Mycoplasmas, the infectious class Pleuropneumoniae (PPLO), are the smallest and simplest prokaryotes ever discovered, lacking a cell wall. They are 0.1 to 0.3 microns in diameter, can pass through filter membranes, and are highly pleomorphic. They are relatively common and difficult to detect bacterial fungi in cultured cells in culture.
Mycoplasma contamination has many adverse effects on cell culture practice and has become a serious problem in cell culture. The impact is maximized, and the mycoplasma contamination rate in cell culture is as high as 15%~35%, which seriously affects the reliability of experimental results.
About 95% of mycoplasma contamination in cell culture is mainly caused by Mycoplasma leijkeri, Mycoplasma spermatophore, Mycoplasma oralis, Mycoplasma fermentation, Mycoplasma hominis and Mycoplasma hyorhinis. In cells, mycoplasma colonies are in the shape of "fried eggs". Cell culture supernatant and cell membrane provide an exogenous environment for the growth of mycoplasma, and mycoplasma parasitism on the cell surface can form cell membranes.
The main sources of mycoplasma contamination include: cell culture medium raw materials (such as culture medium raw materials, serum and other reagents), laboratory personnel, cell culture incubators, liquid nitrogen storage tanks, particulate matter and aerosols in the air, the use of antibiotics, improperly sealed cultured cells, and cells that have been contaminated with mycoplasma.
At the same time, the corresponding explanations of these contaminations include: due to the inability to effectively remove the original body in the downstream closed process, the cell products or products produced by cells are reported in batches; all related equipment, intermediate products or final products that come into contact with the contaminants are contaminated; other normal cells are contaminated by the original body cells; important cells or virus seed banks are scrapped due to the original body contamination; if the laboratory stops production or experimental operations to deal with the original body contamination; the original body is generated, resulting in waste removal; it is difficult to expand the scope of the original body contamination; it poses a threat to the cell laboratory.
Therefore, mycoplasma detection and removal must be performed regularly to ensure that mycoplasma is not present in cell system culture, which is crucial for the normal progress of scientific research.
Mycoplasma detection
Mycoplasma is a relatively common but often difficult to achieve type of contamination. For biologics processes involving cell culture, regulations require that “no mycoplasma contamination is present.”
- The 2020 edition of the Chinese Pharmacopoeia, Part III "Preparation and Quality of Animal Cell Matrices for Production and Testing of Biological Products" stipulates that for cells used in production, the master cell bank (MCB), working cell bank (WCB), and final production cells (EOPC) must be tested for mycoplasma.
- The FDA's Guidance for Industry: Characterization and Identification of Cell Substrates and Other Biological Materials Used in the Production of Infectious Disease Viral Vaccines requires mycoplasma control for raw materials, viral seeds, unprocessed harvest fluids, and other such materials.
- ICH Q5D “Required Derivatization and Characterization of Cell Substrates for the Production of Biotechnological/Biological Products” also mentions mycoplasma control for raw materials, viruses, unprocessed harvest fluids, seeds, and other such materials.
Figure 1 Requirements for mycoplasma infection detection points
Traditional mycoplasma detection methods mainly include culture method and indicator method. However, due to the long or inconsistent detection cycle of these two methods, the company's cell production or product release cycle is extended. With the development of cell and gene therapy, the industry has higher and higher requirements for the timeliness and effectiveness of mycoplasma detection. The existence of the last guarantee period needs to support such a long detection cycle, so the rapid NAT (tandem detection) method is transferred in the queue mycoplasma detection method.
Figure 2 Mycoplasma detection methods listed on the weekly benchmark
Yisen has developed a series of mycoplasma rapid quantitative PCR detection kits based on NAT (mycoplasma detection technology), including the Mycoplasma Rapid Quantitative PCR Detection Kit, the MycAway™ Plus-Color One-Step Mycoplasma Detection Kit, and the GMyc-PCR Mycoplasma Detection Kit.
1. Mycoplasma Real-time Nucleic Acid Quantitative PCR Detection Kit
This product is used for rapid qualitative detection of potential mycoplasma replication in production raw materials, cell banks, viruses, virus or cell harvest fluids, and therapeutic cells. It is mainly used in the research and development, production and release processes of seed biopharmaceuticals.
Product Qualification
Regulatory Compliance: Validated according to the requirements of EP2.6.7, JP G3 and USP 63 pharmacopoeia, in line with the standards of international authorities.
Audit Compliance: Products are manufactured in accordance with ISO13485 quality management system standards and are supported by comprehensive audit documentation.
Quality Assurance: All enzyme raw materials required for the test kit are self-produced and have been industrialized to ensure stable supply.
Accumulation of technical experience: We have a precise technical foundation in the TaqMan method, and the kit is up to 10 CFU/mL or below.
Key product features: Taq enzyme coupling library, with double buffer enhancement, enhances the coupling, consistency and stability of the kit.
Product Features
Multiple detection types: Optimized TaqMan markers can detect up to 183 mycoplasmas.
