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Authors: Ian Mackay, Judy Northill, Alyssa Pyke
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was designed and developed at this laboratory and incorporates a previously published oligoprobe (see below).
The protocol specifically aims to amplify DENV02 viruses and not other dengue viruses. The assay targets the capsid region and is designed as a qualitative test for investigating suspected human cases of DENV-2 infections.
This assay has been superseded by the Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay.
Proper citation: Ian Mackay, Judy Northill, Alyssa Pyke 2018. Dengue virus type 2 (DENV-2) capsid-Thai TaqMan assay (no longer in use; see Guidelines). protocols.io dx.doi.org/10.17504/protocols.io.q4ydyxw Copy
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 2018. Japanese encephalitis virus real-time RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.r23d8gn Copy
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 dx.doi.org/10.17504/protocols.io.74yhqxw 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.ny8dfzw Copy
Authors: Judy Northill, Ian Mackay
Summary: NOT RECOMMENDEDThe sensitivity of the assay has been found to be lower than expected and we no longer recommend it be used.We do recommend the ORF1ab assay (Novel coronavirus (2019-nCoV) real-time RT-PCR ORF1ab 2020) or the E gene assay by Corman et al. (Protocol v2-1)A real-time RT-PCR to designed to detect the "novel Wuhan" betacoronavirus. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. The target region encodes the nucleocapsid (N).Not tested on wild-type virus (as of 25Jan2020), it is expected to be capable of detecting Wuhan virus, bat-like SARS and SARS virus.Limit of detection not yet determined.A single 1 mismatch at probe-binding site identified with the BetaCoV/USA/CA1/2020|EPI_ISL_406034 variant of 2019-nCoV (as of 29JAN2020).Probe is in the 3'-5' (reverse complement) direction.A real-time RT-PCR to designed to detect the "novel Wuhan" betacoronavirus. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. The target region encodes the nucleocapsid (N).Not tested on wild-type virus (as of 25Jan2020), it is expected to be capable of detecting Wuhan virus, bat-like SARS and SARS virus.Limit of detection not yet determined.A single 1 mismatch at probe-binding site identified with the BetaCoV/USA/CA1/2020|EPI_ISL_406034 variant of 2019-nCoV (as of 29JAN2020).Probe is in the 3'-5' (reverse complement) direction.Notes: Assay is optimised (as of 24Jan2020).A final name for this virus has not been decided (as of 25Jan2020).
Proper citation: Judy Northill, Ian Mackay 2020. Novel coronavirus (2019-nCoV) real-time RT-PCR N gene 2020 (Wuhan-N; 2019-nCoV-related test) -NOT RECOMMENDED. protocols.io dx.doi.org/10.17504/protocols.io.bb5piq5n Copy
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 dx.doi.org/10.17504/protocols.io.rs5d6g6 Copy
Authors: Ian Mackay, Judy Northill
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.9tyh6pw Copy
Authors: Judy Northill, Alyssa Pyke, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time RT-PCR targeting the 5' untranslated region of Yellow fever virus.
This protocol was designed and developed at this laboratory.
Proper citation: Judy Northill, Alyssa Pyke, Ian Mackay 2018. Yellow fever virus real-time RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.rszd6f6 Copy
Authors: Ian Mackay, Mitchell Finger, Michael Lyon, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was developed at this laboratory but had not been previously published.
The protocol aims explicitly to amplify hepatitis A virus (HAV) strains and not other virus species. The assay targets the 5' untranslated region and is designed as a qualitative test for investigating suspected human cases of HAV infection.
