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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 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 https://dx.doi.org/10.17504/protocols.io.reqd3dw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bgtnjwme Copy   

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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 https://dx.doi.org/10.17504/protocols.io.rhhd336 Copy   

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Authors: Greg A Smith, Alyssa Pyke, Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was designed and developed at this laboratory by Greg A. Smith and colleagues for Public Helath Virology, Queensland's Department of Health, prior to his departure. It has not been previously published. The protocol aims explicitly to amplify DENV-2 viruses and not other dengue viruses. The assay targets the membrane glycoprotein precursor region and is designed as a qualitative test for investigating suspected human cases of DENV-2 infection. This assay has been superseded by the Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay.

Proper citation: Greg A Smith, Alyssa Pyke, Judy Northill, Ian Mackay 2018. Dengue virus type 2 (DENV-2) prMG-Multiplex TaqMan assay (no longer in use; see Guidelines). protocols.io https://dx.doi.org/10.17504/protocols.io.rkzd4x6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.n38dgrw 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.ntideke Copy   

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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 https://dx.doi.org/10.17504/protocols.io.yxjfxkn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bchvit66 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-1 and is designed as a qualitative screening test for human cases of DENV-1 infection, but not other known DENVs.

Proper citation: Ian Mackay, Judy Northill 2018. Dengue virus type 1 (DENV-1) TaqMan (DENV1-TM2017) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.n7mdhk6 Copy   

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Authors: Alyssa Pyke, Ian Mackay, Frederick Moore, Andrew Van Den Hurk, Judy Northill, Mitchell Finger, Natalie Simpson, Neelima Nair, Peter Burtonclay, Peter Moore, Sarah Wheatley, Sean Moody, Sonja Hall-Mendelin, Elisabeth Gamez, Amanda De Jong, Ben Huang, Carmel Taylor, David Warrilow, Doris Genge, Glen Hewitson, Inga Sultana, Jamie McMahon, Jean Barcelon
Group: protocols.io Ambassadors, Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: We briefly describe a method to inoculate a susceptible cell line with a human patient sample in order to culture the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease, COVID-19.Clinical samples including nasopharyngeal swabs and aspirates were inoculated onto confluent monolayers of African green monkey kidney Vero C1008, clone E6 cells (ATCC®-CRL-1586) grown in Opti-Mem reduced serum growth medium supplemented with 3% foetal bovine serum in polystyrene, flat-sided, screw-cap 3 mL cell culture tubes.This work was conducted in Queensland under PC3 laboratory conditions by experienced scientists.

Proper citation: Alyssa Pyke, Ian Mackay, Frederick Moore, Andrew Van Den Hurk, Judy Northill, Mitchell Finger, Natalie Simpson, Neelima Nair, Peter Burtonclay, Peter Moore, Sarah Wheatley, Sean Moody, Sonja Hall-Mendelin, Elisabeth Gamez, Amanda De Jong, Ben Huang, Carmel Taylor, David Warrilow, Doris Genge, Glen Hewitson, Inga Sultana, Jamie McMahon, Jean Barcelon 2020. Culture of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; f.2019-nCoV). protocols.io https://dx.doi.org/10.17504/protocols.io.bcduis6w Copy   

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Authors: Judy Northill, Ian Mackay
Summary: A real-time RT-PCR 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.Notes: Assay is fully optimised (as of 24Jan2020).A final name for this virus has not been decided (as of 25Jan2020).

Proper citation: Judy Northill, Ian Mackay 2020. Wuhan coronavirus (2019-nCoV) real-time RT-PCR N gene 2020 (Wuhan-N). protocols.io https://dx.doi.org/10.17504/protocols.io.ba86ihze 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-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 https://dx.doi.org/10.17504/protocols.io.n7ndhme 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 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 https://dx.doi.org/10.17504/protocols.io.rdid24e Copy   

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Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
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 2018. Influenza A H3 virus TaqMan assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kmucu6w Copy   

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Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time assay for the detection of Rubella virus RNA from clinical samples. This assay was modified from a published method with oligonucleotides updated, a different kit used, and cycling times adjusted accordingly.

Proper citation: Ian Mackay, Judy Northill 2018. Rubella virus real-time RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.tz9ep96 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 is a handy backup subgenomic sequencing assay for use primarily should the 'Wisdom VP42' protocol fails 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.

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.n5zdg76 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.9tyh6pw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.rszd6f6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.rk3d4yn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.q4ydyxw Copy   

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