Mycoplasma Detection Using Mitochondrial DNA Internal Control
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Solution Overview
Problem
Existing mycoplasma detection methods are limited in sensitivity and reliability, as they primarily detect mycoplasma contamination in cell culture media rather than directly within host cells, and struggle with quantitative analysis due to background interference in PCR amplification.
Innovation Solution
A method involving the extraction of DNA from host cells infected with mycoplasma, followed by PCR amplification using specific primer sets for mycoplasma and mitochondrial DNA, with the latter serving as an internal control to enhance sensitivity and reliability of detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional commercialized detection kits are used to detect mycoplasma in cell culture media, then the detection method is simple and commercially available, but the sensitivity is reduced because it cannot directly confirm mycoplasma infection inside cultured cells
Solution Approach 1:
The patent extracts DNA directly from host cells suspected of mycoplasma infection, separating the detection process from conventional media-based detection. This extraction approach enables direct confirmation of intracellular mycoplasma infection, thereby improving detection sensitivity while maintaining procedural simplicity through standardized DNA extraction protocols
Solution Approach 2:
The patent introduces mitochondrial DNA as an internal control sample that serves as an intermediary reference. This internal control co-amplifies with mycoplasma DNA during PCR, providing a baseline for comparison that enhances the reliability and sensitivity of detection without adding significant procedural complexity
2Measurement precision
If quantitative analysis is performed based on the number of amplification cycles during PCR using conventional kits, then the analysis can be conducted, but the reliability is low due to background interference causing DNA bands to appear even when mycoplasma DNA is low or absent
Solution Approach 1:
The patent implements feedback through internal control analysis, where the amplification results of mitochondrial DNA provide continuous reference information throughout the PCR process. By comparing the intensity of mycoplasma DNA bands against the internal control bands on the same gel, the method achieves reliable quantitative analysis that is insensitive to background interference or variations in amplification efficiency
Solution Approach 2:
The patent utilizes visual intensity comparison of DNA bands after electrophoresis and staining. The quantitative determination of mycoplasma infection is based on comparing the relative intensities of mycoplasma DNA bands versus internal control DNA bands, transforming the abstract concept of amplification cycle count into a reliable visual measurement that resists background interference
3Reliability
If a method of directly lysing cells and confirming mycoplasma DNA in the cell is used, then the reliability of detection can be improved, but the cost and complexity increase compared to existing commercialized kits
Solution Approach 1:
The patent employs mitochondrial DNA amplification as a universal internal control that serves multiple functions simultaneously: it confirms successful DNA extraction, validates PCR reaction conditions, and provides a reference for quantitative comparison. This multi-functional approach achieves reliable detection without requiring multiple separate control experiments, thereby reducing overall cost and complexity
Solution Approach 2:
The patent changes the parameter of detection target from indirect media-based detection to direct cell-based DNA detection. By extracting and amplifying DNA directly from infected cells rather than detecting mycoplasma in culture media, the method achieves superior reliability while maintaining cost-effectiveness through the use of standard molecular biology reagents and protocols
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 allows for improved sensitivity and cost-effectiveness in detecting mycoplasma infection within host cells, enabling quantitative confirmation of infection levels by comparing band amounts between mycoplasma and internal control DNA.
Implementation Method 1
performing polymerase chain reaction (PCR) using the DNA extracted in step i) as a template
Implementation Method 2
confirming a DNA band by electrophoresis of the PCR product performed in step ii)
Data Source
AI summary
The present invention provides a method for detecting mycoplasma, which can directly confirm whether a cell is infected with mycoplasma, by simultaneously amplifying DNA extracted from lysing a host cell infected with mycoplasma, and mitochondrial DNA in the cytoplasm of the host cell. According to the method of the present invention, by directly using the DNA inside the host cell, the DNA of mycoplasma bound around the nucleus of the host cell can be used as an amplification target, thereby increasing detection sensitivity. In addition, since the mitochondrial DNA inside the host cell is used as an amplification target of an internal control sample, whether the sample has been sampled in an appropriate amount can be confirmed in a convenient manner, and furthermore, by comparing the band size between the internal control sample and the mycoplasma DNA, the degree of mycoplasma infection can be quantitatively confirmed.