RT-PCR methods
Detection of influenza A matrix gene by real time Taqman RT-PCR
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Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes
Erica Spackman, Dennis A. Senne, T. J. Myers, Leslie L. Bulaga, Lindsey P. Garber, Michael L. Perdue, Kenton Lohman, Luke T. Daum, David L. Suarez
Development and evaluation of a one-step real-time RT-PCR assay for universal detection of influenza A viruses from avian and mammal species
Alexander Nagy, Veronika Vostinakova, Zuzana Pirchanova, Lenka Cernikova, Zuzana Dirbakova, Miroslav Mojzis, Helena Jirincova, Martina Havlickova, Adam Dan, Krisztina Ursu, Stefan Vilcek, Jitka Hornickova
A universal RT-qPCR assay for “One Health” detection of influenza A viruses
Alexander Nagy, Lenka Černíková, Kateřina Kunteová, Zuzana Dirbáková, Saumya S. Thomas, Marek J. Slomka, Ádám Dán, Tünde Varga, Martina Máté, Helena Jiřincová, Ian H. Brown
A highly sensitive and specific M gene real-time PCR protocol, essentially as described by Spackman et al., provides a simple "yes / no" PCR result for the presence of AIV in clinical specimens. This test detects avian influenza A viruses of all haemagglutinin (HA) subtypes (1-16) by targeting a conserved region of the matrix gene.
Additionally, an updated protocol is provided for the detection of influenza A viruses based on Nagy et al. (2010). This 'Nagy version 2', offers significant improvements, including broader detection of influenza A viruses of all sub-types circulating in all hosts.
Although primarily validated using contemporary viruses of geographical relevance for avian species, the updated assay also shows robust utility for use in diagnostic detection of influenza A viruses from other mammalian hosts, including pigs.
Detection of Eurasian H5 avian influenza virus and High Pathogenicity H5 subtype virus
by real time-PCR
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Novel real-time PCR-based patho- and phylotyping of potentially zoonotic avian influenza A subtype H5 viruses at risk of incursion into Europe in 2017
Mahmoud M Naguib, Annika Graaf, Andrea Fortin, Christine Luttermann, Ulrich Wernery, Nadim Amarin, Hussein A Hussein, Hesham Sultan, Basem Al Adhadh, Mohamed K Hassan, Martin Beer, Isabella Monne, Timm C Harder1
Rapid and sensitive detection of high pathogenicity Eurasian clade 2.3.4.4b avian influenza viruses in wild birds and poultry
Joe James, Amanda H. Seekings, Paul Skinner, Katie Purchase, Sahar Mahmood, Ian H. Brown, Rowena D.E. Hansen, Ashley C. Banyard, Scott M. Reid
The recent global emergence of H5N1 as a dominant circulating HPAI subtype drove the necessity for a rapid assay for its combined detection and pathotype determination.
Through adaptation of the assay described by Naguib et al. (2017) we have validated an alternative assay that enables rapid H5 HPAIV 2.3.4.4b pathotype detection, negating the need to generate cleavage site sequence.
This assay is highly specific for the currently circulating H5 clade 2.3.4.4b that is circulating in Europe, the UK and the USA. This assay has been internally validated and the method published in a peer reviewed manuscript (James et al. 2022). We present this method as an addendum to our pre-existing H5 assay as described in the protocol below.
Eurasian H7 avian influenza real time RT-PCR
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Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses
Marek J. Slomka, Theo Pavlidis, Vivien J. Coward, John Voermans, Guus Koch, Amanda Hanna, Jill Banks, Ian H. Brown
Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes
Erica Spackman, Dennis A. Senne, T. J. Myers, Leslie L. Bulaga, Lindsey P. Garber, Michael L. Perdue, Kenton Lohman, Luke T. Daum, David L. Suarez
This protocol details two different RealTime reverse transcriptase PCR (RRT PCR) detection methods which have been validated and optimised to detect Eurasian H7 avian influenza (Slomka et. al. 2009). Both methods are rapid and sensitive.
The HA2 RRT PCR is the more sensitive of the two and amplifies a product within the HA2 region of the H7 gene. This method modifies a previously published approach to increase specificity to Eurasian H7 AIV isolates (Spackman et al., 2002).
