Table 1.
SNP locations in the obg gene, SNP state, primer sequences, primer volumes and thermal cycler parameters for the two MS-H melt-MAMAs.
Table 2.
SNP locations in the obg gene, SNP state, primer sequences and primer volumes for the two MS-H agarose-MAMAs.
Fig 1.
Sequences of the gBlocks used as positive and validation controls in the study.
The gBlocks (Integrated DNA Technologies Inc., Coralville, IA) contain 330 bp long fragment of the obg gene between nt342 and nt672.
Table 3.
Background information of the Mycoplasma synoviae strains, ts+ MS-H re-isolates and trachea swab samples included in this study.
Fig 2.
Amplification plot and melting-curves of MS-H1 melt-MAMA.
Amplification plot (A) of dilution series of gBlock validation controls showing the sensitivity of the MS-H1 assay. Green line represents negative control. Melting curves (B) show melting temperatures for the temperature sensitive MS-H vaccine strain and non-temperature sensitive MS-H re-isolate (Tm: 80.1°C; blue line) or wild-type strain (Tm: 75.0°C; orange line).
Fig 3.
Amplification plot and melting-curves of MS-H2 melt-MAMA.
Amplification plot (A) of dilution series of gBlock validation controls showing the sensitivity of the MS-H2 assay. Green line represents negative control. Melting curves (B) show melting temperatures for the temperature sensitive MS-H vaccine strain and a wild-type strain (Tm: 76.8°C; blue line) or non-temperature sensitive MS-H re-isolate (Tm: 70.9°C; orange line).
Fig 4.
PCR product sizes of MS-H1 and MS-H2 agarose-MAMAs in agarose gel.
Electrophoresis was performed in 3% agarose gel (MetaPhor Agarose, Lonza Group Ltd., Basel, Switzerland) and a 20-bp DNA ladder (O'RangeRuler 20 bp, Thermo Fisher Scientific Inc.) was used as molecular weight marker (m). In the MS-H1 assay gBlock-B and a wild-type strain yielded 71 bp fragments, while gBlock-A and the temperature sensitive MS-H vaccine strain produced 91 bp fragments. In the MS-H2 assay gBlock-B (non-temperature sensitive MS-H re-isolate homologue) yielded 77 bp fragment, while gBlock-A, the temperature sensitive MS-H vaccine strain and wild-type strains give 96 bp fragments.
Table 4.
Matrix showing the SNP states, melt-MAMA melting temperatures (Tm) and agarose-MAMA PCR fragment sizes in the ts+ MS-H vaccine strain genotype, ts- MS-H re-isolate genotype and wild-type M. synoviae strain genotype.