Problems with MycoAlert (Lonza) - (May/18/2012 )
Hi everyone,
We are currently testing our cell lines with MycoAlert (Lonza) after a recent contamination was found. We have decontaminated the labs. However, I'm having trouble interpreting the results of the test and hope someone out there has experience of this! So, Lonza state that the calculated ratio means <0.9 negative; 0.9-1.2 borderline and <1.2 positive for mycoplasma. Now these are the ratios we have calculated
Cell line 1 Reading A 1.00 Reading B 2.14
Cell line 2 Reading A 22.9 Readinb B 11.3
Neg ctrl Reading A 0.09 Reading B 0.20
Media Reading A 1.14 Reading B 1.6
Does anyone know if these readings are correct? The ratio for cell line 2 seems rather large and from these results it looks like the fresh media is positive but it came out and unopened bottle! We have used DMEM with phenol red - would this interfere with the readings?
Any help would be greatly appreciated!
Sam
Potentially phenol red could intefere with the luminescence, but I don't know how much. Do you prepare the medium in the lab or buy it pre-made? If you make it up yourself, mycoplasma can easily pass through 0.2 um filters and possibly even 0.1 um filters.
Dear Sam80,
Please put my name through the search engine to find my comments on mycoplasma testing. Ask Lonza if the USA Food and Drug Administration (FDA) have validated the test? Also ask them why the ATCC and the Health protection agency (HPA) use solid agar in combination with Hoescht staining and why the ATCC NO LONGER USE PCR AS A TEST FOR MYCOPLASMA'S.
I cannot believe that this is still an issue
Do the right test
Kindest regards
Uncle Rhombus
In regard to comment by Rhombus:
As of 2010 the FDA do actually allow mycoplasma testing by PCR (see attachment). Indeed, for some applications PCR methods are mandated because of non-cultivatable mycoplasma strains (eg cultivar-a of M.hyorhinis).
PCR is also a permissible mycoplasma screening method in Europe (European Pharmacopoeia).
Not that Rhombus needs me to defend him, but he didn't actually say that the FDA hadn't approved PCR as a testing method (only that ATCC no longer uses it, for good reason).
Also, in the document you provided:
"PCR-based assays may be used to detect mycoplasma, provided that such an assay can be shown to be comparable to the agar and broth media procedure and the indicator cell culture procedure. "
The use of PCR to detect mycoplasma comes with the caveat that it must be as good as the culture methods. Don't know if anyone has been able to show that.
Leelee:
You are quite right. The FDA does require all PCR-based methods to be adequately "validated" using traditional (culture etc) comparators. Some PCR methods have been fully validated and are FDA approved ( see Charles River web site).
As far as the ATCC, it is interesting to note that they are now selling their improved "Universal Mycoplasma Detection Kit" (PCR-based. cat 30-1012K). One would hope that since they claim it is for screening cell cultures that it is useful for this purpose.
I agree that PCR screening can be problematic, but for routine screening in research labs, it is simple, cheap and certainly better than ignoring the problem entirely!
klinmed on Wed May 30 12:23:20 2012 said:
Leelee:
You are quite right. The FDA does require all PCR-based methods to be adequately "validated" using traditional (culture etc) comparators. Some PCR methods have been fully validated and are FDA approved ( see Charles River web site).
As far as the ATCC, it is interesting to note that they are now selling their improved "Universal Mycoplasma Detection Kit" (PCR-based. cat 30-1012K). One would hope that since they claim it is for screening cell cultures that it is useful for this purpose.
I agree that PCR screening can be problematic, but for routine screening in research labs, it is simple, cheap and certainly better than ignoring the problem entirely!
Dear Klinmed,
Please refer to the Health Protection agency website below that I cut and pasted 20 minutes ago:
http://www.hpacultures.org.uk/services/mycotestingeradication/mycoplasmatesting.jsp
Please refer to the table on testing, sensitivity, speed, range and FDA approval..........they seem to disagree with you???????????
Having been responsible for mycoplasma testing in cell cultures for the past 35 years, I have seen tremendous advances in culture isolation, the "gold standard" for testing. I agree that for some time certain mycoplasma's could not be grown, but advances in media have almost circumvented this. Hoescht staining in combination with culture will pick up 100% of all known mycoplasma's
However I have seen too many PCR false negatives over the past few years and do not trust PCR. As I stated in my other post nor do the ATCC trust PCR either......that is why they no longer use it as a mycoplasma test (see their website)
As the American tissue culture collection are the LARGEST COMMERCIAL SUPPLIER OF CELL LINES AND THEREFORE THE WORLD AUTHOURITY IN THESE MATTERS whose advice should Protocol online users trust ??????
Kindest regards as always
Uncle Rhombus
klinmed on Wed May 30 12:23:20 2012 said:
As far as the ATCC, it is interesting to note that they are now selling their improved "Universal Mycoplasma Detection Kit" (PCR-based. cat 30-1012K). One would hope that since they claim it is for screening cell cultures that it is useful for this purpose.
I agree that PCR screening can be problematic, but for routine screening in research labs, it is simple, cheap and certainly better than ignoring the problem entirely!
They do sell this kit, yes, but they don't use it themselves.
I suspect that the demand for a quick and cheap testing method is high, so it makes sense to provide something like that.
I whole heartedly agree with you that screening by PCR is better than doing nothing and ignoring the potential problem (as the majority of research labs do). However, I do think it is important (particularly in a forum such as this) to make people aware of the gold standard for mycoplasma testing, and to point out the pitfalls of using other techniques.
At least then they can make an informed decision on what they think is best for their situation.
Hi Sam,
To reply to your original thread, yes, i am seeing the same thing with this kit. I have just completed a piece of work which compares our two in-house assays (Toll like receptor assay and agar culture isolation) with the MycoAlert kit and also sent samples off for external verification to RADIL for PCR testing. So the MycoAlert kit has been trialled against three other completely different methods and we have seen a very high number of suspicious samples thrown up using this kit. We've also had a few false positives and one false negative! It did however manage to pull out all of the true positives which were substantiated with the other three tests.
But in my finding the data you posted is similar to what i saw in that the MycoAlert seems to be over sensitive, which to be honest considering how it's supposed to work i can't figure out. I'm going to be follwoing this up with Lonza though to see if they can shed any light on it.
Best wishes
Clare
Clare_williams1983, did you ever manage to get an answer out of Lonza about the accuracy and precision of mycoalert?
For my 2 cents I think it's very important that you use a sensitive luminometer setup and this is probably why Lonza have produced a large table of recommended machines for the assay (http://bio.lonza.com...uminometers.pdf).
Using a BMG Pherastar with a luminometer optic module, I regularly find my negative and positve controls work as expected in the assay. My only concern is that there is a high false positive rate and that's going to take me some time to determine this value.
The samples that were identified as mycoplasma positive in this assay turned out to be negative by PCR and Hoechst dye test.
Sample Luminescence (A) Luminescence (
B/A.
Blank 3924 332 0.08
Medium (DMEM) 1129 376 0.33
Cell sample#1 2318 27849 12.01
Cell sample#2 2839 10070 3.55
Positive Control 3503 31388 8.96