Cell culture supernatant or cultured cell pellet samples are lysed in cell lysis buffer. Samples are hybridized in a microplate with biotin-labeled capture oligonucleotide probes and digoxigenin-labeled detection probes that are targeted to the 16S ribosomal RNA of the eight most common mycoplasmas. The hybridization solution is then transferred to a streptavidin-coated microplate and the rRNA/probe hybrid is captured. Following a wash to remove unbound material, an anti-digoxigenin alkaline phosphatase conjugate is added. After washing away unbound conjugate, a substrate solution is added. An amplified solution is then added and color develops in proportion to the amount of mycoplasma rRNA in the original sample. Color development is stopped and the intensity of the color is measured using a standard colorimetric plate reader.
