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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020 Resource Report Resource Website |
Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.bgtmjwk6 | Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community | 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) | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 1 | 2020 | Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020. protocols.io https://dx.doi.org/10.17504/protocols.io.bgtmjwk6 | 2021-04-15 09:14:24 | |||
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Enterovirus (EV) D68 real-time RT-PCR (EV-D68-TM2018) Resource Report Resource Website |
Ian Mackay, Judy Northill | DOI:10.17504/protocols.io.xb4fiqw | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | https://www.ncbi.nlm.nih.gov/pubmed/25542461 | 2 | 2019 | Ian Mackay, Judy Northill 2019. Enterovirus (EV) D68 real-time RT-PCR (EV-D68-TM2018). protocols.io https://dx.doi.org/10.17504/protocols.io.xb4fiqw | 2021-04-15 09:14:50 | ||
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Human rhinovirus screening real-time RT-PCR ("modified Lu assay") Resource Report Resource Website |
Ian Mackay | DOI:10.17504/protocols.io.nz4df8w | This is an excellent rhinovirus screening assay which I have used on thousands of 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 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 genotypes.The assay does pick up some enteroviruses due to their genetic similarities in the 5'UTR target region. These can be discriminated using subgenomic sequencing (see VP42 typing assay protocol), or simply described as "respiratory enteroviruses" since there is no specific-specific vaccine or treatment available anyway. The assay was created and first described in 2008 by Xiaoyan Lu and colleagues at the US CDC [Ref 1]. In collaboration with Lu et al., we published a small update that included Lu's modified forward primer design. We routinely use it with this primer (hence the 'mod' text added to the primer name) but without the expensive special base, as outlined in Step 1. | The University of Queensland | 1 | 2018 | Ian Mackay 2018. Human rhinovirus screening real-time RT-PCR ("modified Lu assay"). protocols.io https://dx.doi.org/10.17504/protocols.io.nz4df8w | 2021-04-15 09:14:35 | ||||
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Parechovirus real-time RT-PCR Resource Report Resource Website |
Ian Mackay | DOI:10.17504/protocols.io.khpct5n | The University of Queensland | 2 | 2017 | Ian Mackay 2017. Parechovirus real-time RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.khpct5n | 2021-04-15 09:15:06 | |||||
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Novel coronavirus (2019-nCoV) real-time RT-PCR ORF1ab 2020 (Wuhan-ORF1ab; nCoV-specific test) Resource Report Resource Website |
Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.bb3qiqmw | Public Health Virology, Forensic and Scientific Services | 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). | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 2 | 2020 | Judy Northill, Ian Mackay 2020. Novel coronavirus (2019-nCoV) real-time RT-PCR ORF1ab 2020 (Wuhan-ORF1ab; nCoV-specific test). protocols.io https://dx.doi.org/10.17504/protocols.io.bb3qiqmw | 2021-04-15 09:15:25 | |||
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Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay") Resource Report Resource Website |
Ian Mackay, Judy Northill | DOI:10.17504/protocols.io.xb5fiq6 | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786677/ | 1 | 2019 | 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.xb5fiq6 | 2021-04-15 09:15:26 | ||
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Measles virus TaqMan RT-PCR (no longer in regular use; see Guidelines) Resource Report Resource Website |
Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.rs6d6he | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 1 | 2018 | Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay 2018. Measles virus TaqMan RT-PCR (no longer in regular use; see Guidelines). protocols.io https://dx.doi.org/10.17504/protocols.io.rs6d6he | 2021-04-15 09:15:35 | |||
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Influenza A H3 virus TaqMan assay Resource Report Resource Website |
Ian Mackay, Judy Northill | DOI:10.17504/protocols.io.2qwgdxe | 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. | Public Health Virology, Forensic and Scientific Services, Queensland Health, Public Health Virology, Forensic and Scientific Services | 2 | 2019 | Ian Mackay, Judy Northill 2019. Influenza A H3 virus TaqMan assay. protocols.io https://dx.doi.org/10.17504/protocols.io.2qwgdxe | 2021-04-15 09:15:37 | ||||
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Measles Vaccine Virus Taqman-MGB Resource Report Resource Website |
Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.suceesw | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 2 | 2018 | Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay 2018. Measles Vaccine Virus Taqman-MGB. protocols.io https://dx.doi.org/10.17504/protocols.io.suceesw | 2021-04-15 09:15:13 | |||
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Orthopoxvirus real-time PCR Resource Report Resource Website |
