Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

• Published in: Microbiology (Society for General Microbiology) Vol. 153; no. 8; pp. 2679 - 2688
• Main Authors: Jeffery, Nathan, Gasser, Robin B, Steer, Penelope A, Noormohammadi, Amir H
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.08.2007; Society for General Microbiology
• Subjects: Animals; Bacterial Proteins - genetics; Bacterial Typing Techniques - methods; Base Sequence; Biological and medical sciences; Chickens; DNA, Bacterial - genetics; Fundamental and applied biological sciences. Psychology; Lectins - genetics; Microbiology; Molecular Sequence Data; Mycoplasma Infections - microbiology; Mycoplasma Infections - veterinary; Mycoplasma synoviae; Mycoplasma synoviae - classification; Mycoplasma synoviae - genetics; Mycoplasma synoviae - isolation & purification; Organic Chemicals - metabolism; Polymorphism, Single-Stranded Conformational; Poultry Diseases - microbiology; Sequence Analysis, DNA; Transition Temperature; Mollicutes; Mycoplasmatales; Gene; Classification; Bacteria; Mycoplasma synoviae; Single strand conformation polymorphism; Mycoplasmataceae; Conformation; Strain
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2006/005140-0

1 CSIRO Livestock Industries, F. D. McMaster Laboratory Chiswick, Armidale, New South Wales 2350, Australia 2 School of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia Correspondence Amir H. Noormohammadi amirh{at}unimelb.edu.au Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 5' end of the variable lipoprotein and haemagglutinin gene ( vlhA ), amplicons of 400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A–J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A–J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h. Abbreviations: HRM, high-resolution melting; SSCP, single-strand conformation polymorphism A table of raw data from HRM curve analysis is available with the online version of this paper.