There are several options:
-commercial kit: my lab has had good experiences with the e-myco plus mycoplasma kit, that uses PCR, made by iNtRoN Biotechnology. It’s a conventional PCR method with ready-to-use reagents and comes with a positive control, an extraction control, and an internal control for PCR inhibition. We’ve found it covers a broad spectrum of mycoplasma due to its primer selection for conserved 16S rRNA.
-nested PCR method: We have used a method from this paper with some success: http://ir.hfcas.ac.cn/bitstream/334002/11967/1/Detection%20and%20tentative%20identification%20of%20dominant%20mycoplasma%20species%20in%20cell%20cultures%20by%20restriction%20analysis%20of%20the%2016S-23S%20rRNA%20intergenic%20spacer%20regions.pdf
-a colleague has used a SYBR green rtPCR method, but our lab looked at it and thought it might be expensive and time-consuming, so we haven’t tried it.