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Authors: Judy Northill
Summary: A real-time assay to detect Aichivirus A (AiV-A) in human samples. It is based in the 3'UTR region and has been employed in our laboratory since 2007.
Proper citation: Judy Northill 2019. Aichivirus real-time RT-PCR 2007 method. protocols.io dx.doi.org/10.17504/protocols.io.z3tf8nn Copy
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, though other coronaviruses may still be detected. Assay may be used in resource poor settings where real-time cyclers are not available.Sanger sequencing should 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 dx.doi.org/10.17504/protocols.io.bgz2jx8e Copy
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 dx.doi.org/10.17504/protocols.io.n7pdhmn Copy
Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A real-time RT-PCR to specifically detect SARS-CoV-2 betacoronavirus also called nCoV-2019 or Wuhan seafood market pneumonia virus. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. The target region is within the ORF1ab sequence.NotesAssay is fully optimised (as of 24Jan2020).This test has identified a clinical positive cases of coronavirus disease (COVID-19)
Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020. protocols.io dx.doi.org/10.17504/protocols.io.bgtmjwk6 Copy
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 dx.doi.org/10.17504/protocols.io.vche2t6 Copy
Authors: Judy Northill, Russell Simmons
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 2017. Molluscum contagiosum real-time PCR. protocols.io dx.doi.org/10.17504/protocols.io.kkscuwe Copy
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 dx.doi.org/10.17504/protocols.io.bcmpiu5n Copy
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 dx.doi.org/10.17504/protocols.io.2qugdww Copy
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 dx.doi.org/10.17504/protocols.io.rs7d6hn Copy
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 dx.doi.org/10.17504/protocols.io.rswd6fe Copy
Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This is my preferred, previously published [Ref 1], rhinovirus (RV) and enterovirus (EV) genotyping assay when seeking to identify the genotype of a respiratory picornavirus detected in a clinical sample extract. It is employed after use of a screening real-time RT-PCR has identified a respiratory picornavirus.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, Judy Northill 2019. Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay"). protocols.io dx.doi.org/10.17504/protocols.io.xb5fiq6 Copy
Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A real-time RT-PCR designed to amplify a portion of the ORF1ab gene of sequences from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)The probe and primers were published by the China CDC and are now included in the WHO in-house assay document.This test has been modified for use with our standard reagents and instrumentation.This test has identified clinical positive cases of coronavirus disease 2019 (COVID-19).
Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR CCDC-ORF1ab 2020. protocols.io dx.doi.org/10.17504/protocols.io.bgtnjwme Copy
Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was designed and developed at this laboratory.
The protocol specifically aims to amplify strains of Influenza B VICTORIA virus lineage and not strains of the YAMAGATA virus lineage or other virus species. The assay targets the haemagglutinin (HA) region and is designed as a qualitative lineage-typing test for human cases of seasonal influenza virus type B infections.
FluB-VICT-TM2018 is ideally used alongside its companion protocol, 'Influenza B virus YAMAGATA lineage TaqMan 2018' (FluB-YAMA-TM2018), which aims to target influenza B virus YAMAGATA lineage strains exclusively. The two assays perform best as UNIPLEX protocols; a drop in sensitivity was observed when combined in a DUPLEX format.
Proper citation: Ian Mackay, Judy Northill 2018. Influenza B virus VICTORIA lineage TaqMan 2018 / FluB-VICT-TM2018. protocols.io dx.doi.org/10.17504/protocols.io.reqd3dw Copy
Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR for the detection of Coxiella burnetii DNA targeting the IS1111a gene. The assay is based on a published method by Banazis et al 2010, using a different PCR kit. The oligonucleitide sequences have not been modified however the concentration have been optimised. This method is used for testing DNA from human blood and tissue samples.
Proper citation: Judy Northill, Ian Mackay 2018. Coxiella burnetii real-time PCR - IS1111a method (Q fever). protocols.io dx.doi.org/10.17504/protocols.io.rhhd336 Copy
Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR for Parapoxvirus targeting the DNA polymerase. It is used to screen human samples where Parapoxvirus is suspected.This protocol is based on the published RVSS assay by Das et al 2017. Oligonucleotides have been modified and a different PCR kit is used.
Proper citation: Judy Northill, Ian Mackay 2018. Parapoxvirus real-time PCR. protocols.io dx.doi.org/10.17504/protocols.io.n38dgrw Copy
Authors: Ian Mackay, Judy Northill
Summary: This assay 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.
Proper citation: Ian Mackay, Judy Northill 2019. Influenza A H3 virus TaqMan assay. protocols.io dx.doi.org/10.17504/protocols.io.2qwgdxe Copy
Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol details how to make and use a pair of synthetic template oligonucleotides (STOs) for use as real-time reverse transcription polymerase chain reaction (RT-rPCR) controls. The method can also be used for PCR controls. This binary positive control approach is easy to replicate once mastered, quick to produce, results in enough RNA stock for years of PCR runs, and provides several benefits over the use of a wild-type positive control such as the nucleic acids extracted from virus culture or from a known previously-positive clinical specimen.The method is an adaptation of a previsouly published approach [Ref 4; see Steps section] and has recently been described in some publications.[Ref 5]Our approach to a positive control requires that each PCR run includes two additional reactions. one controls for primer function (PRIMER control). This STO includes hybridisation sites for the specific target assay's primers bouding a generic probe hybridisation target siteone controls for probe function (PROBE control). This STO includes a hybridisation site for the specific target assay's probe(s), bounded by a pair of generic primer hybridisation target sites
Proper citation: Ian Mackay, Judy Northill 2019. Binary synthetic template oligonucleotide positive control for in-house diagnostic real-time RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.yxjfxkn Copy
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 dx.doi.org/10.17504/protocols.io.ntideke Copy
Authors: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
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 2018. Measles Vaccine Virus Taqman-MGB. protocols.io dx.doi.org/10.17504/protocols.io.suceesw Copy
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 dx.doi.org/10.17504/protocols.io.rdhd236 Copy
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