Proper citation: Ian Mackay, Mitchell Finger, Michael Lyon, Judy Northill 2018. Hepatitis A MGB TaqMan. protocols.io dx.doi.org/10.17504/protocols.io.rk3d4yn Copy
Authors: Ian Mackay
Summary: This version is based on a recipe (see forked version) my team and I 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. Further validation of this recipe is ongoing.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"; 2017-]. protocols.io dx.doi.org/10.17504/protocols.io.k2kcycw Copy
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 dx.doi.org/10.17504/protocols.io.kr9cv96 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 YAMAGATA virus lineage and not strains of the VICTORIA 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-YAMA-TM2018 is ideally used alongside its companion protocol, "Influenza B virus VICTORIA lineage TaqMan 2018" (FluB-VICT-TM2018), which aims to target influenza B virus VICTORIA 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 YAMAGATA lineage TaqMan 2018 / FluB-YAMA-TM2018. protocols.io dx.doi.org/10.17504/protocols.io.rdid24e Copy
Authors: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: This real-time TaqMan-MGB RT-PCR protocol aimed to amplify measles virus (MeV) strains and not other viruses.Michael Lyon and Mitchell Finger designed the assay in 2009 using Primer Express software.The method was later published by Greg Smith in 2010 (see below).The assay targets the fusion (F) gene region and is designed as a qualitative test for investigating MeV infection of humans.This was a past assay that we no longer in use. For our favoured Measles virus TaqMan test, please refer to the MeV N TaqMan protocol.
Proper citation: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay 2019. Measles virus TaqMan RT-PCR (F gene; no longer in regular use; see Guidelines). protocols.io dx.doi.org/10.17504/protocols.io.8b9hsr6 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.n7jdhkn 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 designed to detect SARS-CoV-2 and other related sarbecoviruses. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. The target region encodes the nucleocapsid (N).Tested on wild-type SARS-CoV-2 virus , it is expected to be capable of detecting SARS-CoV-2, bat-like SARS and SARS virus (members of the subgenus Sarbecovirus).Limit of detection not yet determined.The performance of the assay has not been tested with low viral load samples or samples from patients who are clinically well.The sensitivity of this assay was improved with the use of the SensiFast™Probe Lo-ROX One-step kit. A single 1 mismatch at probe-binding site identified with the BetaCoV/USA/CA1/2020|EPI_ISL_406034 (GenBank MN994467.1) variant of SARS-CoV-2 (as of 23JUNE2020).Probe is in the 3'-5' (reverse complement) direction.Reverse primers were replaced in March 2020.We also recommend the ORF1ab assay (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020), US-CDC-N1 assay or the E gene assay by Corman et al. (Protocol v2-1)Notes: Assay is optimised.This test has identified clinical positive cases of coronavirus disease (COVID-19)Notes: Assay is optimised.This test has identified 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 N gene 2020 . protocols.io dx.doi.org/10.17504/protocols.io.bhpwj5pe 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.bdcpi2vn Copy
Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol aims to amplify enterovirus (EV) A71 viruses but not other viruses.This protocol was designed by us.The oligonucleotides target the 5'UTR noncoding region. This is a qualitative test for investigating EV-A71 infection of humans.The test has identified both historical EV-A71 strains and contemporary strains and has confirmed these using 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-A71 direct from extracted clinical samples.
Proper citation: Ian Mackay, Judy Northill 2019. Enterovirus (EV) A71 real-time RT-PCR (EV-A71-TM2018). protocols.io dx.doi.org/10.17504/protocols.io.xb3fiqn 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-3 strains and is designed as a qualitative screening test for human cases of DENV-3 infection, but not for infection due to other known DENVs.
Proper citation: Ian Mackay, Judy Northill 2018. Dengue virus type 3 (DENV-3) TaqMan (DENV3-TM2017) assay. protocols.io dx.doi.org/10.17504/protocols.io.n7ndhme Copy
Authors: Judy Northill, Ian Mackay
Group: protocols.io Ambassadors, 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 case 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 (Wuhan-ORF1ab; 2019-nCoV-related test). protocols.io dx.doi.org/10.17504/protocols.io.bchvit66 Copy
Authors: Judy Northill, David Warrilow, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR targeting the DNA-dependent RNA polymerase of Orthopoxviruses.This protocol was designed and developed at this laboratory.
Proper citation: Judy Northill, David Warrilow, Ian Mackay 2018. Orthopoxvirus real-time PCR. protocols.io dx.doi.org/10.17504/protocols.io.n43dgyn Copy
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