MYCOPLASMA testing with PCR - advice sought...

Martin Kennedy mkennedy at chmeds.ac.nz
Wed Jun 1 23:30:07 EST 1994


In article <2sev19$e7r at mserv1.dl.ac.uk>, ahill at crc.ac.uk (Mr. A.F. Hill) writes:
> 
> I remember reading on the net last year sometime about methods for screening
> cell cultures for mycoplasma contamination using PCR.
> 
> Could anyone please post me the faq that was generated (if there was one) or
> enlighten me as to how i might go about setting up an assay.
> Andy Hill

I was interested in this thread last year, and have followed up to the point 
where we now have a working assay going for screening our cell lines.  Here is 
a summary of the thread, and some relevant publications I ferreted out.  We 
now use the primers GPO1 and MGSO from the van Kuppeveld reference given below.
These are useful as a Mycoplasma genus specific set.  I don't really care 
which species I have, I just want to know I don't have any contaminating
my cultures!  We use these routinely on genomic DNA from various mammalian 
cell-lines and as far as we can tell they work well - all our positive 
controls light up, and our best clean lines (as close to negative controls as 
we can get) don't give any bands.

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
		Phone (64-3)364-0880  Fax (64-3)364-0750



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  Spaepen M.  Angulo AF.  Marynen P.  Cassiman JJ.  
  Detection of bacterial and mycoplasma contamination in cell cultures by
  polymerase chain reaction.
  Fems Microbiology Letters.  [JC:fml]  78(1):89-94, 1992 Nov 15.
Abstract
  A fast and simple method to detect bacterial and especially mycoplasma
  contamination in tissue culture by means of polymerase chain reaction
  (PCR) amplification is described. In a first step the universal primer
  pairs P1/P2 (190-bp fragment) and P3/P4 (120-bp fragment) directed to
  different conserved parts of the prokaryotic 16S rRNA gene are used. A
  positive signal after amplification on cell culture DNA with these primers
  provides an indication of bacterial infection. Using the internal primers
  IP1, IP3 and IP'3 complementary to a part of the V4 and V8 variable
  regions of the 16S rRNA gene, in combination with a universal primer,
  cultures contaminated with mycoplasma could be identified. Six mycoplasma
  species, typical contaminants in tissue cultures, were investigated:
  Mycoplasma orale, M. fermentans, M. arginini, M. hyorhinis, M. hominis and
  Aeromonas laidlawii. This mycoplasma test is an easy, specific and
  sensitive assay which should be extremely useful in any tissue culture
  setting.


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  Blanchard A.  Gautier M.  Mayau V.  
  Detection and identification of mycoplasmas by amplification of rDNA.
  Fems Microbiology Letters.  [JC:fml]  65(1):37-42, 1991 Jun 1.
Abstract
  Alignment of published 16S rRNA sequences allowed the definition of a pair
  of oligonucleotides suitable for polymerase chain reaction (PCR). Using
  this pair of PCR primers, several mycoplasmas including the four human
  parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale
  were detected. This DNA amplification was restricted to species of the
  genus Mycoplasma while no cross-reaction was observed with DNA from other
  bacteria and eukaryotic cells. Subsequent analysis of amplified products
  by either specific oligonucleotide hybridization or dideoxy sequencing
  specified the identity of the detected mycoplasmas. This method offers a
  highly discriminating and sensitive assay for the direct detection and
  identification of these microorganisms without the need for prior
  cultivation.


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  van Kuppeveld FJ.  Melchers WJ.  Willemse HF.  Kissing J.  Galama JM.  
  van der Logt JT.  
  Detection of Mycoplasma pulmonis in experimentally infected laboratory
  rats by 16S rRNA amplification.
  Journal of Clinical Microbiology.  [JC:hsh]  31(3):524-7, 1993 Mar.
Abstract
  Recently, an rRNA-based polymerase chain reaction (PCR) has been developed
  for the detection of murine mycoplasmas at both the genus and species
  level (F. J. M. van Kuppeveld, J. T. M. van der Logt, A. F. Angulo, M. J.
  van Zoest, W. G. V. Quint, H. G. Niesters, J. M. D. Galama, and W. J. G.
  Melchers, Appl. Environ. Microbiol. 58:2606-2615, 1992). In this study,
  the diagnostic value of this PCR assay for the detection of Mycoplasma
  pulmonis in infected rats was studied. For this purpose, 25 Wistar rats
  were infected intranasally with M. pulmonis strain M72-138 and
  investigated for the presence of this pathogen by both in vitro isolation
  and PCR. Five rats were monitored longitudinally by screening of throat
  swabs at several time points for up to 248 days postinfection. The
  remaining 20 rats were killed between 3 and 87 days postinfection, and
  organism recovery from both throat and urogenital tract specimens was
  attempted. M. pulmonis could be detected in the throat for up to 248 days
  postinfection but not in the urogenital tract, either by culture or by
  PCR. PCR proved to be the optimal method for testing throat samples. All
  samples in which M. pulmonis was detected by culture were also positive by
  PCR. By PCR, M. pulmonis was also detected in 3.7% of the samples which
  were culture negative and in 9.9% of the samples from which cultures were
  overgrown with bacteria. The results of this study demonstrate the
  suitability of PCR for the detection of mycoplasmal infection in rodents.


