Cell culture contamination control: Detection

Venor®GeM qEP

Mycoplasmas are specifically detected by amplifying a highly conserved rRNA operon, or more specifically, the 16S rRNA coding region in the mycoplasma genome. The mycoplasma-specific amplification is detected at 520 nm (FAM™ channel).The kit includes primer and FAM™ labeled probes which allow the specific detection of all Mollicutes species so far described as contaminants of cell cultures and media components. Eukaryotic DNA is not amplified by this primer/probe system. False negative results due to PCR inhibitors or improper DNA extraction are detected by the internal amplification control which can be added to the PCR master mix. The amplification of the internal amplification control is detected at 610 nm (HEX™ channel).

Important note:

Please see the note section for important information on the use of the supplied positive control

Type of PCR

TaqMan® basedqPCR Assay with FAM™ and HEX™ labeled probes

Recommended Use / Scope

Venor®GeM qEP is used for direct detection of Mollicutes (Mycoplasma, Acholeplasma, Spiroplasma) contamination in cell cultures and cell media components.

Kit Components

lyophilized primer/nucleotide/probe/polymerase/mix in aliquots of 25 tests rehydration bufferlyophilized internal amplification control lyophilized positive control PCR grade water

Package Sizes

• Cat. No. 11-9025 25 tests
• Cat. No. 11-9100 100 tests
• Cat. No. 11-9250 250 tests

Result Evaluation

cycler based, real-time PCR

Required Consumables

PCR reaction tubes Optional for process validation and EP 2.6.7 compliant testing: Internal Control DNA extra (4 vials for 300 µl each of internal amplification control; Cat. No. 11-9905) 10CFU™ Sensitivity Standards available for all EP2.6.7 listed mycoplasma species

Required Lab Devices

qPCR cycler with FAM™ and HEX™ filter variable microliter pipettes benchtop centrifuge for 1.5 ml reaction tubes

Shelf Life and Storage

Components can be stored at2 to8 °C for at least 12 months. After rehydration the reagents must be stored at -18 °C.

EP 2.6.7 Compliance

Yes.Please note that validation data are provided for information purpose only. EP 2.6.7 clearly states “Where commercial kits are used …, documented validation points already covered by the kit manufacturer can replace validation by the user. Nevertheless, the performance of the kit with respect to its intended use has to be demonstrated by the user (e.g. detection limit, robustness, cross-detection of other classes of bacteria.”. Please feel free to contact us if you need further assistance.

Fig. Amplified dilution series of Mycoplasma fermentans, performed on a Corbett RotorGene®6000.

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Venor^®GeM qEP protocol

Quick Visual Protocol

Please note: all protocols off site are the responsibility of the products supplier

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Important notes on the use postive controls in the VenorGem™ QEP kit

The Positive Control comes in one vial. After rehydration, this reagent is stable for frequent thawing/freezing cycles. However we strongly recommend that customers use DNase-free pipette tips and a clean surrounding. One bacteria particle contaminating the reagent is able to digest all the DNA during the thawing phase.

Aliquoting the rehydrated Positive Control can help avoid this as long as the following criterion are met: 1. Make aliquots in PCR tubes (they are usually clean, DNase-free, low-binding tubes, low surface area). 2. Never ever use reaction tubes or other larger vials, as they absorb the minute amounts of DNA 3. keep the thawing time short. Freeze asap.

Required consumables:

PCR reaction tubes

Optional for process validation and EP 2.6.7 compliant testing:

Internal Control DNA extra (4 vials for 300 µl each of internal amplification control; Cat. No. 11-9905)

10CFU™ Sensitivity Standards available for all EP listed mycoplasma species

Required lab devices:

qPCR cycler with FAM™ and ROX™ filter

variable microliter pipettes

benchtop centrifuge for 1.5 ml reaction tubes

Shelf life and storage:

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200