Mycoplasma Detection via Multiplex Real-Time PCR
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Solution Overview
Problem
Current Mycoplasma detection methods, such as culture, DNA staining, and Nested PCR, are inadequate for rapid, sensitive, and accurate detection, particularly in regenerative medicine and cell therapy, due to long culture times, low sensitivity, and risk of false positives.
Innovation Solution
A multiplex real-time quantitative PCR method using specific combinations of forward and reverse primers and probes targeting the 16S rRNA and 23S rRNA genes, along with spacer regions, to detect a wide range of Mycoplasma species with high sensitivity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If culture method is used for Mycoplasma detection, then detection can be performed, but the culture period is too long (28 days)
Solution Approach 1:
The patent replaces the biological culture system with a molecular biology-based PCR system. Instead of relying on Mycoplasma growth in culture media over 28 days, the invention uses DNA extraction followed by real-time quantitative PCR to directly detect Mycoplasma genetic material, reducing detection time to approximately 1 hour while maintaining high reliability through specific primer and probe design
Solution Approach 2:
The patent performs preliminary DNA extraction and amplification setup before detection. By preparing specific primers and probes that target conserved regions of Mycoplasma 16S and 23S rRNA genes in advance, the system enables rapid detection without waiting for culture growth, as the PCR reaction can directly amplify Mycoplasma DNA from clinical samples
2Ease of manufacture
If DNA staining method using indicator cells is used, then detection can be performed, but the sensitivity is low
Solution Approach 1:
The patent changes the detection parameter from visual staining observation to quantitative fluorescence measurement. By using fluorescently labeled probes that emit detectable signals only when bound to Mycoplasma DNA during PCR amplification, the system achieves high sensitivity (detecting as few as 10-100 copies of target DNA) while maintaining ease of operation through automated real-time PCR instrumentation
Solution Approach 2:
The patent replaces the optical staining method with a molecular amplification and fluorescence detection system. Instead of relying on indicator cells to visually display Mycoplasma presence, the invention uses real-time quantitative PCR with fluorescent probes to amplify and detect Mycoplasma DNA, providing significantly higher sensitivity while maintaining operational simplicity
3Measurement precision
If Nested PCR method is used, then detection can be performed, but false-positive due to carry over contamination is likely to occur
Solution Approach 1:
The patent segments the PCR process into separate spatial and temporal components. By using real-time quantitative PCR with fluorescent probe detection, the system monitors amplification in real-time within a closed reaction tube, eliminating the need for post-PCR gel electrophoresis and reducing carryover contamination risks. The segmented approach separates DNA extraction, amplification, and detection into distinct controlled steps
Solution Approach 2:
The patent introduces fluorescent probes as intermediaries between the target DNA and detection system. These probes specifically bind to amplified Mycoplasma DNA sequences and emit fluorescent signals only when bound, providing a reliable intermediary detection mechanism that reduces false positives compared to direct gel electrophoresis methods used in traditional nested PCR
4Measurement precision
If specific primer pairs for single Mycoplasma species are used, then detection accuracy for that species is high, but the number of species that can be detected is limited
Solution Approach 1:
The patent designs universal primers that target highly conserved regions of the 16S and 23S rRNA genes present across all Mycoplasma species. These universal primers can amplify DNA from any Mycoplasma species, while species-specific probes with different fluorescent labels allow differentiation and simultaneous detection of multiple species in a single reaction, achieving both broad versatility and species-specific accuracy
Solution Approach 2:
The patent merges multiple detection capabilities into a single real-time quantitative PCR reaction. By combining universal primers with multiple species-specific fluorescent probes in one reaction tube, the system can simultaneously detect and differentiate multiple Mycoplasma species, merging the functions of what would traditionally require multiple separate PCR reactions
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 enables rapid and accurate detection of multiple Mycoplasma species with a sensitivity of 10 cfu/mL or less, reducing the risk of false positives and improving the reliability of Mycoplasma detection in biological samples.
Implementation Method 1
specific combinations of forward and reverse primers and probes targeting the 16S rRNA and 23S rRNA genes
Implementation Method 2
a probe for detecting a product amplified by use of the primer pair
Data Source
AI summary
An object of the present invention is to provide a detection method for Mycoplasma by which a greater number of Mycoplasma species can be more quickly and easily detected with high sensitivity and accuracy, a set of a forward primer, a reverse primer and a probe for the detection and a kit containing the set. Mycoplasma in a test sample is detected by a multiplex real time quantitative PCR using one or more forward primers, each of which is an oligonucleotide consisting of a nucleotide sequence, which is selected from the group consisting of nucleotide sequences each consisting of continuous 17 to 30 nucleotides in the nucleotide sequence represented by SEQ ID No: 1, and which contains a nucleotide sequence at nucleotide positions 14 to 24 in SEQ ID No: 1; reverse primers, each of which is an oligonucleotide consisting of a nucleotide sequence, which is selected from the group consisting of nucleotide sequences each consisting of continuous 17 to 26 nucleotides in the one or more nucleotide sequences represented by SEQ ID Nos: 14 and 17 to 20; and a probe(s), which is an oligonucleotide, which consists of a nucleotide sequence selected from the group consisting of nucleotide sequences each consisting of continuous 17 to 26 nucleotides in the nucleotide sequence represented by SEQ ID No: 33, or which consists of a complementary nucleotide sequence thereto.