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So it looks like what I want to do is already well worked out.  However, 
various helpful suggestions were also made, summarized below.  Thanks to all 
respondents:

From: dsct at bphvax.biophysics.rochester.edu
To: Mkennedy at chmeds.ac.nz

WE have used both the genprobe kit and the Boeringer manneheim kit. 
WE have used the genprobe kit for years, with good succes. It is easy
to use, and has given us good results (we grow murine/human B-lymphomas).
The Boeringer Manneheim kit is new to us, and we have only used it twice.
It detected the same positive culture as the genprobe kit, and is faster
to use (It is ELISA based). any questions? I would be happy to answer.

					-Gavin Fischer 
					University of Rochester Cancer Center


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From: Michael J Conboy <conboymj at leland.Stanford.EDU>
Message-Id: <199309170615.XAA13841 at elaine44.Stanford.EDU>

Just curious, since ES cells are adherent, why not use Hoesch (sp?)
nuclear stain as described in the cell culture texts?

Mike


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From: <U27111%UICVM.uic.edu.us at UIC.EDU>
To:          <mkennedy at chmeds.ac.nz> (Martin Kennedy)

If your cells are NOT primary cultures... I would like to make the suggestion
you grow them in antibiotic-free media.  I seem to recall reading years ago
mycoplasma tends to come into cultures along with "other' contaminants... thus,
a media with antibiotics can lead to low-level contamination culture which may
also be harboring mycoplasma.  If you can do your cultures free of antibiotic,
you would immediately see a contamination and can discard it.

I don't know if this theory is valid or not today, but I have been growing
cell lines antibiotic -free for years and haven't had a mycoplasma problem
yet.  As a note, I have also read that antibiotics can lower the metabolism of
certain cell lines and/or some metabolites' production.  Tends to depend on
what you are doing and what you are looking at.

Of course primary cultures are a different story entirely and must be tested.

Hope this helps,

-Kathy


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From: Klaus.Matthaei at anu.edu.au
X-Sender: kim227 at cscgpo.anu.edu.au

Hi Martin

We are also concerned of course and would like an easy assay. 

However we have recently tested our C57Bl/6 ES cells and they were
mycoplasma positive.  So we panicked and contacted our collaborators in
Germany.  It turns out that this cell has been positive since its isolation
BUT gives VERY rates of Chimaerism and germline transmission.  The
mycoplasma is held down by Pen/Strep and only blooms on its removal. 
Apparently mycoplasma can chew up your construct and therefore you get very
low rates of transfection.  They also induce chromosomal abnormalities if
allowed to bloom so Pen/strep or other antibiotics are a must.  So thats
why its thought to bea problem.  

On enquiry in Europe it turns out that MANY germline ES cells are
mycoplasma positive.  So maybe it is not as bad a problem as once thought. 
But I will continue to look on with interest and am certainly interested in
its detection.

I will monitor responses to you and please let me know if anything turns up
not on the net.

Regards, Klaus
--------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Think twice - Do once"
--------------------------------------------------------------------------

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From: rbonfigl at waite.adelaide.edu.au (Rod Bonfiglioli)
To: mkennedy at chmeds.ac.nz (Martin Kennedy)

hi,
i know that there is someone at the northern territory university in darwin, nt,
australia who is doing pcr work on mycoplasmas, her name is karen gibb, she has
an email address which i think is k_gibb at bligh.ntu.edu.au
try that address, if you have no luck, get back to me

rod

--
Rod Bonfiglioli, Waite Agricultural Research Institute,
University of Adelaide, South Australia.
e-mail rbonfigl at waite.adelaide.edu.au

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Thanks Rod!


From: Karen Gibb <Karen_Gibb at post.ntu.edu.au>
To: MKENNEDY <MKENNEDY at CHMEDS.AC.NZ>

In response to your enquires re:PCR detection of Mycoplasmas - I'm a plant
mycoplasmalike organism researcher so am not familiar with current animal
mycoplasma protocols. although you may get better advice from someone in the
field, one approach is to see the paper by Lim and Sears J. of Bacteriology
171:5901-5906. They list 16S sequence and one is M. capricolum. I'd recommend a
 set of 20mer primers based on their sequence data, forward primer starting at
position 735 to 755 and the reverse 1224 to 1244. I could give you the primer
sequence from the paper but it'd be better for you to get the article and plan
them yourself if you're going to the expense of buying the primers. DNA express
at colorado state univ fax 303 491-0239 are cheap for primers, US$3.25 per
base. Really good quality and very fast delivery. Then it should be a matter of
a routine DNA extraction from the cell line and a PCR with 20ng of total DNA
(20ng/ul solution). Hope it works, I wish I could refer you to animal people
who do routine PCR work but I only know the plant ones I'm afraid. Hope this
helps though. Regards Karen Gibb

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From: cbksa01 at mailserv.zdv.uni-tuebingen.de (Martin Sauerbeck)
To: mkennedy at chmeds.ac.nz

I am working with human AMNION/cells WISH/cells (ATCC CClL 25) .
to detect mycoplasma contamination I use a DETECTION KIT from BOEHRINGER 
MANNHEIM (Germany), but they should have a branch in the USA aswell.
To fight those nastzy small beeings I use CIBROFLOXACIN that I got from 
BAYER
LEVERKUSEN. 5 to 10 mg/l kuculture medium is fine.

If you need more information, you are wellcome to contact me.

Thanx for summerizing all information in bionet.cellbiol

Yours Martin Sauerbeck
Dept. of Physiol. Chem.
University of Tuebingen
Germany

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From:         Joe <JOFTOPSM at VTVM1.CC.VT.EDU>

Gen-Probe in San Diego has a mycoplasma DNA probe detection kit. Call 800-523-5
001


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-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750



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