RT-PCR methods
Detection of influenza A matrix gene by real time Taqman RT-PCR
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.
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Protocols
Detection of Eurasian H5 avian influenza virus and High Pathogenicity H5 subtype virus by real time-PCR
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.
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Protocols
Eurasian H7 avian influenza real time RT-PCR
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.
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Protocols
One-step real time RT-PCR for detection of H5 and H7 avian influenza virus and cleavage site sequencing
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.
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Protocols
H6 (HA2) avian influenza virus real time PCR
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).
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Protocols
Detection of avian influenza A viruses of subtype N1 by real time RT-PCR
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).
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Protocols
Detection of avian influenza A viruses of subtype N5 by real-time RT PCR
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.
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Protocols
Detection of avian influenza A viruses of subtype N6, N7 and N9 by real time RT-PCR
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)).
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Protocols
Detection of avian influenza A viruses of subtype N8 by real time RT-PCR
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.
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Protocols
Avian Orthoavulavirus Serotype-1 screening real time RT-PCR
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).