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Authors: Ian Mackay, Judy Northill
Group: protocols.io Ambassadors, Public Health Virology, Forensic and Scientific Services
Summary: This test is a modification to the World Health Organization's influenza A H3 TaqMan documented in 'WHO information for molecular diagnosis of influenza virus - update 1' (see file below). New primers were added and the WHO primers discarded.The test targets the hemagglutinin (HA) gene.

Proper citation: Ian Mackay, Judy Northill 2019. Influenza A H3 virus TaqMan assay. protocols.io https://dx.doi.org/10.17504/protocols.io.74yhqxw Copy   

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Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was designed and developed at this laboratory.The assay targets the capsid peptide coding region of DENV 1-4 and is desigend as a qualitative screening test for human cases of DENV infection.

Proper citation: Ian Mackay, Judy Northill 2018. Dengue virus (DENV) universal MGB TaqMan 2017. protocols.io https://dx.doi.org/10.17504/protocols.io.ny8dfzw Copy   

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Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: The protocol aims to specifically amplify Nipah viruses (NiV) and not other viruses.This is a modified version of a published assay. Modifications were to account for mismatches underneath oligo target sites.The assay targets the nucleoprotein (N) gene region and is designed as a qualitative test for investigating NiV infection of humans.

Proper citation: Ian Mackay, Judy Northill 2019. Nipah virus real-time RT-PCR (NiV-TM2018). protocols.io https://dx.doi.org/10.17504/protocols.io.rs5d6g6 Copy   

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Authors: Ian Mackay, Alyssa Pyke, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: The protocol aims explicitly to amplify RRV viruses and not other viruses.Alyssa Pyke designed the original assay which was published in 2011 (see below). Subsequently, Alyssa Pyke modified the reverse primer, and it is this most recent, revised version, described here.The assay targets the E2 gene region and is designed as a qualitative test for investigating RRV infection of humans and arthropods.

Proper citation: Ian Mackay, Alyssa Pyke, Judy Northill 2018. Ross River virus E2 TaqMan 2017 (RRV-E2-TM2017). protocols.io https://dx.doi.org/10.17504/protocols.io.rsyd6fw Copy   

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Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR to detect Orientia tsutsugamushi DNA. This method has been adapted from a publication by Jiang et al 2004, and  the oligonucleotides have been modified and a different PCR kit used.

Proper citation: Judy Northill, Ian Mackay 2018. Orientia tsutsugamushi real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.rhfd33n Copy   

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Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR to broadly detect Rickettsia species DNA. This method has been adapted from a publication by Jiang et al 2012, and  the oligonucleotides have been modified and a different PCR kit used.

Proper citation: Judy Northill, Ian Mackay 2018. Rickettsia real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.rg9d3z6 Copy   

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Authors: Ian Mackay, Judy Northill, Alyssa Pyke, Bruce Harrower
Group: Public Health Virology, Forensic and Scientific Services
Summary: This assay was designed and developed by Alyssa Pyke and Bruce Harrower at this laboratory aided by design software in use at the time. The assay specifically targets the haemagglutinin (HA) region of influenza A(H1)pdm09 virus strains and is designed as a qualitative screening test for human cases of infection, but not for infection due to other known influenza viruses. The assay aims to detect any circulating seasonal H1N1 influenza strains, not only influenza A(H1)pdm09 virus.

Proper citation: Ian Mackay, Judy Northill, Alyssa Pyke, Bruce Harrower 2018. Influenza A(H1)pdm09 virus TaqMan (SwFluH1) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.rdhd236 Copy   

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Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: Mayaro virus (MAYV) is an Alphavirus, transmitted by mosquitos in south and central America.This assay is designed to detect a portion of the nsP1 region of MAYV .

