A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

Fraga, Aline Padilha de; Vargas, Tatiana de; Ikuta, Nilo; Fonseca, André Salvador Kazantzi; Celmer, Álvaro José; Marques, Edmundo Kanan; Lunge, Vagner Ricardo

doi:10.1590/s1517-83822013000200028

Cited by 27 publications

(21 citation statements)

References 16 publications

(32 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been indicated that the use of the MGB probe in combination with NFQ leads to improved specificity in the detection of dual infections by diminishing the background fluorescence obtained through the use of fluorescent quenchers (Farkas et al, 2009). Previous studies have already confirmed that the qPCR assay is a successful tool for sensitive and specific detection of MG in commercial chicken flocks (Raviv and Kleven, 2009;Sprygin et al, 2010;Fraga et al, 2013). The present study describes a new TaqMan RT-PCR assay for the concurrent detection of MG occurring in commercial poultry flocks in Saudi Arabia.…”

Section: Discussionmentioning

confidence: 52%

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

Elbehiry¹,

Aldubaib²,

Marzouk³

2017

AJVS

View full text Add to dashboard Cite

Key words:Mycoplasma Gallisepticum, TaqMan RT-PCR assay, Antimicrobial resistance Mycoplasma Gallisepticum (MG) is considered as one of the most economically important mycoplasmal pathogens among poultry industry worldwide. This pathogen has various strains, therefore their detection using culture method is not sufficient. From this point of view, this study was designed to develop a novel TaqMan ® real-time polymerase chain reaction (TaqMan RT-PCR) assay for direct detection of MG using cytadhesin to encode a surface protein (mgc2) containing a TaqMan FAM-labeled minor groove binder probe that targets this gene and to compare it with conventional polymerase chain reaction and immunological methods. Using serial broth dilution methods against identified MG isolates, minimum inhibitory concentrations of various antimicrobial agents were detected. Throughout Al-Qassim region, Kingdom of Saudi Arabia, 208 specimens were collected from 18 commercial chicken broiler farms where respiratory diseases were present. Furthermore, 180 blood serum samples were investigated for serological diagnosis of MG. Serum plate agglutination and enzyme-linked immunosorbent assay techniques detected positive serological identification in 83 (46.11%) and 97 (53.88%) isolates, respectively. The sensitivity recorded for TaqMan RT-PCR was 10 -3 CFU/ml for MG template DNA. Results of TaqMan RT-PCR revealed 169 positive samples (81.25%), while 108 samples (51.92%) were identified as positive through conventional polymerase chain reaction assay. The results of MIC for 11 antimicrobial agents against 60 identified MG isolates indicated that all groups of MG exhibited a higher degree of sensitivity to tiamulin (93.33%) at low level of MIC 50 and MIC 90 (≤0.032 µg/ml) followed by tylosin (85%) and doxycycline (81.66%) with MIC 50 and MIC 90 ranged from ≤0.032 to 2 µg/ml; In contrast, gentamycin, tilmicosin, erythromycin, ciprofloxacin, oxytetracylcine and enrofloxacin did not show a higher activity at low concentrations. In conclusion, the developed TaqMan RT-PCR exhibited higher sensitivity and applicable accuracy than other molecular and serological techniques and thus could be highly recommended for the management of MG susceptibility and to facilitate implementation of effective treatment and prophylactic measures.

show abstract

Section: Discussionmentioning

confidence: 52%

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

Elbehiry¹,

Aldubaib²,

Marzouk³

2017

AJVS

View full text Add to dashboard Cite

show abstract

“…We found higher prevalence for MG using the RPA test compared to the qPCR assay for both free-living and seized birds, which was expected due to the limited accuracy of RPA [29]. MG-prevalence in poultry and wild birds has usually been higher under RPA than in other tests [71–73].…”

Section: Discussionmentioning

confidence: 95%

“…For the detection of anti-MG antibodies, serum samples were tested by a rapid plate agglutination (RPA) test according to SDA ordinance nº 44, as of November 08, 2001. A real-time polymerase chain reaction assay using a Taqman-labeled probe for the detection of M. gallisepticum (commercial kit MG—NewGene®) DNA [28] was applied, and the positive results were sent for confirmation with a Multiplex real-time PCR [29] at Simbios Biotechnology.…”

Section: Methodsmentioning

confidence: 99%

Assessment of Potential Health and Genetic Impacts in Releasing ConfiscatedParoaria coronataandSaltator similis

Cruz

Fünkler

Zani

et al. 2020

Preprint

View full text Add to dashboard Cite

Illegal capture and trade of wild birds has long been a threat to biodiversity. Translocation—the release of individuals from one location into another—is a useful conservation tool in the management of species. However, both health (such as different pathogens) and adaptive (such as local adaptation), differences among populations must be taken into account, as both can impact the recipient population negatively. Here, we provide health and genetic information to support release planning for two of the most trafficked Brazilian wild bird species (Paroaria coronata and Saltator similis). We focused on two fundamental questions: Are there significant differences in pathogen load between wild and captive populations? Is there significant genetic structure among populations? In total, 223 free-living birds were captured, sampled, and released at the same site. Devices and live decoys characteristics were top factors influencing captures. We tested blood, feces, and oropharyngeal swabs from free-ranging (n=101) and confiscated (n=92) birds for Newcastle disease virus, Salmonella spp., and Mycoplasma gallisepticum. Genetic structure among populations was investigated using mtDNA in a subsample of these birds. We found no evidence for Newcastle disease virus and Salmonella spp. in seized and free-living birds from both species. However, seized P. coronata and S. similis may be potential sources of M. gallisepticum. We found significant but low genetic structure among populations occurring in different Biomes (ΦCT=0.26 for P. coronata; ΦCT=0.13 for S. similis) and no significant structure among populations occurring in the Pampa Biome. These results suggest that while it may be important to screen seized birds for avian pathogens, genetic structure among populations seems to be of lesser concern when planning the release of seized songbirds in the wild.

show abstract

“…The MGB probe with NFQ resulted in increased specificity in the detection of double infections by reducing the background fluorescence that might arise through the use of fluorescent quenchers (Farkas et al 2009). 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).…”

Section: Discussionmentioning

confidence: 99%

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

Ehtisham-ul-Haque¹,

Rahman²,

Khan³

et al. 2015

Vet. Med. - Czech

View full text Add to dashboard Cite

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. ...

show abstract

A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

Cited by 27 publications

References 16 publications

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

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

Assessment of Potential Health and Genetic Impacts in Releasing ConfiscatedParoaria coronataandSaltator similis

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

Contact Info

Product

Resources

About