Mycoplasma detection by PCR
byrne at rwja.umdnj.edu
Thu Sep 5 16:26:46 EST 1991
bhjelle at vaxine.unm.edu (Brian Hjelle) writes:
>In article <9108291447.AA22254 at genbank.bio.net> neuro1 at sys.ife.ethz.ch ("J.P.Magyar") writes:
>>Do you know of any protocol for mycoplasma detection in cell culture
>>using PCR ? Does the method, if any known, detect all mycoplasm types
>>contaminating the cell culture ?
>>Of course I know of DAPI / Hoechst stainig for mycoplasm detection,
>>but detection by PCR could be much more easier.
>>Thanks in advance
>I got interested in this about a year ago and called around until I located
>some honcho mycoplasma guy (at NIH, I think) with this exact question. I
>hope I remember correctly the answer he gave me.
>Apparently, there is a real heterogeneity of mycoplasmas that affect
>cultured cells in various labs. Within a particular lab, the same ones
>tend to recur again and again. However, he discouraged me on no uncertain
>terms because the species are quite divergent (some are animal origin,
>some human). It would be very hard to pick primers until you came across
>one that worked for the species you were seeking.
>Back to Hoechst staining, I guess.
All true, but don't give up. The issue of heterogeneity pervades PCR.
The trick is to define a pair of primers conserved within the strains,
species, or sets of species required by the job. Decades of work with
relatedness of bacterial strains using rRNA sequences has yielded substantial
information appropriate to this task. Don't throw out Hoechst stain yet,
but take a look at Blanchard et al. (FEMS Microbiology Letters 81 (1991)
37-42) for one such approach.
I expect that one day PCR will replace staining and culture for mycoplasma
detection...but maybe not yet
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