You should have an internal control in each sample in order to compare them with the positive control.

Maybe the band you see is the primer's band, which sometimes is shown. But you have to pay attention to the band at 191pb, negative result, and the one at 270pb, positive result. But always compare with your positive and negative controls.

Is it possible that you have got PCR product contamination in the laboratory? If the mycoplasma PCR product from a previous reaction gets out and about and onto pipettes, tubes and surfaces it will make everything mycoplasma PCR positive. If this is the case you could try using Uracil N-glycosylase (UNG) to prevent PCR product carryover. Also you can DNA zap everything to kill DNA off all the surfaces.

I also thought the same thing. It is quite possible that they have cantaminations in their laboratory. It will think decontaminate your pipettes, bench and surfaces. The method that you suggest Hanna (DNA zap) works well.

Yes I suspected cross contamination via pipettes could be the issue - I have deconned everything and will use filter tips from now on for this PCR reaction.

Marina, you're right about the primer band but I can definitely distinguish the smaller primer dimers from the ~300bp amplicon and the amplicon is in all of the controls as well. It's also possible that the PCR reagents are contaminated so I've got fresh ones of these as well.

Thanks everyone for your input - much appreciated.

When getting rid of PCR product contamination go for new everything that you possibly can - even your lab coat. Also, be aware that the decontamination must be good enough to really remove DNA from things you can't replace. A wipe with ethanol will not remove DNA.

I do not know, how was your primers designed?, But I recommend that you sequence the band appears when you use sterile water, is likely to be a primers self-amplification, on the other hand, I use filtered water by nitrocellulose and autoclaved, to avoid any contamination resistant to high temperature.

The time taken to evaluate whether you have cross contamination and where it has come from costs a lot more than getting experts in setting up the tests. The contracting company we use also have no self interest in the tests themselves. We also rely on the soft agar assay which is:

The most sensitive test

You normally get a positive within 3 days of the test initiation

Is FDA approved and has been the excepted method for the past 40 years

And is more reliable than the PCR test which the ATCC (largest commercial supplier of cell lines in the world) no longer use as it is unreliable.

I understand that if you have a 100 cell lines to test then this would cost alot but generally speaking this is not the case in most research groups

Hello Domina,

We have had such results in our lab and the route cause for seeing bands where they are not supposed to be was cross-contamination of PCR reagents, including the water. This is very common in a lab like ours where many different people share the reagents to carry out the test at different times. It will be very hard to tell where the contamination came from.

The solution as echoed above, is to make new aliquots of everything: Taq, dNTPs, Buffer, MgCl2, Primer mixes and Water. Use filter tips to eliminate any possible cross-contamination and ensure you disinfect your pipettes thoroughly; if possible, use only dedicated ones for the procedure.

Since the contamination can occur again in future, ensure you make only small aliquots of everything to avoid fear of waste in case trouble occurs. Mycoplasma PCR is quite specific and it should not give you false positives.

Getting false positive signals with PCR mycoplasma detection is a general problem. With PCR you do not detect intact organisms, you only detect - by definition - (mycoplasmal) DNA, which for example, could be present in FBS.

Therefore, it is recommended to use methods that detect intact, living mycoplasma species. This can be done, for example, with the MycoAlert-Kit from LONZA, that measures mycoplasma-specific kinase activities. Thus, measuring enzyme activities strongly indicates the presence of living cells.

Gerhard Gstraunthaler

Innsbruck Medical University

Thanks for that information Gerhard - good point - I was unaware that Lonza had such a kit. I'll look into it.

Mina

I agree with Michael. That´s the reason why it´s good to outsource testing. Try Multiplexion. They can do Authentication and contamination testing (Mycoplasma, interspecies and viral contaminations) at very affordable prices. http://www.multiplexion.de/en/

I m facing the same problem