Multiplex PCR Assay for Differential MAC Detection
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Solution Overview
Problem
Current methods for detecting Mycobacterium avium complex (MAC) organisms, particularly distinguishing between non-MAP and MAP species, face challenges due to inconsistent classification and the presence of false positives, which complicates differential identification and diagnosis in mammalian host species.
Innovation Solution
The development of specific nucleic acid target sequences and oligonucleotide primers and probes that are unique to non-MAP organisms, such as MAA, MAH, and MAS, allowing for differential detection and exclusion of MAP organisms through nucleic acid amplification and hybridization methods, providing a one-step diagnostic approach.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used to identify MAC organisms, then detection coverage is achieved, but false positives occur due to inability to distinguish between non-MAP and MAP species
Solution Approach 1:
The detection method is segmented into two distinct parts: a first set of primers that amplifies a common MAC region present in all Mycobacterium avium subspecies, and a second set of primers that amplifies a subspecies-specific region unique to non-MAP organisms. This segmentation allows the assay to first confirm MAC presence and then specifically identify non-MAP subspecies, eliminating false positives from MAP organisms.
Solution Approach 2:
The invention applies local quality by targeting specific genomic regions with different properties: a conserved region for general MAC detection and a variable region for subspecies differentiation. The second primer set is designed to bind only to the specific nucleotide sequence found in non-MAP organisms, providing localized specificity within the broader MAC detection framework.
2Ease of manufacture
If subspecies classification is based on infection species rather than nucleic acid sequence, then classification can be performed without genomic data, but classification becomes inconsistent and complicated
Solution Approach 1:
The invention replaces the mechanical/biological observation method (identifying subspecies based on which species they infect) with a molecular/nucleic acid-based method. By substituting the classification mechanism from phenotypic observation to genotypic analysis, the method achieves both ease of application (simple PCR-based detection) and precision (consistent nucleic acid sequence-based classification).
3Adaptability or versatility
If multiple separate assays are used to detect different MAC subspecies, then comprehensive detection is achieved, but diagnostic complexity and time increase
Solution Approach 1:
The invention merges the functionality of multiple separate detection assays into a single multiplex PCR assay. By combining the first primer set (detecting all MAC organisms) and the second primer set (detecting only non-MAP organisms) in one reaction, the method achieves comprehensive subspecies differentiation while simplifying the diagnostic process to a single test rather than multiple sequential assays.
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 solution enables specific and sensitive detection of non-MAP organisms, reducing false positives and improving diagnostic accuracy, facilitating rapid and precise identification in various mammalian host species.
Implementation Method 1
oligonucleotide primers and probes that are unique to non-MAP organisms, allowing for differential detection and exclusion of MAP organisms through nucleic acid amplification and hybridization methods
Implementation Method 2
differential detection and exclusion of MAP organisms through nucleic acid amplification and hybridization methods
Data Source
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AI summary
Disclosed are compositions, assays, methods, diagnostic methods, kits and diagnostic kits for the specific and differential detection of a non-Mycobacterium avium subsp. paratuberculosis (non-MAP) organism, wherein a non-MAP organism is a Mycobacterium avium complex (MAC) organism that does not belong to the Mycobacterium avium subsp. paratuberculosis (MAP) organism, from samples including veterinary samples, clinical samples, food samples, forensic sample, an environmental sample (e.g., soil, dirt, garbage, sewage, air, or water), including food processing and manufacturing surfaces, or a biological sample. Exemplary non-MAP organisms including M. avium subsp. avium (MAA), M. avium subsp. hominissuis (MAH), and M. avium subsp. silvaticum (MAS) can be detected by the present compositions, kits and methods.