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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 dx.doi.org/10.17504/protocols.io.rhfd33n Copy
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 dx.doi.org/10.17504/protocols.io.bchwit7e Copy
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 dx.doi.org/10.17504/protocols.io.kmtcu6n 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 2018. Measles virus TaqMan RT-PCR (no longer in regular use; see Guidelines). protocols.io dx.doi.org/10.17504/protocols.io.rs6d6he Copy
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 dx.doi.org/10.17504/protocols.io.rkzd4x6 Copy
Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol aims to amplify enterovirus (EV) D68 viruses but 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 historical EV-D68 strains and contemporary strains which has been confirmed by subgenomic sequencing of partial 5'UTR-VP2 and partial VP1 regions. 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 2019. Enterovirus (EV) D68 real-time RT-PCR (EV-D68-TM2018). protocols.io dx.doi.org/10.17504/protocols.io.xb4fiqw Copy
Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time RT-PCR to detect the "novel Wuhan" betacoronavirus 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.Not tested on wild-type virus (as of 25Jan2020), it is expected to be capable of only detecting the Wuhan coronavirus.·Limit of detection not yet determined.NotesAssay 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. Novel coronavirus (2019-nCoV) real-time RT-PCR ORF1ab 2020 (Wuhan-ORF1ab; nCoV-specific test). protocols.io dx.doi.org/10.17504/protocols.io.bb3qiqmw Copy
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 dx.doi.org/10.17504/protocols.io.2qxgdxn 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.kmscu6e Copy
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 dx.doi.org/10.17504/protocols.io.rg9d3z6 Copy
Authors: Judy Northill, Ian Mackay
Group: protocols.io Ambassadors, Public Health Virology, Forensic and Scientific Services
Summary: A real-time RT-PCR to detect the "novel Wuhan" betacoronavirus 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.Not tested on wild-type virus (as of 25Jan2020), it is expected to be capable of only detecting the Wuhan coronavirus.NotesAssay 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 ORF1ab 2020 (Wuhan-ORF1ab). protocols.io dx.doi.org/10.17504/protocols.io.bbsginbw Copy
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 dx.doi.org/10.17504/protocols.io.kmucu6w Copy
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 dx.doi.org/10.17504/protocols.io.bcduis6w 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.p4kdquw Copy
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 dx.doi.org/10.17504/protocols.io.ba86ihze 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 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 dx.doi.org/10.17504/protocols.io.bcv9iw96 Copy
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 dx.doi.org/10.17504/protocols.io.rsyd6fw Copy
Authors: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay
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 (no longer in regular use; see Guidelines). protocols.io dx.doi.org/10.17504/protocols.io.2qvgdw6 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-2 strains and is designed as a qualitative screening test for human cases of DENV-2 infection, but not for infection due to other known DENVs.
Proper citation: Ian Mackay, Judy Northill 2018. Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay. protocols.io dx.doi.org/10.17504/protocols.io.n7kdhkw 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-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 dx.doi.org/10.17504/protocols.io.n7mdhk6 Copy
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