Easy to use: Total time for sample preparation and testing is less than 3 hours, compared to the 28 days required for culture methods.
High or: Assay medium as low as 10 CFU/mL, mortality becomes a viable alternative to direct culture methods.
High-quality primers and markers are designed for 16S rRNA, ensuring no cross-reaction with closely related species such as Clostridium and Sesamum.
High safety: The rapid control (PC) in the kit is non-infectious, completely eliminating the risk of contamination.
Strong anti-interference ability: By introducing internal (control IC), sample interference and reaction preparation abnormalities are eliminated, effectively avoiding false negatives.
Figure 3 Workflow of mycoplasma detection using the Mycoplasma Real-time Quantitative PCR Detection Kit
2.MycAway™ Plus-Color One-Step Mycoplasma Detection Kit
It uses a unique air technology that allows for greater color uptake. The change from bluish purple (negative) to sky blue (positive) is easier to see with the naked eye. It also contains contamination components to eliminate the occurrence of false positives.
Figure 4: Mycoplasma detection workflow using the MycAway™ Plus-Color One-Step Mycoplasma Detection Kit
3.GMyc-PCR Mycoplasma Detection Reagent test kit
Different from the traditional PCR detection method, the number of primers increased from one to three pairs, and PCR was performed on the non-region between the mycoplasma 16S and 23S rRNA, which not only reduced the DNA product but also significantly improved the bilaterality of the product, and mycoplasma as low as a single copy could be detected.
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Figure 5. Operation flow of GMyc-PCR mycoplasma detection kit
Mycoplasma Removal
During the cell culture process, regular testing can effectively prevent large-scale mycoplasma contamination. If the cells have been contaminated with mycoplasma, they need to be dealt with in time. The best practice is to high-pressure wash the contaminated cells, discard them, re-infect them with clean cell lines, etc. If the contaminated cells are particularly gone, mycoplasma contamination needs to be carried out, and antibiotics are used in combination to achieve this goal. Because mycoplasmas lack cell walls, traditional antibiotics are usually ineffective. Therefore, choosing the right mycoplasma is key.
Probiotic MycAway™ Mycoplasma Treatment Agent (1000×) is a mixed antibiotic preparation with ingredients that have antibacterial effects on mycoplasmas: quinolones, tetracyclic lactone antibiotics. This mycoplasma removal agent effectively removes mycoplasmas by concentrating DNA synthesis and protein production required for mycoplasma growth without damaging cells. It can maximize cell rescue and minimize losses caused by mycoplasma contamination.
Product Features
Low toxicity: Minimal toxicity to cells ensures that subsequent cell experiments such as transfection and viability assays will not be performed.
High stability: The reagent can be stored at -20℃ for a long time and maintain high efficiency.
Easy to use: simply add the product to excised tissues contaminated with mycoplasma and feed.
Fast-acting: "Immediately effective", results can be seen in as fast as 3 seconds.
Broad spectrum: capable of quantifying most types of protozoa found in the laboratory.
Figure 6. Schematic diagram of the cytotoxicity test of mycoplasma effective reagents
Mycoplasma Prevention
Mycoplasma contamination often occurs during cell culture. However, due to the characteristics of mycoplasma, it is difficult to detect with the naked eye or microscopic equipment. Therefore, it is critical to prevent and control mycoplasma. Adding mycoplasma control reagents to cells can prevent mycoplasma contamination.
At present, the main sources of mycoplasma in the laboratory are: cross-contamination between cells, contamination of the working environment or experimental equipment, improper aseptic operation of the experimenter, contamination of the culture medium or reagents, and mycoplasma infection in the primary tissue or organ. The mycoplasma control series products developed by Yisen are simple to operate and highly stable, and are very suitable for mycoplasma control between cultured cells.
Product Information
|
product |
Catalog Number |
Product Name |
Product Specifications |
|
Sample Pretreatment Kit |
18461ES |
25 tons/100 tons |
|
|
18467ES |
MolPure™ Mag48 Sample Preparation Kit FN |
3×16T/ 6×16T |
|
|
Speed introduction instrument |
80511ES |
48-channel automatic basketball extractor |
48 channels |
|
Mycoplasma Kit |
40619ES |
MycAway ™ Mycoplasma Real-time PCR Detection Kit (2G)
|
25 tons/100 tons |
|
40612ES |
25 tons/100 tons |
||
|
40601ES |
10 times/20 times |
||
|
Mycoplasma reagent |
40607ES |
1ml/5×1ml
|
|
|
Mycoplasma prevention reagents |
40608ES |
MycAway™ Prevention (2000×) - Mycoplasma Prevention Reagent |
1ml/5×1ml
|
|
40605ES |
MycAway™ Spray (Ready-to-use) |
500ml/2×500ml/10×500ml
|
|
|
40609ES |
MycGuard ™ -1 solution (100x) for CO2 incubator water bath disinfection |
100 ml |
|
|
40610ES |
MycGuard ™ -2 solution (500x), for ordinary water bath disinfection |
100 ml |