Mycoplasma Detection Kit (qPCR)
Based on 1 Customer Validation
MCE Mycoplasma Detection Kit (qPCR) is a diagnostic kit that utilizes the real-time quantitative PCR (qPCR) method to detect mycoplasma contamination in biological materials such as cultured cells.
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Storage :
-20°C, 2 years
Keep away from light and avoid repeated freeze-thaw cycles.
Shipping with dry ice.
- Manual (6348KB)
- COA
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
Mycoplasma are small, structurally simple bacteria which lack a cell wall and represent one of the most prevalent and serious sources of cell line contamination. Mycoplasma contamination is invisible, as it causes no discernible change in turbidity or pH even at densities as high as 108 cells/mL, making it particularly insidious. Mycoplasma infections are highly toxic and can induce a range of changes in cell cultures, including reduced growth rates, abnormal morphology, chromosomal abnormalities, and metabolic disturbances. These changes can lead to slowed cell growth, aberrant differentiation, or even cell death, severely disrupting normal cell functions. Mycoplasma contamination not only significantly affects the reliability, repeatability, and consistency of experimental results but can also lead to experimental failure. Therefore, timely and accurate detection of mycoplasma contamination is crucial for ensuring the quality of cell cultures and the accuracy of experimental outcomes.
MCE Mycoplasma Detection Kit (qPCR) is a diagnostic kit that utilizes the real-time quantitative PCR (qPCR) method to detect mycoplasma contamination in biological materials such as cultured cells. The kit includes qPCR premix and specific primer-probe targeting the conserved regions of the mycoplasma, enabling the specific amplification and detection of mycoplasma DNA.
Features of MCE Mycoplasma Detection Kit (PCR)
1. Optimized Premix Formula: The specially optimized reaction buffer introduces dUTP/UDG anti-contamination systems, which can degrade U-containing ssDNA and dsDNA before reverse transcription, effectively preventing cross-contamination caused by PCR products.
2. High Sensitivity: For extracted mycoplasma DNA in samples, the detection limit reaches as low as 10 CFU/mL.
3. Specific Fluorescent Probes: Utilizes FAM and VIC fluorescent probes for the specific amplification of mycoplasma and internal controls, respectively.
4. Broad detection range: Capable of detecting a wide range of species under the class Mollicutes, including various Mycoplasma spp., Acholeplasmaspp. and Spiroplasmaspp.), meeting the requirements of nucleic acid amplification techniques (NAT) in mycoplasma detection in EP 2.6.7 and JP G3.
Protocol
General Protocol
1. Preparation
Thaw all reagents completely on ice or at 4°C, mix gently, and briefly centrifuge. Protect PCR Master Mix from light. Include test samples, a Positive Control, and a no-template negative control in each run.
Internal Control (IC) is used to monitor PCR inhibition and reaction performance. If IC has already been added during sample DNA extraction, replace the IC volume in the qPCR mixture with an equal volume of Mycoplasma-Free Water.
2. qPCR reaction system
| Component | Per reaction |
|---|---|
| PCR Master Mix | 8 μL |
| Primer & Probe Mix | 1.5 μL |
| Internal Control (IC) | 0.5 μL |
| Test sample / Positive Control / Mycoplasma-Free Water | 20 μL |
| Total volume | 30 μL |
Mix gently, dispense into qPCR tubes or a 96-well plate, seal, and briefly centrifuge while avoiding bubbles.
3. qPCR cycling program
| Step | Temperature | Time | Cycles | Signal acquisition |
|---|---|---|---|---|
| UDG pretreatment | 25°C | 10 min | 1 | No |
| Initial denaturation/enzyme activation | 95°C | 10 min | 1 | No |
| Denaturation | 95°C | 15 sec | 45 | No |
| Annealing | 62°C | 30 sec | 45 | No |
| Extension | 72°C | 90 sec | 45 | Yes |
4. Fluorescence channels and interpretation
Use the FAM channel for the mycoplasma target and the VIC channel for the Internal Control. The negative control should show no valid amplification in FAM, while the Positive Control should show a normal FAM amplification curve. A valid FAM amplification curve in a test sample indicates mycoplasma-positive material. If FAM is negative and VIC is normal, the sample is negative. If neither FAM nor VIC amplifies normally, PCR inhibition or an experimental problem should be suspected and the sample should be retested.
Storage
-20°C, 2 years
Keep away from light and avoid repeated freeze-thaw cycles.
Shipping with dry ice.
Components
| Components | HY-K0553-50 rxns |
|---|---|
| PCR Master Mix | 400 μL |
| Primer & Probe Mix | 75 μL |
| Internal Control (IC) | 500 μL |
| Positive Control (PC) | 200 μL |
| Mycoplasma-Free Water | 500 μL |
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