SPECIFICITY AND SENSITIVITY OF POLYMERASE CHAIN-REACTION (PCR) IN COMPARISON WITH OTHER METHODS FOR THE DETECTION OF MYCOPLASMA CONTAMINATION IN CELL-LINES

The polymerase chain reaction (PCR) amplification was used for the det ection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 ce ll lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene s equences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found i n cell cultures). In terms of the positivity or negativity of mycoplas ma infection the results were identical for the agar assay and PCR amp lification. All positive cell lines displayed distinct, unequivocal, o bjectively discernible bands on agarose gels while the non-infected sp ecimens showed no DNA amplification. A simultaneously performed compar ison with four other commonly used detection methods (DNA-RNA hybridiz ation in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced signific antly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10(4) whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection me thods in many respects: simplicity and speed, high specificity and ext reme sensitivity, objectivity and accuracy.