Table 2.

Methods used to detect mycoplasma in cell cultures

Microbiological cultivation*

Electronic microscopy

Biochemical assays

Detection of adenosine phosphorylase activity (6-MPDR)

Enzymatic conversion АТР → АDP detected by luciferase

Chromatographic detection of the transformation of radioactively labeled uridine to uracil with the uridine phosphorylase of mycoplasma

Immunoassays

Immunofluorescence

ELISA

Molecular biology tests

Hybridization analysis

Dot-blot hybridization with specific probes

PCR, RT-PCR *

Microscopic detection

Direct staining of DNA with fluorescent dye (DAPI, Hoechst 33258) *

Fluorescent in situ hybridization (FISH) using probes labeled with fluorescent dyes

^* – officially approved by a number of international expert organizations:

FDA Points to Consider (May 1993), Regularien 21CFR610.30;

USDA federal code #9CFR113.28;

United States Pharmacopoeia, (USP 33/NF 28 '63' and '1226', Mycoplasma tests, 2010); European Pharmacopoeia (EP 2.6.7., Mycoplasmas, 7th ed.; 2012);

Japanese Pharmacopoeia (JP);

ICH Guideline for biotechnological/biological products.