Ultrasensitive Microbial Detection via Selective Lysis and DIANA Binding
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Solution Overview
Problem
Current molecular assays for detecting Borrelia species, such as those causing Lyme disease, face challenges due to low microbial loads in blood samples, leading to insufficient clinical sensitivity and the inability to accurately detect microorganisms at levels below 10 cells/ml, which is crucial for early and accurate diagnosis.
Innovation Solution
The method involves providing a biological sample of at least 5 ml, selectively lysing mammalian cells to separate eukaryotic DNA, isolating microbial genetic materials, amplifying them, and using DNA Invading Artificial Nucleic Acids (DIANAs) complementary to microbial sequences for detection, allowing for the identification of microbial species directly in the sample without culturing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard molecular assays are used for detecting Borrelia species, then the detection process can be performed, but the clinical sensitivity is insufficient and accurate detection below 10 cells/ml cannot be achieved
Solution Approach 1:
The patent applies preliminary action by performing selective lysis of mammalian cells and removal of eukaryotic DNA before microbial DNA extraction. This pre-treatment step removes the overwhelming background of human DNA from blood samples, thereby enabling sensitive detection of low-level Borrelia species without interference from host DNA, directly resolving the contradiction between detection sensitivity and clinical accuracy
Solution Approach 2:
The patent extracts and removes eukaryotic DNA from the sample through selective lysis and purification steps. By taking out the interfering human DNA component, the method isolates microbial genetic materials for accurate detection, thereby achieving both high sensitivity and reliability in detecting low microbial loads
2Loss of time
If blood samples with low microbial loads are used for detection, then early stage infection can be detected, but the background of human DNA overwhelms the signal and limits detection capability
Solution Approach 1:
The patent converts the harmful effect of overwhelming human DNA background into a benefit by using selective lysis conditions that specifically target and remove eukaryotic cells while preserving microbial cells. This transforms the problematic high background interference into an opportunity to enrich the sample for microbial DNA, enabling early detection of low-level infections
Solution Approach 2:
The method performs preliminary removal of eukaryotic DNA through selective lysis and purification before performing the actual detection. This pre-treatment step eliminates the background interference that would otherwise overwhelm the signal, allowing accurate detection of Borrelia species at early infection stages
3Measurement precision
If larger blood sample volumes are used to improve detection sensitivity, then microbial load detection improves, but sample processing complexity and time requirements increase
Solution Approach 1:
The patent extracts and removes eukaryotic DNA through selective lysis and purification, creating an enriched sample with reduced background. This extraction step allows the use of smaller blood sample volumes while maintaining high detection sensitivity, as the removed human DNA would otherwise require proportionally larger input volumes to achieve sufficient microbial DNA for detection
Solution Approach 2:
The method changes the compositional parameters of the sample by selectively removing eukaryotic cells and DNA through controlled lysis conditions. This parameter change (reducing human DNA content) enables sensitive detection with smaller sample volumes, thereby reducing processing complexity while maintaining or improving detection sensitivity
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 ultrasensitive detection of microbial species at low loads, improving clinical sensitivity and specificity, enabling the detection of Borrelia species in blood samples with higher accuracy and reliability, potentially leading to earlier and more effective disease diagnosis.
Implementation Method 1
contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material
Data Source
AI summary
The present disclosure generally relates to the field of ultrasensitive microbial pathogen detection and identification utilizing genomic sequence recognition.