Multiplex Cell Contamination Test

The offered service is designed to detect 28 potential cell culture contamination such as

  • Mycoplasma (Identification of 14 specific strains: M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. genitalium, M. salivarium, M. synoviae, M. pirum, M. gallisepticum, M. pneumoniae, M. yeatsii, Spiroplasma citri and Acholeplasma laidlawii)
  • Squirrel Monkey Retrovirus (SMRV) and Epstein-Barr virus (EBV)
  • Cross-contamination with cells of following origin:
    • Human, Monkey, Mouse, Rat, Chinese hamster, Syrian hamster, Dog, Cat, Rabbit, Guinea pig, Pig, Drosophila

Detection of more than 100 additional Mycoplasma strains by proprietary universal probes. This service detects all Mollicutes mentioned in the European Pharmacopoeia 2.6.7. Identification of Mycoplasma species enables tracing of Mycoplasma contamination sources.

 

Test specifications

Detection of contaminations is done with specific primer sequences in a multiplex PCR targeting viral, bacterial and cellular genome regions, respectively, and subsequent hybridisation using specific oligonucleotide probes (licensed from the DKFZ, patent pending). Even low levels of contamination are detected before they become more widespread. An internal DNA quality control as well as several positive and negative PCR controls are warranting a service at highest standards.

 

To be done by the user

  • Cell line lysate preparation according to the lysate protocol
    • required amount: >15µL
    • tubes: 1.5 ml SafeLock tubes (please do not use screw caps and Parafilm)
  • Complete the
  • Label sample tubes with:
    • the specific sample name as provided in the confirmation Email
  • Ship samples in a padded envelope WITH a copy of the confirmation Email to:

Multiplexion, c/o DKFZ, Dept. F020, INF 242, D-69120 Heidelberg, Germany

 

To be done by Multiplexion

  • Multiplex PCR-based amplification of contaminants
  • Detection of PCR products by specific oligonucleotide probes
  • Transfer of result report to customer by Email (the report can be used as certificate for granting agencies and journals)

 

Turnaround Time

Samples submitted are run in even calendar weeks, with results emailed usually by end of the week.

  • Customers from Heidelberg should drop off samples in the building INF242 between 9AM and 12AM on Monday for inclusion in that week's processing schedule.
  • External customers should ship samples to the lab to be received by Monday for inclusion in that week's processing schedule. Samples received Tuesday through Friday of any given week will be held and processed the following testing week.

 

How to cite the service?

For Grant and Paper Submissions

 

What to do with contaminated cell lines?

  • Different contaminations require different actions. An overview can be found here.
  • You may use the following protocol to eliminate Mycoplasma from your precious cell lines. The protocol was kindly provided by U. Koch (DKFZ, Heidelberg).
  • Problems with contaminations? Our check list for sterile work may help.

 

Please note: This service is FOR RESEARCH USE ONLY. Do not use in diagnostic procedures.

Offer for First Time Users

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Lysate preparation protocol

Download the detailed lysate
preparation protocol.

 

Technical Note

Analytical sensitivity of the
Multiplex cell Contamination Test.

 

Technical Note

Specificity of the
Multiplex cell Contamination Test.

 

Check list for sterile work

Download Multiplexion´s
check list for sterile work.

 

Mycoplasma

The PCR primer sets used in McCT are directed to the highly conserved 16S rRNA coding region in the Mycoplasma genome. This allows for amplification of all Mycoplasma and Acholeplasma species usually encountered as contaminants in cell cultures. Moreover, the McCT assay determines the genotype of Mycoplasma and Acholeplasma species by specific hybridisation, such as M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. synoviae, M. pirum, M. gallisepticum, M. pneumoniae, M. yeatsii, and Acholeplasma laidlawii.

Mycoplasma specification enables the identification of contamination sources:

  mycoplasma species

EBV

The Central Commission for Biological Safety (ZKBS) at the Robert-Koch Institute (RKI) has assessed the risk for cell lines which were immortalised with Epstein-Barr Virus (EBV) also known as Human Herpes Virus 4 (HHV4). Cell lines that are immortalised due to an EBV infection must be used in S2. The cell lines MEC-1/MEC-2, EHEB, Granta 519, JVM-2/JVM-3 and LCL-WEI were assigned to S2. EBV positive cell lines are allowed in S1 only if a sequence defect of a gene essential for lytic replication is detected.

Multiplexion´s Multiplex Cell Contamination Test tests for the EBV DNA polymerase processivity factor in ORF59.

For further information:

  • "Stellungnahme der ZKBS zur Risikobewertung von etablierten humanen Zelllinien, die mithilfe des Epstein-Barr virus (EBV) immortalisiert wurden" (German only)

SMRV

The Central Commission for Biological Safety (ZKBS) at the Robert-Koch Institute (RKI) has recommended testing of cell lines for the Squirrel Monkey Retrovirus (SMRV) that is able to infect a broad range of cell lines. Like all retroviruses, SMRV poses a risk for insertional mutagenesis. In addition, SMRV contamination may lead to a complementation of recombinant, replication-deficient retroviruses in cell lines. Contaminated cells should either be inactivated or used in S2 only. The ZKBS further asks to get notified if a cell line is tested SMRV positive.


For further SMRV information

  • Stellungnahme der ZKBS zur Kontamination von Zelllinien mit dem Squirrel Monkey Retrovirus (German only)