title: A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks.
abstract: Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.
authors.affiliation: Laboratório de Diagnóstico Molecular, Universidade Luterana do Brasil, Canoas, RS, Brazil. ; Simbios Biotecnologia, Cachoeirinha, RS, Brazil.
authors.forename: Aline Padilha
authors.initials: AP
authors.lastname: Fraga
authors.forename: Tatiana
authors.initials: T
authors.lastname: de Vargas
authors.forename: Nilo
authors.initials: N
authors.lastname: Ikuta
authors.forename: André Salvador Kazantzi
authors.initials: AS
authors.lastname: Fonseca
authors.forename: Álvaro José
authors.initials: ÁJ
authors.lastname: Celmer
authors.forename: Edmundo Kanan
authors.initials: EK
authors.lastname: Marques
authors.forename: Vagner Ricardo
authors.initials: VR
authors.lastname: Lunge
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods; D001938:Brazil; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D045686:Mycoplasma gallisepticum / Q000235:genetics / Q000302:isolation & purification*; D045805:Mycoplasma synoviae / Q000235:genetics / Q000302:isolation & purification*; D011200:Poultry; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods*
keywords: Mycoplasma gallisepticum; Mycoplasma synoviae; real-time PCR