Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus

Tignon, Marylène; Gallardo, Carmina; Iscaro, Carmen; Hutet, Evelyne; Stede, Yves Van der; Kolbasov, Denis; Mia, Gian Mario De; Potier, Marie‐Frédérique Le; Bishop, Richard P.; Arias, Marisa; Koenen, F.

doi:10.1016/j.jviromet.2011.09.007

Cited by 121 publications

(129 citation statements)

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“…These PCRs are the most widely used at the national reference laboratory (NRL) level within the EU. New real-time PCRs developed in recent years have been shown to provide greater sensitivity for detecting animals that have survived infection (38,39). Other assays, such as antigen detection enzymelinked immunosorbent assay (ELISA), which allows for largescale testing of samples, are also available at the NRL level but have been reported as having lower analytical sensitivity than that of PCR tests (34).…”

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Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs

et al. 2015

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A frican swine fever (ASF) is a complex and lethal viral disease affecting swine and has a significant socioeconomic impact on both the developed and developing world. It has a major negative effect on national, regional, and international trade and constrains pig production in affected areas. The devastating acute form of the disease is characterized, among other features, by functional and congestive-hemorrhagic disorders of the digestive and respiratory systems and causes around 100% mortality in infected pigs (1). Both European wild boars (Sus scrofa) and feral pigs are susceptible and exhibit clinical signs and mortality rates similar to those of domestic pigs. In contrast, African wild pigs (Phacochoerus and Potamochoerus spp.) are resistant to the disease (2-10).The causative agent of the disease, the ASF virus (ASFV), is a large double-stranded DNA virus and the only member of the Asfarviridae family, genus Asfivirus (11, 12). The virus genome is 170 to 192 kb long (13-17). ASF is endemic in sub-Saharan Africa, where it was first described in 1921 (18). Several outbreaks have occurred since then in Europe and South and Central America. In most non-African countries, the disease has been successfully eradicated, the only exception being Sardinia (Italy), where the disease is still endemic (19,20). In April 2007, the disease spread from East Africa to the Republic of Georgia (21), and outbreaks occurred in Armenia, Azerbaijan, and the Russian Federation (22). The ongoing spread of ASFV into adjacent eastern European countries, such as Ukraine (23,24) and Belarus (25), and the situation in Russia affecting both wild boars and domestic pigs placed neighboring areas in the European Union (EU) at risk for the spread of ASFV. The first cases of ASF in wild boars in Lithuania and Poland were reported in early 2014 in areas bordering . According to the World Organisation for Animal Health (OIE), during 2014, nearly 260 ASF cases or outbreaks in wild boars and domestic pigs were detected in EU countries (Latvia, Lithuania, Estonia, and Poland). This situation, combined with the uncertainty present in Belarus, has created a permanent risk of reintroducing ASF into the EU via wild boars or the illegal trade of contaminated pork products and waste (31).No vaccine is available to prevent ASF infection. The control and eradication measures applicable are based on classical disease control methods, including surveillance, epidemiological investigation, tracing of pigs, and stamping out in infected holdings. Citation Gallardo C, Nieto R, Soler A, Pelayo V, Fernández-Pinero J, MarkowskaDaniel I, Pridotkas G, Nurmoja I, Granta R, Simón A, Pérez C, Martín E, Fernández-Pacheco P, Arias M. 2015. Assessment of African swine fever diagnostic techniques as a response to the epidemic outbreaks in eastern European Union countries: how to improve surveillance and control programs.

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Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs

et al. 2015

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“…Todas adotaram como alvo para as reações o gene p72, aquele que apresenta maiores nível de sequências conservadas e diversidade de genótipos virais, se comparado com outros genes também preconizados em técnicas moleculares (Tignon et al 2011). A determinação de iniciadores foi obtida pelo alinhamento de várias sequências depositadas no GenBank, oriundas de vírus de até 11 genótipos diferentes.…”

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“…As técnicas utilizaram, ainda, a construção de plasmídeos contendo sequências miméticas do gene p72, justificando que a ausência de outras sequências do genoma viral representaria ganho na especificidade das qPCR. Tignon et al (2011) e Zsak et al (2005 fizeram validações mais rigorosas de seus ensaios ao incluir avaliações clíni-cas após a infecção experimental de suínos com amostras virais crescidas em culturas de células.…”

Section: Discussionunclassified

“…A utilização de plasmídeos com as sequências de interesse do gene p72 possibilitou a praticidade das técnicas, o controle da concentração de DNA empregada e a contaminação experimental de amostras de tecidos sem, contudo, interferir no processo de extração de DNA adotado. A partir da técnica para PSA-EU de Tignon et al (2011), no entanto, não foi desenvolvida a multiplex, com detecção simultânea do gene da beta-actina, um importante normalizador celular. Em vez disso, as reações qPCR para beta-actina foram realizadas em paralelo.…”

Section: Discussionunclassified

“…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. As abordagens diagnós-ticas sobre a detecção dos genes da proteína VP72 foram aprofundadas por Tignon et al (2011), em que a técnica de qPCR foi ampliada para um formato multiplex, que detecta, simultaneamente, a presença de genes da beta-actina de artiodáctilos para controle endógeno de extração de DNA em tecidos. Tal técnica foi aprovada por laboratórios de referência da União Europeia (EU), com métodos de validação bastante rigorosos.…”

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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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Trace amounts of African swine fever virus DNA detected in insects collected from an infected pig farm in Estonia

Herm

Tummeleht

Jürison

et al. 2019

Veterinary Medicine & Sci

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Background African swine fever (ASF), a severe multi‐systemic disease in pigs, was introduced into Estonia in 2014. The majority of outbreaks have occurred during the summer months. Given that ASFV is transmitted in a sylvatic cycle that includes the transmission by African soft ticks and that mechanical transmission by flying insects was shown, transmission by other arthropod vectors need to be considered. Objectives Here, we report the results of a pilot study on flying insects caught on an outbreak farm during epidemiological investigations. Methods In brief, 15 different insect species (flies and mosquitoes) were collected by random catch using an aerial net. Nucleic acids derived from these samples or their pools were tested for African swine fever virus (ASFV) DNA by real‐time PCR. Results and Conclusions Viral DNA was detected in small quantities in two samples from flies and mosquitoes. Given the slow spread of virus within the farm, the impact of these findings seems rather low, but a role in local transmission cannot be ruled out. However, given the very low number of insects sampled, and taken into the account that viral isolation was not performed and insects outside the farm were not investigated, future investigations are needed to assess the true impact of insects as mechanical vectors.

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Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus

Cited by 121 publications

References 36 publications

Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs

Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs

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

Trace amounts of African swine fever virus DNA detected in insects collected from an infected pig farm in Estonia

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