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Cat. No.: MQPDK-100 (for 100T)

Cat. No.: MQPDK-500 (for 500T)

Description

Mycoplasma qPCR Detection Kit is a kit that allows for the high-sensitivity detection of mycoplasma contamination in cultured cells and other biological materials using probe-based qPCR (Quantitative PCR) without the need to extract DNA or RNA from the samples. It can directly use cell culture supernatant or serum samples for detection. This kit can quickly, effectively, and sensitively detect mycoplasma contamination.

Mycoplasma is the smallest and simplest prokaryote. Mycoplasma has the following characteristics:

• Mycoplasma lacks a cell wall structure, so many common antibiotics targeting the cell wall, such as penicillin or β-lactam antibiotics, are ineffective against mycoplasma.
• Mycoplasma sizes range between bacteria and viruses, approximately 0.2-0.8μm, so some mycoplasma can pass through 0.22μm filters, making conventional filtration ineffective.
• Many mycoplasmas rely on the host for nutrients due to their limited biosynthetic capabilities, often adhering to or dispersing between cell surfaces.

These characteristics of mycoplasma pose a risk of contamination during cell culture, and mycoplasma contamination has become a global issue. Mycoplasma contamination can severely affect cell status, altering gene expression and metabolic characteristics, leading to slowed cell growth, abnormal differentiation, and death, significantly impacting cell function. These factors can seriously affect the reliability, reproducibility, and consistency of experimental results, making the detection of mycoplasma contamination very important.

Bacterial, yeast, or fungal contamination in cell culture can be seen under an optical microscope, but mycoplasma contamination is usually invisible under an optical microscope and must be detected using specific methods. Common methods for detecting mycoplasma contamination include mycoplasma isolation culture, ELISA, luminescence-based biochemical detection, and DNA fluorescence staining detection. Most of these methods involve relatively cumbersome steps, low sensitivity, or long detection times. In contrast, qPCR is convenient and straightforward, and after PCR amplification, it can determine the presence of mycoplasma contamination without the need for electrophoresis analysis.

This kit uses specific primers and fluorescent probes (FAM-labeled) designed based on the conserved 23S rRNA operon coding region sequence of the mycoplasma genome. It can directly use cell culture supernatant or serum as a template for detection. This kit can amplify and detect 39 types of mycoplasma. It specifically amplifies mycoplasma DNA and does not amplify bacterial, fungal, or eukaryotic cell DNA.

The kit provides all the universal components needed for qPCR experiments, including Fast Taq DNA Polymerase, primers, probes, PCR Buffer, dNTPs, stabilizers, Nuclease-free Water, and magnesium ions. The kit also includes Internal Control DNA (VIC-labeled) to be added to each PCR reaction to check for PCR inhibitors. Additionally, the kit provides a Positive Control to verify that the kit itself is working correctly.

This kit is suitable for fluorescent quantitative PCR instruments with FAM and VIC detection channels, such as ABI’s 7500, 7900, StepOne, QuantStudio series, Roche’s LightCycler480, SmartCycler, Bio-Rad’s CFX96, Chromo4, Opticon 2, iCycler IQ, iQ5, Qiagen’s Rotor Gene 6000, and Shanghai Hongshi’s SLAN-96P.

The kit has been optimized to directly detect mycoplasma in cell culture supernatant, serum, and other samples without the need for separate DNA extraction. The detection method used in this kit is highly sensitive, with a detection limit calculated from the positive control of approximately 2-20 copies/μl.

If mycoplasma contamination is detected, it is recommended to replace the contaminated cells with uncontaminated ones for culture. If it is necessary to prevent or remove mycoplasma, specialized mycoplasma prevention or removal reagents can be used, such as Mycoplasma Removal Reagent, Mycoplasma Removal Reagent Plus, Mycoplasma Prophylactic and Elimination Reagent I, Mycoplasma Prophylactic and Elimination Reagent II, and MycoErase™ Mycoplasma Remover.

For routine 96-well plate qPCR detection (recommended reaction volume of 20µl) or 384-well plate qPCR detection (recommended reaction volume of 10µl), the small package of this product can perform 100 and 200 tests respectively, while the medium package can perform 500 and 1000 tests respectively.

Components

• Probe qPCR Mix (2X)
• Primer Probe Mix
• Internal Control DNA
• Positive Control
• Nuclease-free Water

Storage

Store at -20°C, valid for one year. Probe qPCR Mix (2X) and Primer Probe Mix should be stored at -20°C and protected from light. Avoid repeated freezing and thawing.

Precautions

• Ensure the reagent is completely thawed before use. Mix gently by inverting the tube, avoiding the formation of bubbles.
• Probe qPCR Mix (2X) and Primer Probe Mix contain fluorescent dyes. Avoid strong light exposure when storing the product or setting up PCR reactions to prevent fluorescence quenching.
• Testing has shown that repeated freezing and thawing up to 10 times does not significantly affect the product’s performance, but it is still recommended to avoid repeated freezing and thawing as it may degrade the product’s performance.
• qPCR detection is highly sensitive. Perform the detection in a standard PCR laboratory. The PCR setup area should be free from potential amplification product contamination. Do not open PCR plates or tubes in the PCR setup area. Seal PCR products and handle them according to post-amplification requirements to avoid contaminating the lab environment with high-concentration PCR products.
• This kit can detect common mycoplasma species contaminating cells. For important cells suspected of contamination by unidentified mycoplasma species, it is recommended to use our Luminescent Mycoplasma Detection Kit for dual detection. Strict mycoplasma detection requires using two or more different methods to effectively avoid false positives and false negatives.
• Use filter tips when preparing the PCR system to minimize the risk of contamination leading to false positives.
• This product is for scientific research use only by professionals and is not intended for clinical diagnosis or treatment, food, or drugs, and should not be stored in ordinary residences.
• For your safety and health, wear a lab coat and disposable gloves when handling.

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

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