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.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. ...

Serological, rapid molecular characterization and antibiogram resistance for field isolates of Mycoplasma Gallisepticum in chicken in Saudi Arabia

“…A higher sensitivity detection (lower CT values) was achieved with the TaqMan FAM TM reporter dye in this study. Others (Ehtisham-ul-Haque et al, 2015) have reported the results for FAM to be stronger that those obtained with the NED TM reporter dye. Additionally, the low detection value of NED as a reporter dye is well corroborated in past experience.…”

“…Traditionally, diagnosis of MG infection in the poultry industry depended upon serological screening such as serum plate agglutination (SPA) and enzyme-linked immunosorbent assay (ELISA) hemagglutination inhibition (HI), which historically are considered the most reliable tools for identifying subclinical infection in the flock (Barua et al, 2006;Purswell et al, 2012;Kaboli et al, 2013); however, they sometimes lack the required specificity and sensitivity (Carli, and Eyigor, 2003;Kaboli et al, 2013). Therefore, in the last decade, rapid polymerase chain reaction (PCR) methods with high specificity and sensitivity for MG have been very useful for laboratory diagnosis of infected birds (Ehtisham-ul-Haque et al, 2015;Raviv and Kleven, 2009). Polymerase chain reaction based on identification of the mgc2-cytadhesin encoding surface protein gene for MG is the most widely used methods for the detection, typing and determination of the source of infection (Gerchman et al, 2009).…”

“…Nevertheless, some problems are associated with PCR, including the existence of inhibiting substances in the samples, the danger of environmental contamination and the probability of the recognition of nonviable Mycoplasma (Kempf, 1998;Kleven, 1997;Carli and Eyigor, 2003).The progress in diagnostic PCR technology has greatly resolved the problems with the time, specificity, and sample size. Thus, the TaqMan RT-PCR avoids the need for post amplification processing, which saves time and labor compared to traditional PCR methods (Glew et al, 2000;Kawahara et al, 2008;Ehtisham-ul-Haque et al, 2015). The TaqMan RT-PCR is a simpler technique on which to base a multiplex PCR assay in order to detect mixed infections in a single PCR reaction.…”

“…The TaqMan RT-PCR is a simpler technique on which to base a multiplex PCR assay in order to detect mixed infections in a single PCR reaction. Until now, only a limited number of TaqMan RT-PCR-based diagnostic techniques have been reported that target the mgc2 and gapA genes for the detection of MG (Grodio et al, 2008;Raviv and Kleven, 2009;Sprygin et al, 2010;Ehtisham-ul-Haque et al, 2015). To improve the specificity of TaqMan RT-PCR procedures, TaqMan minor groove binder (TM-MGB) probes are preferred because their small size enables binding in the minor groove of doublestranded DNA, thus stabilizing the duplex, which results in high melting temperature (Tm) values (Kutyavin et al, 2000;Guo et al, 2009;Ehtishamul-Haque et al, 2015) and greater accuracy in measurements because of low background fluorescence (Chiu and Ou, 1996;Farkas et al, 2009).…”

“…The chronic nature of Mycoplasma infections illustrates a failure of the host immune system to deal efficiently with these pathogens. The antigenic dissimilarity of surface proteins permits MG to get away from the host immune system through the generation of escape variants (Papazisi et al, 2003;Ehtisham-ul-Haque et al, 2015). In addition, intracellular attack by MG and its continued existence within eukaryotic cells may contribute to this organism's resistance to the host's immune response and to various antimicrobial agents (Winner et al, 2000;Papazisi et al, 2003).…”