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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: Judy Northill
Summary: This RT-PCR will detected Aichivirus A from human samples. It spans the junction region of 3C and 3D and is used for genotyping.

Proper citation: Judy Northill 2019. Aichivirus 3C3D RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.2i7gchn 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, 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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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 https://dx.doi.org/10.17504/protocols.io.r23d8gn 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, 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 https://dx.doi.org/10.17504/protocols.io.bgz2jx8e 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 envelope gene of sequences from the Betacoronavirus sub-genus Sarbecovirus.The probe and primers were published by Corman et al., and we have slightly modified the protocol, increasing the concentration of the reverse primer, using a different kit and different cycling conditions.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 E gene 2020. protocols.io https://dx.doi.org/10.17504/protocols.io.bcv9iw96 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: NOT RECOMMENDED FOR SCREENINGThe 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 (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 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 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).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 (members of the subgenus Sarbecovirus).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 SARS-CoV-2 (as of 29JAN2020).Probe is in the 3'-5' (reverse complement) direction.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).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 (members of the subgenus Sarbecovirus).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 SARS-CoV-2 (as of 29JAN2020).Probe is in the 3'-5' (reverse complement) direction.Notes: Assay is 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 N gene 2020 (Wuhan-N; 2019-nCoV-related test) -NOT RECOMMENDED. protocols.io https://dx.doi.org/10.17504/protocols.io.bchwit7e Copy   

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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 https://dx.doi.org/10.17504/protocols.io.xb3fiqn Copy   

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