Duplex PCR to differentiate between Mycoplasma synoviae and Mycoplasma gallisepticum on the basis of conserved species-specific sequences of their hemagglutinin genes

Abstract

We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum, respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.

FIG. 1.

(a) Specificity of the duplex PCR tested with various ATCC mollicutes. Lanes 1 and 13, 1-kb DNA ladder; lanes 2 to 5, M. synoviae and M. gallisepticum primers amplify individually and, respectively, DNA from M. synoviae WVU 1853, M. gallisepticum S6, M. synoviae NCTC 10124, and M. gallisepticum PG31; lanes 6 to 10, both M. synoviae and M. gallisepticum primers used, respectively, in duplex PCR with DNA from M. imitans, M. meleagridis, M. iowae, M. gallinarum, and M. iners; lane 11, M. synoviae and M. gallisepticum DNA amplified with the two pairs of M. synoviae and M. gallisepticum primers (duplex positive control); lane 12, extracted DNA from poultry blood with M. synoviae and M. gallisepticum primers (duplex internal negative control). (b) Ethidium bromide-stained agarose gel confirming the specificity results obtained after the first-round amplification of DNA from M. synoviae and M. gallisepticum and other mollicutes by using the inner M. synoviae and M. gallisepticum primers in duplex nested PCR. Lane 1, 50-bp DNA ladder; lanes 2 and 4, M. synoviae and M. gallisepticum inner primers amplify, respectively, in duplex nested PCR DNA from M. synoviae WVU 1853-M. gallisepticum S6 and M. synoviae NCTC 10124-M. gallisepticum PG31 (duplex nested positive control); lane 3, extracted DNA from poultry blood with M. synoviae and M. gallisepticum primers (duplex internal negative control); lanes 5 to 9, both M. synoviae and M. gallisepticum inner primers used, respectively, with DNA from M. imitans, M. meleagridis, M. iowae, M. gallinarum, and M. iners.

FIG. 2.

Electrophoretic profiles of M. gallisepticum and M. synoviae DNA fragments obtained from 28 clinical samples by duplex PCR. We used 5-μl samples of amplified DNA. Lane 1, 1-kb ladder; lanes 2, 5, 9, 13, 14, 21, and 25, pMGAF-pMGAR and MS1.2F-MS1.2R primers amplifying M. gallisepticum DNA from clinical samples; lanes 3, 4, 6, 10 to 12, 15 to 20, 22 to 24, and 26 to 29, pMGAF-pMGAR and MS1.2F-MS1.2R primers amplifying M. synoviae DNA from clinical samples; lanes 7 and 8, pMGAF-pMGAR and MS1.2F-MS1.2R primers used with extracted DNA from M. gallisepticum- and M. synoviae-negative cultures.