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Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most pathogenic and economically important mycoplasma pathogens that infect chickens. The development of rapid and innovative molecular diagnostic techniques is of pivotal importance for their effective control. The aim of the present study was to develop a novel duplex TaqMan real-time PCR assay for the simultaneous detection of MG and MS. This duplex real-time PCR assay incorporates TaqMan (FAM/NED) labelled minor groove binder (MGB) probes that target the cytadhesin encoding surface protein (mgc2) gene and the haemagglutinin surface protein (vlhA) gene of MG and MS, respectively. The assay also contained a TaqMan exogenous internal positive control (Exo IPC), to avoid false negative results that might happen due to failure in DNA extraction/PCR inhibition. The TaqMan MGB probe-based duplex RT-PCR incorporating Exo IPC was then applied to DNA from culture isolates for the simultaneous detection of MG (mgc2 gene) and MS (vlhA gene). For duplex RT-PCR the sensitivity recorded was 10 -3 CFU/ml and 10 -2 CFU/ml for MG and MS template DNA, respectively. The specificity of the real-time PCR assay was 100% for MG-and MS-specific probes in detecting both single as well as double infections. In conclusion, the use of TaqMan MGB probes for the detection of mgc2 and vlhA genes confers extra specificity and the incorporation of Exo IPC simplifies the assay, allowing the detection of double infections with low-copy target DNAs.Keywords: avian Mycoplasma spp.; duplex real-time PCR; MGB probe; mgc2 gene; vlhA gene; internal positive control Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most pathogenic and economically significant mycoplasma pathogens that infect chickens. The three major approaches currently used for the diagnosis of avian mycoplasmas are isolation and identification, detection of antibodies and molecular detection of the organisms by polymerase chain reaction (Raviv and Kleven 2009). PCR based on detection of mgc2-cytadhesin encoding surface protein gene for MG (Garcia et al. 2005), and vlhA-haemagglutinin surface protein gene of M. synoviae (Hong et al. 2004) are the most widely used methods for the detection, typing and determination of the source of infection. Real-time PCR (RT-PCR) avoids the need for post amplification processing, which saves time and labour compared to conventional PCR methods (Kawahara et al. 2008). TaqMan RT-PCR is a simpler method on which to base a multiplex PCR assay to detect mixed infections in a single PCR reaction. So far, only a limited number of TaqMan based diagnostic RT-PCR assays have been reported that target the mgc2 gene (Grodio et al. 2008;Raviv and Kleven 2009;Sprygin et al. 2010) for detection of MG, and the vlhA gene for detection of MS. The duplex RT-PCR-based detection of MG and MS using the 269Veterinarni Medicina, 60, 2015 (5): 268-273 Original Paper (OIE 2008). The avian mycoplasma RT-PCR assays previously described by Grodio et al. (2008) and Sprygin et al. ...

Species-specific DNA probes for Mycoplasma gallisepticum (MG) were compared with serologic and isolation procedures as a routine diagnostic tool on field specimens acquired from chicken flocks experiencing egg-production losses and suspected of MG infection. The MG DNA probe clearly identified MG directly from tracheal specimens within 2 days, unlike the 7 to 10 days required for culture procedures. Cross-reaction of MG with M. synoviae continues to be a stumbling block in the serum plate agglutination test.