DNA amplification methods for diagnosis and epidemiological investigations of avian mycoplasmosis

Rapid, sensitive and specific tests that detect nucleic acid from pathogenic mycoplasmas are very attractive for the laboratory detection of infected flocks, and methods for direct detection of the four main pathogenic mycoplasmas have been developed. Moreover, most avian mycoplasma species can be differentiated, according to their unique restriction fragment length polymorphism (RFLP) patterns generated with different restriction enzymes. However, this method is limited to the identification of pure cultures of avian mycoplasmas as other bacteria may be amplified by the set of primers chosen. Another application of PCR-RFLP is the ability to distinguish between very closely related species such as M. gallisepticum and M. imitans. In order to characterise isolates below the species level, PCR-based subtyping methods have been introduced. One of them, arbitrarily primed-PCR, results in strain-specific arrays of DNA fragments that can distinguish even closely related strains of a given species. This method was successfully used to investigate the molecular epidemiology of vaccine strains or of Mycoplasma gallisepticum conjunctivitis in songbirds. Major issues in the development of DNA amplification tests concern the selection of the appropriate target for amplification, specimen collection, DNA preparation and detection of amplification reaction inhibitors. Detection of amplified products is most commonly performed after gel electrophoresis or probe-based methods. Careful consideration to the design and work flow of the facility are necessary to avoid false-positive results.