Lakhasly

The present study appears to be first of its kind in India to standardize
real-time TaqMan PCR for rapid, specific and accurate detection
of MG infection in poultry and its comparison with conventional PCR
using lipoprotein and 16 S rRNA genes of MG, respectively. In our earlier
study, we have used this set of 146 samples to screen Mycoplasma gallisepticum
(MG) and Mycoplasma synoviae (MS) to develop duplex PCR
and compare it with monoplex PCR (Yadav et al., 2022b). The standardized
MG real-time PCR assay in this study was found to be highly
specific when tested with 8 different avian microbes known to inhabit
the avian respiratory tract. The MG real-time PCR assay exclusively
amplified MG DNA. The specificity of the standardized MG real-time
PCR in this study is in concurrence with other MG real-time PCR assays
developed earlier (Carli and Eyigor, 2003; Mekkes and Feberwee,
2005; Callison et al., 2006; Raviv and Kleven, 2009). Detection limit of
the standardized real-time PCR was 0.0625 µg/ml with the pure culture
of field isolate of MG (PT-71; accession no. KX759104.1). Taking the
results of conventional PCR as a gold standard, sensitivity and specificity
of the standardized real-time PCR was found to be 100% and 89.8%,
respectively. While standardization of real-time PCR, results showed an
excellent linear correlation between the MG DNA dilutions and the Ct
values. Previous studies reported the detection limits in colony-forming
units (CFU), copy number and color changing-units (CCU) (Carli and
Eyigor, 2003; Mekkes and Feberwee, 2005; Callison et al., 2006; Jarquin
et al., 2009; Raviv and Kleven, 2009; Sprygin et al., 2010). In this
context, a direct comparison of detection limits between the studies
cannot be made. The real-time PCR detected 10 times lower concentrations
of MG DNA as compared to the conventional PCR, indicating
that low concentration of MG may be missed by conventional PCR assay
(Mekkes and Feberwee, 2005). The greater sensitivity of real-time PCR
may be partially attributable to the use of TaqMan probe for the
detection of products rather than visualization of bands after conventional
gel electrophoresis. In our study, the conventional PCR positive
samples were also found positive in real-time PCR. However, real-time
PCR positive samples do not ensure that it will be positive in conventional
PCR, as the TaqMan based PCR are highly sensitive and can even
detect low copy number of pathogens than conventional PCR. Lipoprotein
gene used to standardize the real-time PCR assay is found to be
the most dominant immunogens in mycoplasmas which have a role in
virulence-associated functions, such as colonization, invasion, and
evasion of host defense (Razin et al., 1998; Browning et al., 2011). Also,
lipoproteins are extremely abundant in the cell membrane of mycoplasmas
(Atalla et al., 2015). Previous studies also reported that TaqMan
PCR was more accurate, sensitive, and specific than conventional PCR
(Leal et al., 2013; Gomes et al., 2017; Yadav et al., 2022c).
Although the culture is considered to be the gold standard for the
detection of Mycoplasma species, but this method is laborious, time-
consuming, expensive and fastidious (Nascimento et al., 1991; Mekkes
and Feberwee, 2005; Müs ¸tak and Kolukirik, 2021). PCR-based methods
have replaced culture method for the rapid and accurate detection of
Mycoplasma species (Marois et al., 2002; Mekkes and Feberwee, 2005;
Fraga et al., 2013; Khalifa et al., 2013; Fujisawa et al., 2019; Yadav et al.,
2022b). Real-time PCR has distinct advantages over conventional PCR,
such as higher reliability, rapidity and prevention of false positive result
due to environmental contamination during post-amplification analysis
(Sprygin et al., 2010).