High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Goodman, Laura B.; Anderson, Renee; Slater, Marcia; Ortenberg, Elen; Renshaw, Randall W.; Chilson, Brittany D.; Laverack, Melissa; Beeby, John; Dubovi, Edward J.; Glaser, Amy L.

doi:10.3791/54781-v

Cited by 2 publications

(5 citation statements)

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“…Preamplified products were loaded onto custom nanoscale PCR plates (OpenArray; Applied Biosystems) printed in a 18 × 3 format. 46 The pathogens targeted included canine respiratory coronavirus (CRCoV, betacoronavirus), CDV, CAV, canine parainfluenza virus (CPiV), influenza A, Mycoplasma cynos , and Streptococcus zooepidemicus . The CAV assay is optimized for detection of CAV-2.…”

Section: Methodsmentioning

confidence: 99%

“…Myocardial samples were tested with a comprehensive panel of validated polymerase chain reaction (PCR) assays to screen for possible causes of canine myocarditis in our region (northeastern United States), encompassing reported causes of canine (CPV-2, canine distemper virus [CDV], CHV, Borrelia burgdorferi , West Nile virus [WNV], Bartonella spp , Rickettsia spp , and Neospora caninum ) and human myocarditis (adenovirus, herpesviruses, and influenza virus), as well other agents common in the canine population. 46…”

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confidence: 99%

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The Causes of Canine Myocarditis and Myocardial Fibrosis Are Elusive by Targeted Molecular Testing: Retrospective Analysis and Literature Review

et al. 2019

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Myocarditis can cause death or permanent heart damage. As epidemiologic and etiopathologic data for canine myocarditis are lacking, we performed a retrospective study using nucleic acid extracted from archived (2007 to 2015) tissues from myocarditis cases and control dogs without myocardial lesions. Heart tissue from pediatric/juvenile and adult dogs was tested with a comprehensive panel of conventional and real-time polymerase chain reaction (PCR) assays targeting recognized agents of canine myocarditis based on a literature review and informed by the comparative epidemiology of human myocarditis. The PCR screen, which included canine parvovirus 2 (CPV-2), canine distemper virus, canine herpesvirus, Borrelia spp, West Nile virus, adenovirus, parainfluenza virus, pneumovirus, respiratory coronavirus, influenza virus, Bartonella spp, Rickettsia spp, Mycoplasma spp, and Neospora caninum, did not detect agents in 35 of 66 cases (53%; 95% confidence interval [CI], 41%-65%) and was frequently negative in adults (21/26); by comparison, agents were not detected in 27 of 57 controls (47%; 95% CI, 35%-60%). Canine distemper virus, herpesvirus, adenovirus, coronavirus, parainfluenza virus, Mycoplasma haemocanis, and N. caninum were occasionally detected in both cases and controls; thus, PCR detection was not considered to indicate causation. We previously reported that CPV-2 continues to be associated with myocarditis in young dogs despite widespread vaccination; in adults, CPV-2 was detected in 2 of 26 cases and 4 of 22 controls. As several agents were similarly detected in cases and controls, it is unclear if these are cardiopathogenic, incidental, or latent. West Nile virus was detected at the analytic limit in 1 adult case. We did not detect Borrelia spp, Bartonella spp, Rickettsia spp, or influenza A virus in the myocarditis cases. These data demonstrate the limitations of current targeted diagnostic tests and the need for additional research to identify unknown agents and develop testing strategies for canine myocarditis.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

The Causes of Canine Myocarditis and Myocardial Fibrosis Are Elusive by Targeted Molecular Testing: Retrospective Analysis and Literature Review

et al. 2019

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“…This nanoscale PCR workflow has been described previously. 11 Custom nanoscale PCR amplification plates arranged in an 18×3 gene expression format were ordered with the M. cynos tuf oligonucleotides listed above, in addition to oligonucleotides for other pathogen and control tests. 11 The forward and reverse primers were also included in a pre-amplification/reverse-transcription pool in combination with the other respiratory panel primers at 9 μM (Integrated DNA Technologies, Coralville, IA).…”

Section: Methodsmentioning

confidence: 99%

“…11 Custom nanoscale PCR amplification plates arranged in an 18×3 gene expression format were ordered with the M. cynos tuf oligonucleotides listed above, in addition to oligonucleotides for other pathogen and control tests. 11 The forward and reverse primers were also included in a pre-amplification/reverse-transcription pool in combination with the other respiratory panel primers at 9 μM (Integrated DNA Technologies, Coralville, IA). This pool was combined with random primers at 300-nM final concentration (New England Biolabs, Ipswich, MA), TaqMan Fast virus 1-step master mix (ABI; Thermo Fisher Scientific), and 7 μL of template in a 10-µL total volume.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of a novel Mycoplasma cynos real-time PCR assay

Tallmadge¹,

Anderson²,

Mitchell³

et al. 2019

J VET Diagn Invest

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Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3-97.9% (r 2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1-122.5% (r 2 = 0.9784). The assay performed very well over a range of template input, from 10 9 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

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High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Cited by 2 publications

References 4 publications

The Causes of Canine Myocarditis and Myocardial Fibrosis Are Elusive by Targeted Molecular Testing: Retrospective Analysis and Literature Review

The Causes of Canine Myocarditis and Myocardial Fibrosis Are Elusive by Targeted Molecular Testing: Retrospective Analysis and Literature Review

Characterization of a novel Mycoplasma cynos real-time PCR assay

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