Proper citation: Judy Northill, Ian Mackay 2019. Mayaro virus real-time RT-PCR - 2016 method. protocols.io https://dx.doi.org/10.17504/protocols.io.2qxgdxn Copy   

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Authors: Ian Mackay, Claire Y. T. Wang, Katherine E Arden
Summary: This is my preferred, previously published [Ref 1], rhinovirus (RV) and enterovirus (EV) genotyping assay when seeking to characterize the genotypes of respiratory picornavirus identified after use of a screening real-time RT-PCR to test nucleic acid extracts from clinical samples.I have not confirmed that it can detect every single RV genotype but I do know that it detects many from each of the three RV species (Human rhinovirus A, Human rhinovirus B and Human rhinovirus C) as well as at least some Human enterovirus (EV) genotypes.The assay picks up EVs due to the shared genetic similarities in the 5'UTR target region. EVs can be discriminated using subgenomic sequencing (see  VP42 typing assay protocol), or simply described as 'respiratory EVs' since there is no specific-specific vaccine or treatment available anyway. This is a robust primary subgenomic sequencing assay. It is more sensitive than any VP1 protocols because it targets more conserved primer target sites. It produces a more reliable typing result than does the 5'UTR region alone. 

Proper citation: Ian Mackay, Claire Y. T. Wang, Katherine E Arden 2018. Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay"). protocols.io https://dx.doi.org/10.17504/protocols.io.nz7df9n Copy   

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Authors: Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time RT-PCR using an MGB probe, this assay detects Japanese encephalitis virus (JEV) from human and mosquito samples.The assay targets the 3'UTR region of known JEV strains.

Proper citation: Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay 2017. Japanese encephalitis virus real-time RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.kmscu6e Copy   

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Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was designed and developed at this laboratory. The assay specifically targets the 3' UTR region of DENV-4 strains and is designed as a qualitative screening test for human cases of DENV-4 infection, but not for infection due to other known DENVs.

Proper citation: Ian Mackay, Judy Northill 2018. Dengue virus type 4 (DENV-4) TaqMan (DENV4-TM2017) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.n7pdhmn Copy   

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Authors: Ian Mackay
Summary: I and my team used this assay between 2008-2015; we dubbed it the "Nix assay". It targets the 5'UTR and employs quite degenerate oligos.In silico sequence alignments indicated the olignucleotides could theoretically detect aft least HPeV 1-7, 17 and 18.However during a period of assay comparison, another assay (see link below), the "Benschop assay" (J.Clin.Virol. 2008. 41(2):69-74), was found to produce more sigmoidal and higher curves and 1-5 cycle improvements to CT values when compared among the sample sampe extract set.

Proper citation: Ian Mackay 2017. Human Parechovirus A real-time RT-PCR ["Nix assay"; 2008-2015]. protocols.io https://dx.doi.org/10.17504/protocols.io.krpcv5n Copy   

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Authors: Ian Mackay, Judy Northill
Group: protocols.io Ambassadors, Public Health Virology, Forensic and Scientific Services
Summary: This protocol aims to amplify enterovirus (EV) D68 viruses and not other viruses.This protocol is modified from a previously published method cited below. Details of the modification are included in the method.The oligonucleotides target the 5'UTR noncoding region. This is a qualitative test for investigating EV-D68 infection of humans.The test has identified both the original EV-D68 strains and contemporary strains which has been confirmed by subgenomic sequencing of partial 5'UTR-VP2 and partial VP1 sequencing. Culture of the virus is not required as this assay is capable of detecting EV-D68 direct from extracted clinical samples.

Proper citation: Ian Mackay, Judy Northill 2018. Enterovirus (EV) D68 TaqMan 2018 (EV-D68-TM2018). protocols.io https://dx.doi.org/10.17504/protocols.io.vche2t6 Copy   

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Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A nested RT-PCR targeting the RdRp region of the sub-genus Sarbecovirus. The primers are modified from the pan-coronavirus RT-PCR published by Hu et al. 2017 to be more specific to SARS-CoV-2.Assay may be used in resource poor settings where real-time cyclers are not available.Sanger sequencing can be used to confirm SARS-CoV-2 where WGS is not available or where WGS fails due to poor quality sample.

Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp nested RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bcmpiu5n Copy   

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Authors: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay
Summary: This previously unpublished protocol aims to amplify genotype A measles virus (MeV) strains but not non-mealses viruses.Mitchell Finger and Michael Lyon developed this in-house test in 2010.The assay targets the intergenic region between the M (matrix) and F (fusion) genes, designed as a qualitative test for investigating measles vaccine virus (MVV) strains.Numbering indicates the oligonucleotide location on the sequence with MeV strain Edmonston (Moraten vaccine), complete genome, GenBank accession number AF266287.

Proper citation: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay 2019. Measles Vaccine Virus Taqman-MGB. protocols.io https://dx.doi.org/10.17504/protocols.io.2qugdww Copy   

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Authors: Ian Mackay, Claire Y. T. Wang
Summary: I and my team used this assay between 2008-2015; we dubbed it the "Harvala assay". It produces an amplicon that includes the 3' end of VP3 and the 5' end of VP1, spanning the junction.In silico sequence alignments indicated the highly degenerate olignucleotides could theoretically detect af least HPeV 1-7, 17 and 18.

Proper citation: Ian Mackay, Claire Y. T. Wang 2017. Human Parechovirus A conventional nested genotyping RT-PCR ["Harvala assay"; 2008-2015]. protocols.io https://dx.doi.org/10.17504/protocols.io.ksacwae Copy   

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Authors: Judy Northill, Russell Simmons, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR method for the detection of Molluscum contagiosum virus from human samples. This assay targets the MC021L gene and detects both subtype 1 and 2 of the virus.

Proper citation: Judy Northill, Russell Simmons, Ian Mackay 2017. Molluscum contagiosum virus real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.kmtcu6n Copy   

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Authors: Michael Lyon, Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: This was a past assay that we no longer in use. For our favoured Measles TaqMan test, please refer to the MeV N TaqMan protocol.The protocol aims to amplify measles virus (MeV) strains and not other viruses.Michael Lyon designed the original version of this unpublished in-house assay in 1999. It was superseded in 2003 but occassionally brought out as needed for troubelshooting. The superseding test was a first generation MGB-TaqMan protocol called Measles MGB TaqMan FAST.The assay targets the fusion (F) gene region and is designed as a qualitative test for investigating MeV infection of humans.

Proper citation: Michael Lyon, Judy Northill, Ian Mackay 2018. Measles virus fusion (F) gene TaqMan (no longer in regular use; see Guidelines). protocols.io https://dx.doi.org/10.17504/protocols.io.rs7d6hn Copy   

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Authors: Ian Mackay, Katherine E. Arden
Summary: This is a very handy, previously published [Ref 1 and 2], rhinovirus (RV) screening and genotyping assay which I and many others have used on many sample extracts, mostly originating from acutely ill paediatric patients, spanning well over a decade's worth of collection dates.I have not confirmed that it can detect every single RV genotype but I do know that it detects many from each of the three RV species (Human rhinovirus A, Human rhinovirus B and Human rhinovirus C) as well as at least some Human enterovirus (EV) genotypes.The assay picks up EVs due to the shared genetic similarities in the 5'UTR target region.This assay produces a useful backup subgenomic sequencing target for use primarily should the 'Wisdom VP42' protocol fail to amplify a product for sequencing. It is worth noting that these primers span a target region that can produce confusing results. Recombination within the 5' end of the genome of some members of the genus Enterovirus can lead to 5'UTR sequences generated by RV-C genotypes that appear to be RV-As (see the figure below in which green RV-Cs appear in what is expected to be the red RV-A branch of the phylogenetic tree). 

Proper citation: Ian Mackay, Katherine E. Arden 2018. Human rhinovirus screening conventional RT-PCR ("Gama assay"). protocols.io https://dx.doi.org/10.17504/protocols.io.n7gdhjw Copy   

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Authors: Ian Mackay, Ina Smith, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: The protocol aims explicitly to amplify BFV viruses and not other viruses. The assay targets the E2 gene region and is designed as a qualitative test for investigating BFV infection of humans and arthropods.

Proper citation: Ian Mackay, Ina Smith, Judy Northill 2018. Barmah Forest virus TaqMan 2017 (BFV-TM2017). protocols.io https://dx.doi.org/10.17504/protocols.io.rswd6fe Copy   

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