Development of a duplex real-time TaqMan PCR assay with an internal control for the detection ofMycoplasma gallisepticumandMycoplasma synoviaein clinical samples from commercial and backyard poultry

Abstract

In this study, we report the development and validation of a duplex real-time polymerase chain reaction (PCR) assay with an internal control using TaqMan-labelled probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae (duplex MGMS PCR). The MGMS PCR was highly specific with a sensitivity of 7 and 1 colony-forming units/ml for M. gallisepticum and M. synoviae, respectively, using dilution of pure culture that corresponds to 34 and 29 DNA copies per reaction. Validation of the assay was completed with 260 and 27 pooled samples (tracheal swabs) from commercial chickens and turkeys, respectively, with potential M. gallisepticum and M. synoviae involvement and 42 samples (palatine cleft swabs) from backyard geese and ducks. Using isolation as the gold standard, the MGMS PCR was more sensitive than isolation and the analytical sensitivity was 0.944 and 0.958 for M. gallisepticum and M. synoviae, respectively. In comparison with a gapA-based assay (gapA PCR) and a 16S rRNA-based assay (16S PCR) for M. gallisepticum and M. synoviae, respectively, the results agreed for 94.5% and 96.6%, respectively. The use of the internal control allowed monitoring of proper extraction and inhibition of amplification that was detected in 12 samples. The duplex MGMS PCR was shown to be superior to the presently reported real-time PCR assays in terms of combination of sensitivity, specificity and capacity of detection of more than one target in a single tube. In conclusion, the duplex MGMS PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens, turkeys and backyard poultry including ducks and geese.

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

“…Recently, Fraga et al (2013) observed a problem in specificity when using conserved 16SrRNA gene primers for MS which detected Mycoplasma cloacale as well as MG and MS mixed infection in multiplex RT-PCR. Such non-specificity issues have not been reported for species-specific (mgc2 and vlhA genes) primers/probes (Raviv and Kleven 2009;Sprygin et al 2010). We determined specificity in the present study using in silico BLASTn analysis.…”

“…We determined specificity in the present study using in silico BLASTn analysis. Furthermore, the method described by Sprygin et al (2010) incorporated avian Reo virus (S3 gene) as an internal control (IC) in a Real-time PCR assay (Table 1). In contrast, we successfully incorporated commercially available (ready to use) TaqMan ® Exo IPC to avoid false negative results that might happen due to failure in DNA extraction/PCR inhibition.…”

“…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 doi: 10.17221/8179-VETMED TaqMan method has only been reported by Sprygin et al (2010) as shown in Table 1.…”

“…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 doi: 10.17221/8179-VETMED TaqMan method has only been reported by Sprygin et al (2010) as shown in Table 1. To increase the specificity of RT-PCR procedures, TaqMan minor groove binder (MGB TM ) probes are preferred because their small size enables binding in the minor groove of double-stranded DNA, thus stabilising the duplex, which results in high melting temperature (Tm) values (Kutyavin et al 2000;Guo et al 2009), and greater accuracy in measurements because of low background fluorescence (Farkas et al 2009).…”

“…Moreover, according to the requirements of the Office International des Epizooties (OIE), each PCR assay must incorporate an internal control (IC) (OIE 2008). The avian mycoplasma RT-PCR assays previously described by Grodio et al (2008) and Sprygin et al (2010) contained ICs. This study was aimed at developing a duplex TaqMan MGB-probe based RT-PCR, incorporating a TaqMan exogenous internal positive control (TaqMan ® Exo IPC) for simultaneous detection of MG and MS. We propose that the developed assay will serve as a simplified and improved molecular detection method compared to the previously developed monoplex or duplex TaqMan RT-PCR methods for MG and MS.…”