A simplified duplex real-time PCR incorporating TaqMan minor groove binder (MGB) probes and an exogenous internal positive control for the simultaneous detection of Mycoplasma gallisepticum and Mycoplasma synoviae cultures

Abstract

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.