SPECIES IDENTIFICATION OF AVIAN MYCOPLASMAS BY POLYMERASE CHAIN-REACTION AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS

Polymerase chain reaction (PCR) and restriction fragment length polymo rphism (RFLP) analysis were used to detect and differentiate four path ogenic species (Mycoplasma gallisepticum, M. iowae, M. meleagridis, an d M. synoviae ) and ten nonpathogenic species of avian mycoplasma. A s equence of 1026 base pairs within the gene for 16S ribosomal RNA (16S rRNA) from avian mycoplasmas was successfully amplified by PCR with ol igonucleotide primers (M16SPCR5' and M16SPCR3') common to all al ain m ycoplasmas tested. Restriction endonucleases (REs) with unique restric tion sites, selected by computet-assisted analysis of known sequences of the amplified segment of arian mycoplasmas, were then used to diges t the PCR products. After electrophoresis of the resulting RE fragment s, the RFLP patterns were compared. Combinations of up to six REs (Hpa I, HhaI, HaeIII, HphI, FokI, and NlaIV) produced unique RFLP patterns by which the 14 species of avian mycoplasmas could be differentiated. The newly classified avian species, M. imitans was also investigated b y this method; M. imitans and M. gallisepticum gave identical RFLP pat terns with the REs used in this study. The results obtained by the PCR and RFLP analysis were in agreement with current methods for species identification of avian mycoplasmas.