Preclinical Diagnosis of African Swine Fever in Contact-Exposed Swine by a Real-Time PCR Assay

Zsák, László; Borca, Manuel V.; Risatti, Guillermo R.; Zsak, Aniko; French, Richard A.; Lu, Zhiqiang; Kutish, G. F.; Neilan, John G.; Callahan, Johnny D.; Nelson, William M.; Rock, Daniel L.

doi:10.1128/jcm.43.1.112-119.2005

Cited by 147 publications

(142 citation statements)

References 29 publications

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“…30 The oropharynx is an important site for FMDV replication. 22 In addition, ASFV and CSFV replication occur in the tonsil and pharyngeal region resulting in virus secretions from the nasal pharyngeal 5,6,8,31 and oropharyngeal compartments. 26 All 3 disease agents are highly infectious and may be spread by direct or indirect contact via oronasal and oropharyngeal routes.…”

Section: Research-article2015mentioning

confidence: 99%

“…The mRT-qPCR consisted of previously well-characterized and published assays 1,4,21,31 that, at the time of this publication, were in use as single-plex RT-qPCR or qPCR at the USDA-NVSL-FADDL and within the U.S. National Animal Health Laboratory Network (NAHLN). These assays were combined into the multiplex format (Table 2), and the linear dynamic range and relative analytical sensitivity of the mRT-qPCR assay was determined using serial dilutions of pooled purified nucleic acid (purNA) from culture stock viruses.…”

Section: Multiplex Rt-qpcr Linear Dynamic Range Relative Analytical mentioning

confidence: 99%

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Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

et al. 2015

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Abstract. African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations.

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Section: Research-article2015mentioning

confidence: 99%

Section: Multiplex Rt-qpcr Linear Dynamic Range Relative Analytical mentioning

confidence: 99%

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

et al. 2015

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“…A OIE adota em seus manuais a preconizada por King et al (2003), cuja técni-ca foi padronizada com base em sequências do gene da proteína VP72, do capsídeo viral, observadas em diversas amostras virais, de várias partes do mundo e de diferentes grupos genotípicos. Zsak et al (2005) também adotaram a proteína VP72 como alvo, desenvolvendo iniciadores próprios, e testando a viabilidade da técnica em avaliações pré-clínica, poucos dias após a infecção experimental em suínos. Tal técnica é indicada pelo Departamento de Agricultura dos Estados Unidos (USDA), com vistas ao estabelecimento de diagnósticos rápidos e precisos, em situações adversas e em grandes planteis, mesmo antes de manifestações clínicas da doença.…”

Section: Introductionunclassified

Estudo comparativo e validação de três técnicas de PCR em tempo real (qPCR) para diagnóstico de Peste Suína Africana

et al. 2016

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Resumo: Este estudo verificou o desempenho de três técnicas de PCR quantitativa (Real-Time) para o diagnóstico de Peste Suína Africana, uma doença exótica no Brasil, a partir de amostras de tecidos. As três técnicas escolhidas baseiam-se na amplificação de sequências do gene da proteína viral VP72 e são preconizadas, cada uma, por laboratórios oficiais da OIE (PSA-OIE), dos Estados Unidos (PSA-USDA) e da União Europeia (PSA-EU), respectivamente. Oligonucleotídeos iniciadores e sondas de hidrólise marcadas com fluoróforos foram sintetizados conforme a literatura de referência consultada. Sequências-alvo do DNA viral foram inseridos em plasmídeo sintético, os quais serviram de controle positivo para a padronização das técnicas e otimização de reagentes, determinação dos limites de detecção e testes de verificação de desempenho. Para aferição de repetibilidade e reprodutibilidade das técnicas, as técnicas padronizadas foram repetidas em dias diferentes, por um segundo analista, com alteração no mix comercial de reagentes utilizado e em um equipamento diferente, e também por outro laboratório. Realizaram-se, ainda, provas de sensibilidade analítica com amostras de DNA viral de referência e especificidade analítica e diagnóstica, com amostras negativas. As técnicas de PSA-EU e PSA-USDA apresentaram-se mais vantajosas quanto ao consumo de iniciadores. Não houve diferenças significativas nos resultados quantitativos variando-se os dias dos ensaios, os analistas, os equipamentos e o mix de reagentes. As três técnicas apresentaram alta especificidade analítica e diagnóstica e sensibilidade diagnóstica. As três técnicas de qPCR mostraram-se eficazes para serem adotadas por um mesmo laboratório para emissão de diagnósticos oficiais de Peste Suína Africana.

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“…ASF is an economically important swine disease. The clinical signs, which can vary between hyperacute and acute, are typical hemorrhagic symptoms similar to those of classical swine fever, salmonellosis, or erysipelas (Agüero et al, 2003;Zsak et al, 2005;Pérez-Filgueira et al, 2006). ASF is highly contagious and has a high mortality rate in swine populations without vaccination (Hess, 1981;Ley et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

Phylogeographic analysis of African swine fever virus based on the p72 gene sequence

Muangkram¹,

Sukmak²,

Wajjwalku³

2015

Genet. Mol. Res.

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ABSTRACT. African swine fever virus (ASFV) outbreak has been considered as an emerging and re-emerging disease for almost a century. Diagnostically, simple polymerase chain reaction and sequencing-based molecular detection could be employed for both viral identification and genotyping. This study established a novel phylogenetic analysis and epidemiology comparison based on 205 bp of p72 gene sequences. Based on this partial p72 fragment, an updated list of 44 different genotypes from a total of 516 ASFV sequences compiled from GenBank was generated. Nucleotide diversity was 0.04325 ± 0.00231. The analysis of spatial genetic variation divided the ASFV populations of the African continent into four clades (clade A: central and upper eastern Africa; clade B: eastern Africa; clade C: eastern and southern Africa; and clade D: southern Africa). These results and the developed protocol could serve as useful molecular tools for ASFV diagnosis from degraded DNA or putrefied samples, and also 4567 Phylogeographic analysis of African swine fever virus ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (2): 4566-4574 (2015) provide the phylogeographic perspective to identify the origin of viral outbreaks, facilitating the decision planning to limit their spread.

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Preclinical Diagnosis of African Swine Fever in Contact-Exposed Swine by a Real-Time PCR Assay

Cited by 147 publications

References 29 publications

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

Estudo comparativo e validação de três técnicas de PCR em tempo real (qPCR) para diagnóstico de Peste Suína Africana

Phylogeographic analysis of African swine fever virus based on the p72 gene sequence

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