Protecting your cultures

Tissue cell cultures can become contaminated by mycoplasma species without you knowing, which can be a serious problem. We offer mycoplasma testing using PCR based kits:

                MycoDtect kit from Greiner Bio-One and

                EZ-PCR from Biological Industries USA. 

Screening for mycoplasma contamination is recommended on a regular basis, and before cell banking and cryopreservation.

Johns Hopkins Nucleic Acid Technologies Lab

offers two different kits to better fit your needs, below are the features for each kit. Please take the time to read carefully!


MycoDtect TM from Greiner Bio-One

DNA is extracted from the cell culture supernatant using a DNEasy Blood & Tissue kit automated with a QIAcube (Qiagen). A MycoDtect internal control is added prior to isolation to monitor the DNA extraction process and template use for the amplification. Using a highly conserved Cy5 fluorescent-labeled primer pair the DNA is polymerase chain amplified (PCR) for the 16S-23S rRNA intergenic spacer region of any mycoplasma species present. A PCR control within the PCR MasterMix allows for validation of the PCR. The labeled products are then hybridized to complementary sequences on the MycoDtect chip. Each mycoplasma species, the universal probe, and the internal and PCR controls are each detected by five measuring points on the chip. Non-specifically bound probes are removed by washing. The bound labeled probes are detected by stimulation with monochromatic light and analyzed using CheckReport software. The MycoDtect assay will detect both viable and non-viable mycoplasma, it will not distinguish between the two. 

MycoDtect was tested by Greiner Bio-One with a broad range of closely related bacteria. None of the tested bacteria showed a signal with the specific probes, but some hybridized to the universal probe. For a detailed list, please  inquire.

Advantages

This kit uses microarray technology to identify 9 mycoplasmas at the species level plus a universal probe to detect Mycoplasma. This is helpful for troubleshooting source of contamination.

Disadvantages

Longer turn-around-time. Time- consuming, laborious. More expensive in comparison with EZ-PCR kit.

Suggestion to consider: This kit offers identification of the 9 most common Mycoplasma at species level. If knowing the source of a contamination is key for you. Consider selecting this kit.

EZ-PCR Mycoplasma Detection Kit from Biological Industries USA

DNA is extracted from the cell culture supernatant using a DNEasy Blood & Tissue kit automated with a QIAcube (Qiagen). After extraction, DNA quantification is done to avoid false negatives.

This assay uses PCR to amplify a target sequence within the highly-conserved 16S rRNA coding region of the mycoplasma genome.

A highly-specific and optimized primer set allows the detection of over 90 species of Mycoplasma, Acholeplasma, and Spiroplasma. The kit includes two positive controls, the internal amplification control and the positive DNA template control. The first one is a plasmid containing a non-mycoplasma-specific DNA sequence and it is simultaneously amplified in all PCR reactions. The second positive control is a highly conserved, mycoplasma-specific DNA sequence. PCR products are run in agarose gel.

For a detailed list, please  inquire. 

Advantages

Faster turn-around-time, less labor intensive and less expensive than MycoDtect.

Disadvantages

Results are negative or positive with no speciation. 

Suggestion to consider: If you only want to know if your sample is negative or positive. This kit offers this results.

Sample Preparation

Samples should be submitted from growing culture or as frozen ampoules. We accept cells, cell culture supernatant or frozen stocks.

Instructions for Growing Cultures: We recommend passage of cells in antibiotic-free media twice. Do not change the medium or dilute with fresh medium during the last 72 hours before testing. Cells should be 50-80% confluent. Submission of cells at higher confluency may inhibit the PCR assay due to the accumulation of PCR inhibitors. Samples grown in antibiotics or given fresh medium prior to testing may have artificially low mycoplasma levels if present or may give a false negative result. 

Cell Pellets: 10E5 to 10E6 cells 

Adherent cells: Use a cell scraper to dissociated cells from the substrate. Do not treat with trypsin or other enzymatic agents for dissociation. Collect 5 to 7 ml of medium in a T25 flask or 8 to 10 ml in a T75 flask. Centrifuge to pellet cells, then remove supernatant. 

Suspension culture: Count cells. Remove suspension culture to a 15ml conical tube. 

Supernatant: Collect 5-10ml of supernatant medium of adherent cells or suspension cultures and submit in a 15ml conical tube. Paraffin the top if shipping. 

Frozen Stock: Submit ampoule.

Turnaround Time

Samples submitted for mycoplasma testing are run once a week on Wednesday, with results emailed Friday of the same week or following Monday. Turnaround time may vary depending on cumulative sample submission.

Johns Hopkins Customers

Johns Hopkins customers should drop off samples in Blalock 1004 by 4PM on Tuesday for inclusion in that week’s processing schedule. Samples received after 11AM Wednesday through Friday of any given week will be held and processed the following week. JH Customers: Drop samples off in Blalock 1004 with the GeneSifter order number, Monday – Friday from 9 AM – 4:00 PM

External Customers

External customers should ship samples to the lab to be received by 11AM Wednesday for inclusion in that week’s processing schedule. 

GRCF, Johns Hopkins University, 725 N. Wolfe Street, PCTB G20, Baltimore, MD 21205

Phone (443) 287-7948

Email the courier tracking number and request form to  FAF@jhmi.edu.


Pricing

MYCOPLASMA TEST only

EZ-PCR Kit:

$70/sample.

MicoDtect Kit:

$80/sample. 

Non-Academic customers will be charge %20 more for a service.


EZ-PCR Mycoplasma test and Str profile analysis

GenePrint 10: $170/sample (Standard Service).

GenePrint 10: $215/sample (Standard Service plus Profile Search).

PowerPlex 18D: $200/sample (Standard Service).

PowerPlex 18D: $245/sample (Standard Service plus Profile Search).

NOTE: DNA isolation fee is included in in the mycoplasma test.

Non-Academic customers will be charge %20 more for a service.

Mycodtect mycoplasma test and str profile service

GenePrint 10: $180/sample (Standard Service).

GenePrint 10: $225/sample (Standard Service plus Profile Search).

PowerPlex 18D: $210/sample (Standard Service).

PowerPlex 18D: $255/sample (Standard Service plus Profile Search).

NOTE: DNA isolation fee is included in in the mycoplasma test.

Non-Academic customers will be charge %20 more for a service.

How to order

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