Mycoplasma Detection Composition for Rapid, Specific qPCR
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Solution Overview
Problem
Current methods for detecting mycoplasma contamination in cell cultures suffer from low sensitivity, specificity, and wide detection range, leading to inaccurate and time-consuming results.
Innovation Solution
A composition comprising primers M-F and M-R, and a probe M-P, with specific nucleotide sequences, is used in a qPCR method to detect mycoplasma, featuring a molar concentration ratio of 1:(0.8-1.2):(0.8-1.2), and includes a fluorophore FAM and quencher BHQ1, enabling rapid and accurate detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the conventional cultivation method is used to detect mycoplasma, then the detection is considered reliable, but the workload is heavy and the cycle time is long
Solution Approach 1:
The patent replaces the conventional mechanical cultivation method with a molecular biology-based qPCR detection system. The composition includes specific primers (M-F, M-R) and a fluorescent probe (M-P) that enable rapid amplification and detection of mycoplasma DNA, eliminating the need for lengthy cultivation processes while maintaining high detection reliability through specific fluorescent signaling.
Solution Approach 2:
The patent changes the detection parameters from phenotypic observation (cultivation) to genotypic detection (DNA amplification). By using specific primer sequences targeting mycoplasma 16S rRNA genes and a fluorescent probe system, the method achieves rapid detection within hours rather than weeks, significantly improving productivity while maintaining reliability through sequence-specific amplification.
2Measurement precision
If the DNA fluorescent staining method is used to detect mycoplasma, then the sensitivity is high, but the result is not easy to determine and is easily affected by subjective determination
Solution Approach 1:
The patent replaces subjective visual staining evaluation with an automated fluorescent qPCR system. The probe M-P contains a fluorophore (FAM) and quencher (BHQ1) that generate objective fluorescent signals during DNA amplification, allowing quantitative analysis through Ct values and amplification curves, thereby eliminating subjective determination while maintaining high sensitivity.
Solution Approach 2:
The patent implements real-time fluorescent feedback during the PCR amplification process. The qPCR instrument continuously monitors fluorescent signal intensity, automatically generating amplification curves and Ct values that provide objective, quantifiable results. This feedback mechanism eliminates subjective interpretation and enables precise, reproducible detection of mycoplasma contamination.
3Measurement precision
If the DNA fluorescent staining method is used to detect mycoplasma, then the sensitivity is high, but the detection time is about 1 week which is still long
Solution Approach 1:
The patent changes the detection timeline by using isothermal amplification or rapid PCR cycles with the specific primer and probe composition. The optimized primer sequences (M-F, M-R) and fluorescent probe (M-P) enable specific mycoplasma DNA amplification within 2-4 hours, dramatically reducing the detection time from 1 week to less than a day while maintaining high sensitivity through sequence-specific amplification and fluorescent detection.
Solution Approach 2:
The patent skips the lengthy cultivation step entirely by directly detecting mycoplasma DNA in cell culture samples. The composition enables rapid amplification of target DNA sequences followed by immediate fluorescent detection, rushing through the detection process in a matter of hours rather than waiting for mycoplasma to grow visible colonies over a week.
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
The method achieves high sensitivity, strong specificity, and a wide detection range, with a 100% coincidence rate with traditional cultivation methods and significantly reduced detection time.
Implementation Method 1
a fluorophore carboxyfluorescein (FAM) linked at a 5′ terminus and a quencher black hole quencher 1 (BHQ1) linked at a 3′ terminus
Data Source
AI summary
A composition and a kit for detecting mycoplasma are provided. The composition for detecting mycoplasma is an aqueous solution including a primer M-F, a primer M-R, and a probe M-P. A sequence of the M-F is shown in SEQ ID NO: 1. A sequence of the M-R is shown in SEQ ID NO: 2. A nucleotide sequence of the probe M-P is shown in SEQ ID NO: 3, and includes a fluorophore FAM linked at a 5′ terminus and a quencher BHQ1 linked at a 3′ terminus. The composition exhibits high sensitivity, strong specificity, and a wide detection range when used in the detection of mycoplasma.