CS RRT PCR is less sensitive and amplifies a product which spans the H7 cleavage site. Sequencing of this CS amplicon allows successful pathotyping of the H7 AIV as either LPAI or HPAI.
One-step real time RT-PCR for detection of H5 and H7 avian influenza virus and cleavage site sequencing
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Identification of Sensitive and Specific Avian Influenza Polymerase Chain Reaction Methods Through Blind Ring Trials Organized in the European Union
M. J. Slomka1 V. J. Coward, J. Banks, B. Z. Löndt, I. H. Brown, J. Voermans, G. Koch, K. J. Handberg, P. H. JØrgensen, M. Cherbonnel-Pansart, V. Jestin, G. Cattoli, I. Capua, A. Ejdersund, P. Thorén, G. Czifra
Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses
Bernd Hoffmann, Donata Hoffmann, Dinah Henritzi, Martin Beer, Timm C. Harder
Development and evaluation of a one-step real-time RT-PCR assay for universal detection of influenza A viruses from avian and mammal species
Nagy A, Vostinakova V, Pirchanova Z, Cernikova L, Dirbakova Z, Mojzis M, Jirincova H, Havlickova M, Dan A, Ursu K, Vilcek S, Hornickova J
This protocol details the requirements to determine and analyse the nucleotide sequences of H5 and H7 Avian Influenza at the cleavage site. Determination of the nucleotide sequence and deduced amino acid sequence of specific regions of the genomes allows the pathogenicity of the viruses to be estimated.
The PCR conditions outlined have been successful in generating amplicons from RNA directly extracted from clinical specimens. Results from the APHA and collaborating EU laboratories have revealed differences in the suitability of various primer pairs for H5 and H7 amplicon production (Slomka et al. 2007).
Choice of primer pair will depend on the required sensitivity and robustness of the method and are discussed within.
H6 (HA2) avian influenza virus real time PCR
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Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes
Erica Spackman, Dennis A. Senne, T. J. Myers, Leslie L. Bulaga, Lindsey P. Garber, Michael L. Perdue, Kenton Lohman, Luke T. Daum, David L. Suarez
Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses
Bernd Hoffmann, Donata Hoffmann, Dinah Henritzi, Martin Beer, Timm C. Harder
Development and evaluation of a one-step real-time RT-PCR assay for universal detection of influenza A viruses from avian and mammal species
Nagy A, Vostinakova V , Pirchanova Z , Cernikova L , Dirbakova Z , Mojzis M , Jirincova H , Havlickova M Dan A , Ursu K , Vilcek S , Hornickova J
This protocol details the requirements for detection of European H6 avian influenza virus (AIV) by Real-Time reverse transcription-PCR (RRT-PCR). This is a rapid, sensitive and specific method for detecting Eurasian H6 AIV directly from clinical samples, with no need of growing first to high titre in embryonated eggs.
The primers and probe used in this protocol were designed from conserved regions within the haemagglutinin 2 (HA2).
Detection of avian influenza A viruses of subtype N1 by real time RT-PCR
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Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection
Sunchai Payungporn, Salin Chutinimitkul, Arunee Chaisingh, Sudarat Damrongwantanapokin, Chantanee Buranathai, Alongkorn Amonsin, Apiradee Theamboonlers , Yong Poovorawan
This protocol details the requirements for the detection of N1 avian influenza viruses by Real-Time reverse transcription-PCR (RRT-PCR). This is a rapid, sensitive and specific method for detecting N1 AIV viruses in swabs and tissues.
Primers and probes sensitive to N1 conserved regions of the neuraminidase (NA) gene were adapted from a multiplex assay (Payungporn et al. 2006) to provide the single-target approach detailed here (Slomka et al. 2012).
