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Asses mycoplasma contamination


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6 replies to this topic

#1 dvddecarvalho

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Posted 26 February 2009 - 11:45 AM

Dear all,
I need some help.
How can I asses for micoplasma contamination in my cell culture?
Are there any kit for that? Can I do PCR?

Thanks,

#2 hodzic

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Posted 26 February 2009 - 01:04 PM

There is kit by Applied Biosystems "MicroSEQ® Mycoplasma Detection Assay". It is a real-time PCR based kit (multiplex) that is able to detect 90 different Mycoplasma species. In addition you can use ELISA, but requires monoclonal antibodies, as well as PCR-ELISA.
Good luck

#3 DRT

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Posted 26 February 2009 - 03:12 PM

In the absence of PCR facilities :D I use a kit from Cambrex called Mycoalert. It is very easy to use (fluorescence detection) and allows me to screen all of my cell lines in one hit on a 96well plate. The down side is that it is costly, which in effect means that I do not test as often as I ought.

#4 bob1

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Posted 26 February 2009 - 03:23 PM

The three most common methods are fluorescent microscopy, ELISA, and PCR. There are kits available for all three.

However, and this is important, the gold standard is to do fluorescent micorscopy. It is the only reliable method as both the PCR and ELISA are prone to both false positives and false negatives and are not very sensitive. The ATCC has moved away from using PCR for these reasons and has switched back to microscopy.

#5 dvddecarvalho

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Posted 27 February 2009 - 06:15 AM

Ok,
Thank you all.
Can I just stain nucleus with flourescent labels or similars?

Cheers.

#6 rhombus

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Posted 01 March 2009 - 02:32 AM

Dear all,
I need some help.
How can I asses for micoplasma contamination in my cell culture?
Are there any kit for that? Can I do PCR?

Thanks,


How many times do I have to post on this forum about the right way to test for mycoplasma contamination!!!!!!!.

"Can I do PCR"....the answer is NO NO NO.

The ATCC, the world's largest supplier of cell lines, DO NOT NOW USE PCR AS A DETECTION METHOD....WHY?.....because it is so unreliable. PLEASE CHECK THEIR WEBSITE.

The 2 FDA approved methods (PCR is NOT FDA approved) are Hoescht Staining in combination with the Agar Growth Test.....when you have a vaccine or drug, these 2 methods are used, NOT NOT NOT PCR.

You should regularly:

TEST all cells, both cell lines and primaries
Quarantine all cells that come into your lab...apart from those from the ATCC/ECACC
DO NOT REGULARLY USE ANTIBIOTICS in your media....these mask cryptic contaminations.
Use quality FCS/FBS that has been tested the right way.

Please accept this advice in the manner it is given.....30 years of cell culture experience in a group that has had many Nature and Science papers, and is a world leader in their particular field of research.


Kindest regards

Rhombus

#7 dvddecarvalho

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Posted 01 March 2009 - 06:48 AM

Ok Rhombus,
Thanks for your reply.

I am just new here and I didn`t see your previous posts about mycoplasma contamination.

Dvd




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