Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.
Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here.
Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia. Electronic address: aghorashi@csu.edu.au.
Seyed A
SA
Ghorashi
Janet M
JM
Bradbury
Naola M
NM
Ferguson-Noel
Amir H
AH
Noormohammadi
D000818:Animals; D015373:Bacterial Typing Techniques; D017931:DNA Primers / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005060:Europe; D005798:Genes, Bacterial / Q000235:genetics*; D014644:Genetic Variation; D009175:Mycoplasma Infections / Q000175:diagnosis; D045686:Mycoplasma gallisepticum / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics
D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S
Genotyping; High resolution melting curve analysis; MG; Mycoplasma gallisepticum; PCR; Strain differentiation