, which occurs much faster. This substitution fundamentally changes the detection mechanism from direct observation of bacterial growth to observation of phage-induced plaques.
2Productivity
If direct culture method is used, then mycobacteria can be detected, but the method is slow and not suitable for rapid diagnosis
Solution Approach 1:
The patent applies preliminary action by pre-incubating the sample with bacteriophage before plating. This allows the phage to infect any present mycobacteria in advance, so that when the mixture is plated and incubated, plaque formation occurs rapidly. The preliminary phage infection step accelerates the overall process while maintaining reliability through the specific phage-host interaction.
Solution Approach 2:
The patent changes the detection parameter from observing mycobacterial growth (slow) to observing phage plaque formation (fast). By shifting the measurable outcome from bacterial colony formation to viral plaque formation, the method achieves faster detection speeds while maintaining specificity through the unique phage-host relationship. The parameter change transforms a slow biological process into a rapid virological assay.
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 method provides a rapid, reliable, and reproducible means to detect viable mycobacteria in human samples, enabling discrimination between diseased and asymptomatic individuals and assessing Crohn's disease severity, while guiding therapy and identifying latent infections.
Implementation Method 1
admixing a bacteriophage with a sample under conditions suitable to allow the bacteriophage to infect any viable target mycobacterial cell present in the sample
Implementation Method 2
admixing at least a portion of the admixture of step c) following the removal of step d) with bacteria capable of supporting replication of the bacteriophage
Implementation Method 3
quantifying the number of bacteriophage plaques that form
Data Source
AI summary
The present invention provides a semi-quantitative phage-based assay for Mycobacterial infection, in which the phage assay plaque count correlates to the number of viable Mycobacterial organisms in the subject sample, and methods of use thereof for diagnosing, treating or monitoring Mycobacterial infection.