CpG Island qPCR for Microorganism Detection
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Solution Overview
Problem
Current methods for detecting and quantifying microorganisms, such as PCR-based techniques, face limitations in sensitivity and specificity, particularly in measuring bacterial/viral loads and are prone to false positive results, which hinders early detection and diagnosis of diseases associated with microorganisms.
Innovation Solution
A method involving quantitative polymerase chain reaction (qPCR) that amplifies target nucleic acid sequences within CpG islands of microorganisms, regardless of methylation status, using specific oligonucleotide primers and probes, allowing for improved detection and quantification of microorganisms like Epstein-Barr virus (EBV) and other pathogens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR-based techniques are used for detection, then microorganism presence can be detected, but sensitivity and specificity are insufficient and false positive results occur
Solution Approach 1:
The patent applies local quality by selecting specific target regions (CpG islands) within the microorganism genome that have unique characteristics. These CpG islands contain specific sequences that are highly conserved and unique to certain microorganisms, allowing for more precise and specific detection compared to amplifying entire genomes or non-specific regions.
Solution Approach 2:
The patent changes the detection parameter from conventional PCR to quantitative real-time PCR (qPCR), which allows for precise quantification of nucleic acid sequences. This parameter change enables measurement of the actual amount of microorganism DNA present, improving both sensitivity and specificity while reducing false positives through quantitative analysis.
2Measurement precision
If culture-based procedures are used for detection, then microorganism growth can be observed, but results take days to become available
Solution Approach 1:
The patent replaces the mechanical/biological system of culture-based growth observation with a molecular amplification system (qPCR). Instead of waiting for microorganisms to grow culturally over days, the method directly amplifies and detects specific nucleic acid sequences in hours, dramatically reducing detection time while maintaining or improving accuracy.
Solution Approach 2:
The patent performs preliminary action by extracting and amplifying nucleic acids directly from clinical samples without requiring prior culture growth. This eliminates the time-consuming culture step while still achieving accurate detection through direct molecular analysis of the microorganism's genetic material.
3Ease of operation
If serologic tests are used for detection, then antibody presence can be measured, but false negative and false positive rates are high
Solution Approach 1:
The patent extracts the detection from the host's immune response (antibodies) and directly targets the microorganism's nucleic acid. By taking out the detection target from the patient's serum antibodies to the microorganism's own genetic material, the method eliminates cross-reactivity issues that cause false positives and negatives in serologic tests.
Solution Approach 2:
The patent uses nucleic acid amplification as an intermediary between sample collection and detection. Instead of directly measuring antibodies that may cross-react, the method uses specific CpG island sequences as intermediaries that are unique to the target microorganism, providing more specific and accurate detection.
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 enhances sensitivity and specificity in detecting microorganisms, enabling better prediction of disease presence and treatment outcomes by accurately quantifying nucleic acid sequences, thereby improving diagnostic accuracy and treatment efficacy.
Implementation Method 1
A method involving quantitative polymerase chain reaction (qPCR) that amplifies target nucleic acid sequences within CpG islands of microorganisms
Implementation Method 2
amplifies target nucleic acid sequences within CpG islands of microorganisms, irrespective of the methylation status of the CpG island
Data Source
Figure 1A
Figure 1C~1D
Figure 1E
AI summary
The present invention provides a method for detecting and/or quantifying the presence of a target nucleic acid sequence of a microorganism in a sample obtained from a subject, including amplifying the target sequence in a CpG island of the nucleic acid of the microorganism, irrespective of the methylation status of the CpG island. The invention is embodied by a method for detecting and/or quantifying the presence of a target nucleic acid sequence of Epstein-Barr virus (EBV) by amplifying a target sequence in the BamHI-W region of EBV in cell free DNAs (cfDNAs) obtained from a subject. The present invention also provides a kit to be used for the method of the invention.