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Cat. No.: MCLK-25 (for 25T)

Cat. No.: MCLK-100 (for 100T)

Description

GoodColor™ Mycoplasma Colorimetric LAMP Assay Kit is a reagent kit used to detect the presence of Mycoplasma contamination in cell cultures and other biological materials by utilizing Loop-Mediated Isothermal Amplification (LAMP), a technique that amplifies Mycoplasma DNA through strand displacement and detects contamination through color changes in the reaction system. This kit employs dU incorporation and UDG enzyme to prevent amplicon contamination, ensuring rapid, efficient, and highly sensitive detection of Mycoplasma contamination. The assay utilizes visual color changes for result determination, eliminating the need for gel electrophoresis and allowing results to be assessed by simple visual observation.

Mycoplasma, the smallest and simplest prokaryotes, possesses distinct characteristics. Mycoplasma lack a cell wall, rendering common antibiotics targeting cell walls, such as penicillin or β-lactam antibiotics, ineffective against them. With a size ranging between bacteria and viruses, approximately 0.2-0.8 μm, some Mycoplasma can pass through a 0.22 μm filter, rendering standard filtration ineffective. Due to limited biosynthetic capabilities, many Mycoplasma rely on host cells for nutrition, often adhering to or spreading between cell surfaces. These traits pose a risk of Mycoplasma contamination during cell culture, a global issue.

Mycoplasma contamination can profoundly affect cell states, altering gene expression and metabolic traits, leading to compromised growth, abnormal differentiation, and cell death, thereby impacting cell functionality. Such effects can substantially undermine experimental reliability, reproducibility, and consistency, emphasizing the crucial need for Mycoplasma detection.

While bacterial, yeast, or fungal contaminations in cell cultures are visible under an optical microscope, Mycoplasma contamination is typically invisible and requires specific detection methods. Common methods for detecting Mycoplasma contamination include Mycoplasma isolation culture, ELISA, luminescence assays, conventional nested PCR, qPCR, biochemical assays, and DNA fluorescent staining. Among these methods, Mycoplasma isolation culture is intricate, ELISA lacks high sensitivity and specificity, conventional nested PCR is time-consuming and requires gel electrophoresis, qPCR and luminescence assays demand specialized equipment, while isothermal amplification only necessitates a constant temperature reaction and can determine Mycoplasma contamination based on color changes without gel electrophoresis analysis.

Isothermal amplification technology is an extracellular nucleic acid amplification technique, maintaining a constant reaction temperature and rapidly amplifying DNA through specific enzymes and primers with varying activity levels. GoodColor™ Mycoplasma Colorimetric LAMP Assay Kit employs Loop-Mediated Isothermal Amplification (LAMP), involving the design of 4-6 specific primers targeting 6 regions of the target gene. Bst DNA Polymerase initiates DNA synthesis, forming a dumbbell-shaped complementary strand, further entering a loop amplification phase through continuous strand displacement, yielding a mixture of DNA with varying stem-loop and cauliflower-like structures. LAMP requires only isothermal conditions (e.g., 60-65°C) for 30-60 minutes to complete nucleic acid amplification. In contrast to conventional PCR, LAMP eliminates the need for template denaturation, temperature cycling, gel electrophoresis, and UV observation, offering simplicity, speed, high sensitivity, and strong specificity.

Components

25T

• LAMP Master Mix with UDG (2X)    250μl
• Bst DNA Polymerase    25μl
• Myco Positive Control    50μl
• Nuclease-free Water    200μl
• Mineral Oil    500μl

• LAMP Master Mix with UDG (2X)    1ml
• Bst DNA Polymerase    100μl
• Myco Positive Control    200μl
• Nuclease-free Water    1ml
• Mineral Oil    2ml

Features

• This assay kit offers high sensitivity and short reaction times. The sensitivity of this kit is higher by 2-5 orders of magnitude compared to traditional PCR methods, with a detection limit of approximately 20-200 copies/μl calculated using positive control plasmids. Furthermore, the detection reaction can be completed within just one hour.
• The assay kit demonstrates strong specificity and broad applicability across various Mycoplasma species. Based on the conserved 16S rRNA gene sequences of Mycoplasma, the kit has been designed with multiple specific LAMP primers, allowing direct detection using biological materials like cell culture supernatants or serum as templates. This kit can amplify DNA from around 45 different Mycoplasma species, encompassing the commonly encountered types responsible for cell contamination. The assay selectively amplifies Mycoplasma DNA and does not amplify DNA from bacteria, fungi, or eukaryotic cells.
• Employing visual color change technology, the assay eliminates the need for gel electrophoresis. Results can be determined through naked-eye observation of color changes. The assay kit has optimized indicator dyes; upon significant amplification of Mycoplasma DNA fragments, the color of the liquid in the reaction tube changes from violet or bluish-purple to light blue or deep sky blue, indicating a positive Mycoplasma result.
• This assay kit employs contamination prevention technology to effectively avoid contamination. By utilizing dU and a thermosensitive UDG enzyme, the kit allows for the incorporation of dU into amplification products. Incubating at 30°C for 5-15 minutes before isothermal amplification can effectively eliminate product contamination issues associated with the amplification process. During the isothermal amplification, the thermosensitive UDG enzyme is deactivated, ensuring that it does not interfere with subsequent isothermal amplification detection.
• This assay kit includes a positive control, Myco Positive Control, which can be used to verify the proper functioning of the kit. The Myco Positive Control does not contain Mycoplasma and poses no risk of Mycoplasma contamination. It is recommended to include the positive control in every testing session.
• This assay kit can directly detect samples suspected of Mycoplasma contamination without the need for separate DNA extraction.

Storage

Store at -20°C, valid for one year. The LAMP Master Mix with UDG (2X) should be stored away from light. Avoid repeated freeze-thaw cycles as much as possible.

Precaution

• Before use, ensure that the reagents are completely thawed and gently mix by inverting the tubes. Try to avoid bubble formation during the mixing process.
• Based on testing, this product has shown no significant impact on performance after undergoing 10 freeze-thaw cycles. However, it's still advisable to minimize repeated freeze-thaw cycles, as they might cause a decrease in product performance.
• Isothermal amplification is an extremely sensitive detection method, so it's recommended to conduct testing in a standard PCR laboratory. The setup for isothermal amplification reactions should be performed in an area that minimizes the risk of contamination from various potential amplification products. Although this assay kit uses UDG enzyme for contamination prevention, please do not open the PCR tube cap. After isothermal amplification, seal the amplification products as required to prevent highly concentrated amplification products from contaminating the laboratory environment due to aerosols and other factors.
• This product contains a color indicator. If there is a significant color difference between the sample added and the colors corresponding to negative and positive samples before the reaction, it might be necessary to dilute the sample, treat it with a DNA extraction kit, or adjust the sample's pH to near-neutral.
• While this assay kit can detect common Mycoplasma species responsible for cell contamination, for important cells suspected of potential contamination by Mycoplasma species that cannot be identified, it is recommended to use two or more methods based on different principles to detect simultaneously, effectively avoiding false positives and false negatives.
• This isothermal amplification system requires high concentrations of divalent ions; the sample must not contain high concentrations of metal ion chelators like EDTA.
• It is recommended to use filter-tipped pipette tips to set up the isothermal amplification system, which can maximize the prevention of contamination-induced false positives.
• This product is intended for scientific research by professionals only. It must not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in residential settings.
• For your safety and health, please wear lab coats and disposable gloves during operation.

Only for research and not intended for treatment of humans or animals

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