Use of an alkaline phosphatase-labelled probe for the detection of Mycoplasma synoviae in chickens

Short nucleotides directly labelled to alkaline phosphatase (SNAP probes) a re an interesting alternative to digoxigenin-labelled probes (DIG probes), because they reduce the number of steps necessary in dot blots for the dete ction of DNA or amplificate. This study examined the questions whether a SN AP probe might not only save time, but also increase the sensitivity of ano ther PCR-based DNA probe test using a digoxigenin probe. Amplificates obtai ned by multispecies polymerase chain reaction (PCR), with either purified g enomic DNA or DNA extracted from tracheal swabs taken in chicken flocks; we re detected by both methods. The results for the clinical specimens were co mpared to culture. Under stringent conditions, the specificity and sensitiv ity obtained with the SNAP probe mere comparable to the results obtained wi th the DIG probe. The quatities 10 fg (SNAP probe) and 100 ig (DIG probe) o f purified Mycoplasma synoviae DNA mere detected after amplification, but m ore positive clinical specimens were detected with the DIG probe. Under non -stringent conditions sensitivity with purified DNA did no change, but the coloration of the dots improved markedly, and more positive specimens could be detected with the SNAP probe than with the DIG probe, truly positives a s confirmed by culture. Because cross-reaction with Mycocplasma galliseptic um and Mycoplasma imitans, two species with DNA that was also recognized by the multispecies primers, occurred under non-stringent conditions, it ii a s concluded that, to take the full advantage of SNAP probes, their use in c ombination with species-specific primer pairs is recommended. PCR as a meth od for mycoplasma detection is however, always accompanied with serological and cultural methods. When a M. synoviae mono-infection is likely by serol ogical results, non-stringent dot blot conditions and use of the SNAP probe will ease and improve the detection of mycoplasma.