MYCOPLASMA DETECTION BY PCR ANALYSIS

The polymerase chain reaction (PCR) method was used to detect mycoplas ma contamination in a panel of 42 continuous cell lines. According to the microbiological cultivation assay on agar, 29 cell lines were chro nically infected and 13 cell lines were negative. Sets of outer and in ner primers (nested double-step PCR) were applied which anneal to DNA sequences coding for conserved regions of the 16S rRNA. These oligonuc leotides allow for the amplification of DNA regions found in at least 25 mycoplasma species (including the ones most commonly found in cell cultures), but do not cross-hybridize with DNA from eukaryotic cells. Mycoplasma-positive cell lines showed distinctive bands in ethidium br omide-stained gels, both after the first round of amplification as wel l as after the second PCR; all agar-negative cell lines were also unam biguously negative in the PCR assay. Thus, neither false-positive nor false-negative results occurred. Provided that the proper PCR working conditions are scrupulously observed, the PCR amplification has severa l outstanding advantages: high sensitivity, specificity, reliability, objectivity, speed, and simplicity.