Hierarchical PCR Primer Screening for Onychomycosis Pathogen Identification
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Solution Overview
Problem
Current methods for detecting onychomycosis, a fungal infection of the nail, are inadequate in accurately identifying the specific fungal pathogens, particularly in differentiating between dermatophytes, Candida, and saprophytes, which are crucial for effective treatment and management.
Innovation Solution
A method involving the use of secondary and primary clade-specific primers for PCR to determine the presence of onychomycotic fungi by amplifying specific nucleotide sequences, such as 18S and 28S ribosomal RNA, and internal transcribed spacer regions, allowing for the differentiation between dermatophytes, Candida, and saprophytes in a sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for onychomycosis, then the detection process is simple, but the accuracy in identifying specific fungal pathogens is insufficient
Solution Approach 1:
The patent divides the fungal pathogen detection into hierarchical segments: first identifying the broad fungal category (dermatophyte, Candida, or saprophyte) using secondary clade-specific primers, then progressively narrowing down to primary clade members and finally to specific species using species-specific primers. This segmentation allows accurate pathogen identification while managing complexity through a structured, multi-stage approach rather than attempting to detect all species simultaneously.
Solution Approach 2:
The patent adds a hierarchical dimension to the detection process by organizing primers into multiple levels (secondary clade, primary clade, species-specific). This dimensional organization transforms a potentially overwhelming single-step detection problem into a manageable multi-level classification system, where each level provides progressively more specific information about the pathogen identity.
2Adaptability or versatility
If a single PCR method is used, then the detection process is quick, but it cannot differentiate between different types of onychomycotic fungi
Solution Approach 1:
The detection process is segmented into sequential stages: first using secondary clade-specific primers to categorize the fungus into one of three broad groups (dermatophyte, Candida, or saprophyte), then using primary clade-specific primers for further classification, and finally using species-specific primers for precise identification. This segmentation enables comprehensive pathogen differentiation while optimizing time by only proceeding to subsequent stages when needed based on initial results.
Solution Approach 2:
The patent performs preliminary classification using secondary clade-specific primers before proceeding to more specific detection methods. This preliminary action quickly identifies the broad category of the pathogen, allowing the diagnostic process to stop early if the information is sufficient, or to proceed to more specific testing only when necessary, thereby reducing overall detection time while maintaining comprehensive differentiation capability.
3Measurement precision
If broad-spectrum primers are used, then all fungal types can be detected, but specific pathogen identification is lost
Solution Approach 1:
The primer set is segmented into three distinct levels of specificity: secondary clade-specific primers that detect broad fungal categories (maintaining wide detection range), primary clade-specific primers for intermediate classification, and species-specific primers for precise pathogen identification. This segmentation allows the system to maintain both broad adaptability and high precision by selecting the appropriate primer level for each diagnostic need.
Solution Approach 2:
The patent implements a nested hierarchical structure where species-specific primers are nested within primary clade-specific primers, which are in turn nested within secondary clade-specific primers. Each level of nesting contains progressively more specific information, allowing the system to maintain both broad detection capability (outer layers) and precise identification (inner layers) simultaneously, with each level building upon the previous level.
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 precise identification of the fungal pathogens, improving diagnostic accuracy and guiding targeted treatments for onychomycosis.
Implementation Method 1
The method may include the steps of i) screening a sample using a first and second sets of secondary clade-specific primers to determine the presence or absence of a secondary clade member among a plurality of secondary clade members that include (a) a dermatophyte, (b) a candida, and (c) a saprophyte, and ii) after determining the presence of the secondary clade member, screening the sample to determine the presence or absence of an onychomycotic fungus using primary clade-specific primers that are specific to a primary clade member that belongs to the secondary clade member. In some embodiments, the screening step i) includes performing a first polymerase chain reaction (PCR) using the first set of secondary clade-specific primers in a first reaction mixture, and performing a second PCR using the second set of secondary clade-specific primers in a second reaction mixture.
Data Source
AI summary
Provided herein is a method of detecting an onychomycotic fungus in a sample, wherein the onychomycotic fungus belongs to a secondary clade member including one or more primary clade members. The method may include the steps of i) screening a sample using a first and second sets of secondary clade-specific primers to determine whether a secondary clade member among a plurality of secondary clade members is present or absent in the sample, where the plurality of secondary clade members includes (a) a dermatophyte, (b) a candida, and (c) a saprophyte, and ii) after determining the presence of the secondary clade member, screening the sample to determine whether an onychomycotic fungus is present or absent in the sample using primary clade-specific primers that are specific to a primary clade member that belongs to the secondary clade member. Also provided is a kit that finds use in implementing the present method.