Types of Mycoplasma testing

We recommend that a combination of methods is used to determine whether a sample is free of mycoplasma.  This is because the different methods have different levels of detection sensitivity and some strains of mycoplasma do not grow in vitro. These can be detected by DNA stain and PCR, the three different mycoplasma detection methods that are routinely used by our scientists:

1. qPCR

This assay consists of a closed tube qPCR detection format and uses TaqMan fluorogenic probes and primers derived from the 16SrRNA region; these were designed manually in-house and consist of three sense primers and four TaqMan fluorogenic probes targeted to anneal to specific regions common in many bacteria.

Specificity of the assay is conferred by the 53 anti-sense primers used, which overlap a broadly conserved region in the 16SrRNA gene of mycoplasma but differ significantly from other (non-mycoplasma) bacterial sequences to enable differentiation from phylogenetically related bacteria. The assay also incorporates an inhibition control that is detected in parallel to the mycoplasma target in a duplex, single-tube reaction.

2. Indirect Hoechst Stain

This test is used to detect mycoplasma by Hoechst stain using an indicator cell line (Vero ECACC 84113001) and meets the requirements of the FDA Points to Consider 1993.

The test sample is inoculated onto Vero cells which have been grown on coverslips in tissue culture dishes. Both negative and positive controls (Mycoplasma hyorhinis NCTC 10112 and Mycoplasma orale NCTC 10130) are also set up. All samples are then incubated for 3-5 days after which the samples are fixed to the coverslip and stained using the fluorescent dye (Hoechst 33258). The fixed samples are then examined under UV epi fluorescence at x100 magnification. Positive samples are identified by their characteristic particulate or filamentous pattern of fluorescence on the cell surface or if contamination is heavy in the surrounding area. Negative samples should demonstrate no evidence of fluorescing DNA outside of the cell membrane against the fluorescing Vero cell nuclei.

3. Culture Isolation

Often regarded as the gold standard method for mycoplasma detection, the culture isolation detection method uses a combination of selective growth media and incubation conditions to positively enrich for any mycoplasma present in a sample. Most mycoplasma contaminants can be detected by growth on standardised agar, with the exception of certain strains of M.hyorhinis.

Colonies of mycoplasma exhibit a distinctive "fried egg" morphology when viewed under a plate microscope. The assay requires a 28 day test interval before a definitive result can be obtained.

This test meets the requirements of the FDA Points to Consider 1993.