Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses

Slomka, Marek J.; Pavlidis, T.; Coward, Vivien; Voermans, J.J.M.; Koch, G.; Hanna, Amanda; Banks, Jill; Brown, Ian H.

doi:10.1111/j.1750-2659.2009.00083.x

Cited by 115 publications

(129 citation statements)

References 39 publications

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“…Often these assays are combined with a differentiation of the NA subtypes N1, N2, or N7 [26]. So far, only few studies have focused on the generic detection of the N9 gene [27] or the H7 gene [16,28]. In addition, a one-step H7-specific reverse transcription loopmediated isothermal amplification (LAMP) assay has been described for the identification of H7 viruses and shown to be more sensitive than conventional RT-PCR systems [29].…”

Section: Discussionmentioning

confidence: 99%

Nucleic acid-based detection of influenza A virus subtypes H7 and N9 with a special emphasis on the avian H7N9 virus

et al. 2014

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In 2013, a novel influenza A virus of subtype H7N9 was transmitted from avian sources to humans in China, causing severe illness and substantial mortality. Rapid and sensitive diagnostic approaches are the basis of epidemiological studies and of utmost importance for the detection of infected humans and animals. We developed various quantitative reverse transcriptase PCR (RT-qPCR) assays for (i) the generic detection of the haemagglutinin (HA) gene of H7 viruses or the neuraminidase (NA) gene of N9 viruses, and (ii) the specific detection of HA and NA of the novel avian H7N9/2013 virus. The sensitivity of the newly developed assays was compared with previously published PCRs, and the specificity of all RT-qPCRs was examined using a panel of 42 different H7 and 16 different N9 isolates. Furthermore, we analysed the performance of the RT-qPCR assays with dilution series and diagnostic samples obtained from animal experiments. Our study provides a comprehensive set of RT-qPCR assays for the reliable detection of the novel avian H7N9 virus, with high sensitivity and improved and tailored specificity values compared with published assays. Finally, we also present data about the robustness of a duplex assay for the simultaneous detection of HA and NA of the avian influenza H7N9/2013 virus.

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Section: Discussionmentioning

confidence: 99%

Nucleic acid-based detection of influenza A virus subtypes H7 and N9 with a special emphasis on the avian H7N9 virus

et al. 2014

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“…Slomka et al (2009) described the collection of feathers from a turkey and chicken that had been infected experimentally with H7N1 HPAI at 48 h post inoculation when clinical signs were apparent, and the AI RRT-PCR showed these to be harbouring the virus at titres that were very similar to the highest titres detected in swabs obtained from H7N1 HPAIinfected galliformes. In the present study, feathers were collected from chickens and ducks at four H5N1 HPAIinfected locations when clinical signs were apparent, and in such disease scenarios the testing of feathers may be a highly practicable approach for HPAI RRT-PCR diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…M-gene RRT-PCR was as described by Slomka et al (2009) using the primers and probe originally designed by Spackman et al (2002), and will be referred to as the ''wet'' M-gene RRT-PCR. 2.…”

Section: Virus Isolationmentioning

confidence: 99%

Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam

Slomka

Tong

et al. 2012

Avian Pathology

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Forty-six chickens and 48 ducks were sampled from four Vietnamese poultry premises in 2009 infected with H5N1 highly pathogenic avian influenza (HPAI) clade 2.3.2 and 2.3.4 viruses, which also differed by cleavage site (CS) sequences in their haemagglutinin (HA) genes. All clinical specimens (n0282), namely tracheal and cloacal swabs plus feathers, were tested by five Eurasian reverse-transcriptase AI RealTime polymerase chain reaction (RRT-PCR) methods. Bayesian modelling showed similar high sensitivity for the validated H5 HA2 RRT-PCR and a new modified M-gene RRT-PCR that utilizes lyophilized reagents. Both were more sensitive than the validated ''wet'' M-gene RRT-PCR. Another RRT-PCR, which targeted the H5-gene CS region, was effective for clade 2.3.4 detection, but severely compromised for clade 2.3.2 viruses. Reduced sensitivity of the H5 CS and ''wet'' M-gene RRT-PCRs correlated with mismatches between the target and the primer and/or probe sequences. However, the H5 HA2 RRT-PCR sensitively detected both clade 2.3.2 and 2.3.4 viruses, and agreed with N1 RRT-PCR results. Feather testing from diseased chicken and duck flocks by AI RRT-PCRs resulted in the most sensitive identification of H5N1 HPAI-infected birds. Evolution of new H5N1 HPAI clades remains a concern for currently affected Asian countries, but also for more distant regions where it is important to be prepared for new incursions of H5N1 HPAI viruses. Genetic evidence for adamantane resistance and sensitivity was also observed in isolates from both clades.

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“…Antibodies from egg yolk were extracted with chloroform, as described by Mohammed [12]. Turkey cloacal and oropharyngeal swabs and tissue samples were tested with a pan-influenza A real-time RT-PCR (rRT-PCR) [13], and subtype specific rRT-PCRs for H5, H7 and H1N1pdm09 [14–16]. HA gene sequences from positive samples were based on PCR fragments generated as previously described [17].…”

Section: Methodsmentioning

confidence: 99%

Human to animal transmission of influenza A(H1N1)pdm09 in a turkey breeder flock in Norway

Sjurseth

Gjerset

Bragstad

et al. 2017

Infection Ecology & Epidemiology

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Introduction: Routine surveillance samples disclosed seropositivity to influenza A virus (IAV) in a Norwegian turkey breeder flock. Simultaneous reports of influenza-like symptoms in farm workers and a laboratory confirmed influenza A(H1N1)pdm09 (H1N1pdm09) infection in one person led to the suspicion of a H1N1pdm09 infection in the turkeys. Animals and methods: H1N1pdm09 infection was confirmed by a positive haemaggutinin inhibition test using H1N1pdm09 antigens, and detection of H1N1pdm09 nucleic acid in reproductive organs of turkey hens. The flock showed no clinical signs except for a temporary drop in egg production. Previous reports of H1N1pdm09 infection in turkeys suggested human-to-turkey transmission (anthroponosis) during artificial insemination. Results and discussion: The flock remained seropositive to IAV and the homologous H1N1pdm09 antigen throughout the following 106 days, with decreasing seroprevalence over time. IAV was not detected in fertilised eggs or in turkey poults from the farm, however, maternally derived antibodies against H1N1pdm09 were found in egg yolks and in day-old poults. Genetic analyses of haemagglutinin gene sequences from one of the infected farm workers and turkeys revealed a close phylogenetic relationship, and confirmed human-to-turkey virus transmission.

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Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses

Cited by 115 publications

References 39 publications

Nucleic acid-based detection of influenza A virus subtypes H7 and N9 with a special emphasis on the avian H7N9 virus

Nucleic acid-based detection of influenza A virus subtypes H7 and N9 with a special emphasis on the avian H7N9 virus

Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam

Human to animal transmission of influenza A(H1N1)pdm09 in a turkey breeder flock in Norway

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