Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region

Steer, Penelope A.; Kirkpatrick, Naomi C.; O’Rourke, Denise; Noormohammadi, Amir H.

doi:10.1128/jcm.00557-09

Cited by 23 publications

(37 citation statements)

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“…These viruses were identified as FAdV-1, FAdV-8b and FAdV-11 by virus neutralization and polymerase chain reaction-high resolution melt curve (PCR-HRM) genotyping (Steer et al, 2011), and their genotypes confirmed by sequencing the loop 1 region of the hexon gene, which showed they were identical in this region to GenBank sequences GU120272, GU120267 and GU120269, respectively. The viruses were propagated, as described previously (Steer et al, 2009(Steer et al, , 2011, from the infected hepatic or bursal tissue of broiler chickens from flocks experiencing outbreaks of IBH.…”

Section: Methodsmentioning

confidence: 99%

Chronological analysis of gross and histological lesions induced by field strains of fowl adenovirus serotypes 1, 8b and 11 in one-day-old chickens

et al. 2015

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Fowl adenoviruses (FAdVs) cause diseases in domestic chickens, including inclusion body hepatitis (IBH), with immunosuppression believed to play a role in their pathogenesis. To gain a better understanding of the pathogenesis and chronology of disease caused by FAdVs, the gross pathology, histopathology and dissemination of virus were examined at several different time points, after inoculation of one-day-old specific pathogen-free chickens with FAdV-1, FAdV-8b or FAdV-11 via the ocular route. FAdV-8b had a slightly greater virulence than FAdV-11, but both were primary pathogens. The presence and severity of hepatic lesions were used to define the three stages of the disease: incubation (1-3 days post-inoculation, PI), degeneration (4-7 days PI) and convalescence (14 days PI). Both viruses were detected in the liver, kidney, bursa, thymus and gizzard of most birds during the degenerative stage, and persisted in the gizzard into convalescence. The FAdV-1 isolate was found to be apathogenic, but virus was detected in the bursa and/or gizzard of several birds between 2 and 7 days PI. This is the first study examining the chronology of gross and microscopic lesions of pathogenic and apathogenic FAdVs in association with viral presence in multiple tissues. It was concluded that both FAdV-8b and FAdV-11 are primary pathogens, and that these strains may play a role in immunosuppression.

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Section: Methodsmentioning

confidence: 99%

Chronological analysis of gross and histological lesions induced by field strains of fowl adenovirus serotypes 1, 8b and 11 in one-day-old chickens

et al. 2015

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“…The methodology has been widely used in medical research applications including detection of cancer gene mutations (Simi et al 2008) and characterization of clinical pathogens (Mancini et al 2010). The technique has also found wider applications, including classification and identification of poultry pathogens (Ghorashi et al 2010;Steer et al 2009), discrimination of cultivars/genotypes and identification of novel alleles of plants (Hofinger et al 2009;Mackay et al 2008), and authentication of plant and food products (Ganopoulos et al 2011).…”

Section: Applications Of Hrm Analysis In Plant Fungal Diagnosticsmentioning

confidence: 99%

High-Resolution Melting approaches towards plant fungal molecular diagnostics

et al. 2014

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Reliable and early molecular detection of phytopathogenic fungi is crucial in an era where the expansion of global trade in plant material is undoubtedly increasing the risk of invasive outbreaks, with devastating effects in crop production. Genetic variation within and between fungal species or strains is also important for screening isolates regarding various resistance attributes. Until today many approaches have been employed in fungal diagnostics which are either laborand time-consuming or costly and of reduced sensitivity. Here, we demonstrate and review recent advances on high-resolution melting (HRM) analysis as a rapid, accurate and powerful tool, capable of differentiating even closely related fungal isolates. HRM technique is based on monitoring the melting of PCR amplicons, using saturating concentrations of a fluorescent intercalating dye that binds to double-stranded DNA. Additionally, we discuss the four case studies inferring applications of HRM analysis towards either genotyping of closely related fungal species or screening for fungicide resistance. We focus on the promising results of these studies, giving some technical considerations and describing the advantages of the application of this approach. Finally, we discuss current prospects and applications for research and development related to this innovative HRM technique in plant fungal diagnostics.

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“…DNA was amplified using 100 pg of plasmid DNA or 10 ng of genomic DNA as a template in a reaction mixture including 200 μM of each dNTP, 0.3 μM of each primer, 1× PCR buffer without Mg 2+ , 1.5 mM of MgCl 2 , and 0.4 unit of Taq polymerase at 94°C for 2 min followed by 50 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s for Expand Hi Fi; in a reaction mixture including 200 μM of each dNTP, 0.3 μM of each primer, 1× PCR buffer, and 0.5 unit of Taq polymerase at 95°C for 15 min followed by 50 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s for HotStarTaq; in a reaction mixture including 200 μM of each dNTP, 0.3 μM of each primer, 1× PCR buffer without Mg 2+ , 1.5 mM of MgCl 2 , and 0.4 unit of Taq polymerase at 94°C for 1 min, followed by 50 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s for Platinum Taq; or in a reaction mixture including 200 μM of each dNTP, 0.3 μM of each primer, 1× PCR buffer, and 0.5 unit of Taq polymerase at 50 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 30 s for Ex Taq with GeneAmp PCR system 2700 (Applied Biosystems, Foster City, CA). After the PCR reaction, SYTO9, EvaGreen, LCGreen Plus, or Resolight was added into the reaction mixture in the presence of the indicated concentrations of MgCl 2 , and DNA melting was monitored from 75°C to 99°C after incubation at 95°C for 1 min and 50°C for 1 min using a Rotor-Gene 6000 (Qiagen) as described previously (Steer et al, 2009). DNA melting curves were corrected by normalization at the start and end points of the curves, and the confidence percentages (C%) for the curves were provided based on HRM algorithm using Rotor-Gene operating software ver.1.7.75 (Menon et al, 2007;Hewson et al, 2009).…”

Section: Hrm Analysismentioning

confidence: 99%

Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer

Aihara¹,

Yamamoto²,

Nishioka³

et al. 2012

Gene

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Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region

Cited by 23 publications

References 0 publications

Chronological analysis of gross and histological lesions induced by field strains of fowl adenovirus serotypes 1, 8b and 11 in one-day-old chickens

Chronological analysis of gross and histological lesions induced by field strains of fowl adenovirus serotypes 1, 8b and 11 in one-day-old chickens

High-Resolution Melting approaches towards plant fungal molecular diagnostics

Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer

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