The best method is (3) PCR.

Why PCR is preferred

• PCR assays amplify mycoplasma-specific DNA, usually regions of the 16S rRNA gene, from culture supernatant or cell pellets.

• They are highly sensitive, specific, rapid (few hours), and can detect low-level contamination long before morphological changes appear.

• Because of this, PCR-based kits are widely regarded as the routine “gold standard” screening method for mycoplasma in research and production cell lines.

Why the other options are not ideal

(1) Southern hybridization

• Southern blotting detects specific DNA, but it is laborious, slow, and low-throughput.

• It is not used routinely for mycoplasma testing in cell culture laboratories.

(2) ELISA

• Standard ELISAs detect proteins or antibodies, not directly mycoplasma DNA.

• While specialized probe‑based microplate assays exist, generic ELISA is not the primary or most sensitive method for mycoplasma detection compared with PCR.

(4) Western hybridization (Western blot)

• Western blotting probes for specific proteins.

• Mycoplasma detection relies more on nucleic-acid–based methods; there are no common routine Western assays for mycoplasma in cell culture.

Therefore, among the listed techniques, PCR (option 3) is the best and most widely used method to check for mycoplasma contamination in mammalian cell lines.