Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region
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.
BencinaD.,
Drobnic-ValicM.,
HorvatS.,
NaratM.,
KlevenS. H.,
DovcP.2001; Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin. FEMS Microbiol Lett 203:115–123
CharvalosE.,
PeteinakiE.,
SpyridakiI.,
ManetasS.,
TselentisY.1996; Detection of ciprofloxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single-strand conformation polymorphism and direct DNA sequencing. J Clin Lab Anal 10:129–133
FanH. H.,
KlevenS. H.,
JackwoodM. W.1995; Studies of intraspecies heterogeneity of Mycoplasma synoviae, M. meleagridis , and M. iowae with arbitrarily primed polymerase chain reaction. Avian Dis 39:766–777
GarciaM.,
JackwoodM. W.,
LevisohnS.,
KlevenS. H.1995; Detection of Mycoplasma gallisepticum, M. synoviae , and M. iowae by multi-species polymerase chain reaction and restriction fragment length polymorphism. Avian Dis 39:606–616
GasserR. B.,
HuM.,
Abs EL-OstaG. Y.,
ZarlengaD. S.,
PozioE.2004; Nonisotopic single-strand conformation polymorphism analysis of sequence variability in ribosomal DNA expansion segments within the genus Trichinella (Nematoda: Adenophorea. Electrophoresis 25:3357–3364
HongY.,
GarciaM.,
LeitingV.,
BencinaD.,
Dufour-ZavalaL.,
ZavalaG.,
KlevenS. H.2004; Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene vlhA . Avian Dis 48:606–616
KissI.,
MatizK.,
KaszanyitzkyE.,
ChavezY.,
JohanssonK. E.1997; Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay. Vet Microbiol 58:23–30
KlevenS. H.1997; Mycoplasma synoviae infection. In Diseases of Poultry pp 220–228 Edited by
CalnekB. W.,
BarnesH. J.,
BeardC. W.,
McDougaldL. R.,
SaifY. M.
Ames, IA: Iowa State University Press;
KrypuyM.,
NewnhamG. M.,
ThomasD. M.,
ConronM.,
DobrovicA.2006; High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer 6:295
LauermanL. H.,
HoerrF. J.,
SharptonA. R.,
ShahS. M.,
van SentenV. L.1993; Development and application of a polymerase chain reaction assay for Mycoplasma synoviae . Avian Dis 37:829–834
MarkhamJ. F.,
ScottP. C.,
WhithearK. G.1998; Field evaluation of the safety and efficacy of a temperature-sensitive Mycoplasma synoviae live vaccine. Avian Dis 42:682–689
MonisP. T.,
GiglioS.,
SaintC. P.2005; Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal Biochem 340:24–34
MorrowC. J.,
BellI. G.,
WalkerS. B.,
MarkhamP. F.,
ThorpB. H.,
WhithearK. G.1990a; Isolation of Mycoplasma synoviae from infectious synovitis of chickens. Aust Vet J 67:121–124
MorrowC. J.,
MarkhamJ. F.,
WhithearK. G.1998; Production of temperature-sensitive clones of Mycoplasma synoviae for evaluation as live vaccines. Avian Dis 42:667–670
NoormohammadiA. H.,
MarkhamP. F.,
WhithearK. G.,
WalkerI. D.,
GurevichV. A.,
LeyD. H.,
BrowningG. F.1997; Mycoplasma synoviae has two distinct phase-variable major membrane antigens, one of which is a putative hemagglutinin. Infect Immun 65:2542–2547
NoormohammadiA. H.,
MarkhamP. F.,
KanciA.,
WhithearK. G.,
BrowningG. F.2000; A novel mechanism for control of antigenic variation in the hemagglutinin gene family of Mycoplasma synoviae . Mol Microbiol 35:911–923
OdellI. D.,
CloudJ. L.,
SeippM.,
WittwerC. T.2005; Rapid species identification within the Mycobacterium chelonae-abscessus group by high-resolution melting analysis of hsp65 PCR products. Am J Clin Pathol 123:96–101
RobinsonB. S.,
MonisP. T.,
DobsonP. J.2006; Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis. Appl Environ Microbiol 72:5857–5863
SilveiraR. M.,
FiorentinL.,
MarquesE. K.1996; Polymerase chain reaction optimization for Mycoplasma gallisepticum and M. synoviae diagnosis. Avian Dis 40:218–222
SykesJ. E.,
StuddertV. P.,
BrowningG. F.1998; Detection and strain differentiation of feline calicivirus in conjunctival swabs by RT-PCR of the hypervariable region of the capsid protein gene. Arch Virol 143:1321–1334
ThompsonJ. D.,
HigginsD. G.,
GibsonT. J.1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
TokueY.,
SuganoK.,
SaitoD.,
NodaT.,
OhkuraH.,
ShimosatoY.,
SekiyaT.1994; Detection of novel mutations in the gyrA gene of Staphylococcus aureus by nonradioisotopic single-strand conformation polymorphism analysis and direct DNA sequencing. Antimicrob Agents Chemother 38:428–431
YoderH. W.Jr,
DruryL. N.,
HopkinsS. R.1977; Influence of environment on airsacculitis: effects of relative humidity and air temperature on broilers infected with Mycoplasma synoviae and infectious bronchitis. Avian Dis 21:195–208
Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region