Detection of avian influenza A viruses of subtype N5 by real-time RT PCR
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Development and Application of Real-Time PCR Assays for Specific Detection of Contemporary Avian Influenza Virus Subtypes N5, N6, N7, N8, and N9
Joe James, Marek J. Slomka, Scott M. Reid, Saumya S. Thomas, Sahar Mahmood, Alexander M. P. Byrne, Jayne Cooper, Christine Russell, Benjamin C. Mollett, Eric Agyeman-Dua, Steve Essen, Ian H. Brown, Sharon M. Brookes
This protocol details two different versions of the method for detection of subtype N5 avian influenza virus (AIV) by reverse transcriptase RealTime (RRT-PCR). These methods are used for detection of N5 AIV in swabs, tissues and egg amplified material.
Both versions of the method amplify a region of neuraminidase (NA) gene. The first version uses primers and probes for detection of N5 AIVs in global circulation (Hoffmann et al. 2016). The second version, referred to as APHA-2016 N5 RRT-PCR, uses primers and probes modified for optimal detection of Eurasian H5N5 HPAIVs which emerged in 2016 (James et al. 2019).
This second version is recommended in the first instance as it both detects more recently circulating HXN5 HPAIVs and uses fewer primers.
Detection of avian influenza A viruses of subtype N6, N7 and N9 by real time RT-PCR
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Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses
Bernd Hoffmann, Donata Hoffmann, Dinah Henritzi, Martin Beer & Timm C. Harder
This protocol details two versions of a method required to detect avian influenza viruses (AIVs) of subtype N6, N7 or N9 by independent Real-Time reverse transcription-PCR (RRT-PCR) in swabs, tissues and egg amplified material. Both versions of the method amplify a region within the neuraminidase (NA) gene.
In the first instance, version 1 is recommended for detection of HxN6 and H5N6 viruses circulating in Asia (Hoffman et al. 2016) while version 2 is recommended for detection of N7 and N9 viruses (as the primers and probes associated with this version were designed against more contemporaneous viruses (James et al. 2019)).
Detection of avian influenza A viruses of subtype N8 by real time RT-PCR
Sources
Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses
Bernd Hoffmann, Donata Hoffmann, Dinah Henritzi, Martin Beer and Timm C. Harder
Development and Application of Real-Time PCR Assays for Specific Detection of Contemporary Avian Influenza Virus Subtypes N5, N6, N7, N8, and N9
Joe James, Marek J. Slomka, Scott M. Reid, Saumya S. Thomas, Sahar Mahmood, Alexander M. P. Byrne, Jayne Cooper, Christine Russell, Benjamin C. Mollett, Eric Agyeman-Dua, Steve Essen, Ian H. Brown, Sharon M. Brookes
This protocol details two different versions of the method for detection of subtype N8 avian influenza virus (AIV) by Real-Time reverse transcription-PCR (RRT-PCR). These methods are used for detection of N8 AIV in swabs, tissues and egg amplified material.
Both versions of the method amplify a region of neuraminidase (NA) gene of subtype N8 AIVs. The first version uses primers and probes for detection of N8 AIVs in global circulation (Hoffmann et al 2016).
The second version, referred to as APHA-2016 N8 RRT-PCR, uses primers and probes modified for optimal detection of Eurasian H5N8 HPAIVs which emerged 2014-2016 (James et al. 2019). The second version should be used in the first instance as it is designed to detect more recently circulating HXN8 HPAIVs.
Avian Orthoavulavirus Serotype-1 screening real time RT-PCR
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Development of an avian avulavirus 1 (AAvV-1) L-gene real-time RT-PCR assay using minor groove binding probes for application as a routine diagnostic tool
David A. Sutton, David P. Allen, Chad M. Fuller, Jo Mayers, Benjamin C. Mollett, Brandon Z. Londt, Scott M. Reid, Karen L. Mansfield, Ian H. Brown
This protocol details the rapid detection of avian paramyxovirus type 1 (APMV-1) using Real-Time reverse transcription PCR (RRT-PCR) on RNA extracted from chick embryo amplified samples and clinical material (e.g., avian tissues, swabs, and fluids).
RRT-PCR approaches are less time consuming than gel based-analysis, has a reduced risk of sample contamination during the procedure and produces a quantitative result which is used to confirm the presence or absence of APMV-1 RNA.
The PCR is based on a 160 base pair conserved region of APMV-1 L polymerase gene ensuring all 6 lineages can be detected (Sutton et al. 2019).