Detection of Mycoplasma gallisepticum by fluorescence in situ hybridization

Published sequences of a Mycoplasma gallisepticum (MG) specific DNA fragment were used to prepare a MG-specific DNA probe. Biotin-16-dUTP was added to the polymerase chain reaction (PCR) mix to obtain a labeled 732 bp-long amplicon of the MG reference strain PG31. With the aid of several avidin-CY5 layers the hybridization product was visualized by a confocal laser scanning microscope. The newly developed fluorescence in situ hybridization (FISH) protocol was tested on ten Mycoplasma gallisepticum reference strains and fourty-three Mycoplasma field isolates. The FISH method was compared with PCR and immunofluorescence (IF) and proved to be superior to IF and equal to PCR in specificity, without the risk of contamination.