( roduced PCR-based methods these compet...)A New PCR-based Mycoplasma Detection Method

Inasmuch as these targets share a large degree of sequence homology, both within the mycoplasma genus and with other prokaryotes, a mycoplasma-specific procedure needed to be developed. Unfortunately, this required two sequential PCRs using nested primersa cumbersome and time-consuming procedure.

We sought to develop a PCR-based method to detect a single copy gene. In our approach, we identified a target gene whose evolution rate was sufficiently rapid to contain regions of sequence divergence that could be used to speciate mycoplasmas. Once the target gene was identified, we examined the outer portions for regions of sequence conservation within the mycoplasma genus that could serve as targets for consensus PCR primers. We also evaluated the potential priming sites for their low degree of homology with other prokaryotic sequences and their complete lack of homology with eukaryotic sequences.

These resulting primers yielded a single 874-bp PCR product when used to amplify DNA from the four mycoplasma species commonly associated with cell culture infections. Restriction analysis of the PCR product produced distinctive fingerprints to diagnose mycoplasma species. Consequently, primary analysis of the PCR product and secondary restriction analysis could be conducted in parallel on a single agarose gel.

PCR Primers Amplified

We initially evaluated how well the mycoplasma-consensus primers amplified an 874-bp target sequence from crude extracts of mycoplasma/acholeplasma samples obtained from the American Type Culture Collection (ATCC). We tested the comprised mycoplasma/acholeplasma species most commonly encountered in infected cultures. To approximate the conditions used in testing tissue culture supernatants, each samp
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