Judy Northill, David Warrilow, Ian Mackay | DOI:10.17504/protocols.io.n43dgyn | Public Health Virology, Forensic and Scientific Services | A real-time PCR targeting the DNA-dependent RNA polymerase of Orthopoxviruses.This protocol was designed and developed at this laboratory. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 3 | 2018 | Judy Northill, David Warrilow, Ian Mackay 2018. Orthopoxvirus real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.n43dgyn | 2021-04-15 09:15:46 | |||
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Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay Resource Report Resource Website |
Ian Mackay, Judy Northill | DOI:10.17504/protocols.io.n7kdhkw | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 1 | 2018 | Ian Mackay, Judy Northill 2018. Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.n7kdhkw | 2021-04-15 09:15:18 | |||
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Parapoxvirus real-time PCR Resource Report Resource Website |
Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.p4kdquw | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | http://journals.sagepub.com/doi/abs/10.1177/1040638716680676 | 2 | 2018 | Judy Northill, Ian Mackay 2018. Parapoxvirus real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.p4kdquw | 2021-04-15 09:15:29 | ||
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Wuhan coronavirus (2019-nCoV) real-time RT-PCR ORF1ab 2020 (Wuhan-ORF1ab) Resource Report Resource Website |
Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.bbsginbw | protocols.io Ambassadors, Public Health Virology, Forensic and Scientific Services | 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). | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 1 | 2020 | Judy Northill, Ian Mackay 2020. Wuhan coronavirus (2019-nCoV) real-time RT-PCR ORF1ab 2020 (Wuhan-ORF1ab). protocols.io https://dx.doi.org/10.17504/protocols.io.bbsginbw | 2021-04-15 09:15:34 | |||
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Japanese encephalitis virus real-time RT-PCR Resource Report Resource Website |
Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay | DOI:10.17504/protocols.io.kr9cv96 | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | https://doi.org/10.1089/vbz.2013.1373 | 2 | 2017 | Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay 2017. Japanese encephalitis virus real-time RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.kr9cv96 | 2021-04-15 09:15:41 | ||
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Human Parechovirus A real-time RT-PCR ["Nix assay"; 2017-] Resource Report Resource Website |
Ian Mackay | DOI:10.17504/protocols.io.k2kcycw | 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. | The University of Queensland | 4 | 2017 | Ian Mackay 2017. Human Parechovirus A real-time RT-PCR ["Nix assay"; 2017-]. protocols.io https://dx.doi.org/10.17504/protocols.io.k2kcycw | 2021-04-15 09:16:07 | ||||
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Measles virus TaqMan RT-PCR (F gene; no longer in regular use; see Guidelines) Resource Report Resource Website |
Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.8b9hsr6 | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 3 | 2019 | 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 https://dx.doi.org/10.17504/protocols.io.8b9hsr6 | 2021-04-15 09:16:12 | |||
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Dengue virus (DENV) universal MGB TaqMan 2017 Resource Report Resource Website |
Ian Mackay, Judy Northill | DOI:10.17504/protocols.io.n7jdhkn | Public Health Virology, Forensic and Scientific Services | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 4 | 2018 | Ian Mackay, Judy Northill 2018. Dengue virus (DENV) universal MGB TaqMan 2017. protocols.io https://dx.doi.org/10.17504/protocols.io.n7jdhkn | 2021-04-15 09:16:12 | |||
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR N gene 2020 Resource Report Resource Website |
Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.bhpwj5pe | Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community | 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) | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 1 | 2020 | Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR N gene 2020 . protocols.io https://dx.doi.org/10.17504/protocols.io.bhpwj5pe | 2021-04-15 09:16:13 | |||
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Measles virus TaqMan RT-PCR (no longer in regular use; see Guidelines) Resource Report Resource Website |
Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.2qvgdw6 | 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. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 2 | 2019 | Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay 2019. Measles virus TaqMan RT-PCR (no longer in regular use; see Guidelines). protocols.io https://dx.doi.org/10.17504/protocols.io.2qvgdw6 | 2021-04-15 09:16:16 | ||||
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Novel coronavirus (2019-nCoV) real-time RT-PCR N gene 2020 (Wuhan-N; 2019-nCoV-related test) -NOT RECOMMENDED Resource Report Resource Website |
Judy Northill, Ian Mackay | DOI:10.17504/protocols.io.bb5piq5n | 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). | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | 3 | 2020 | 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 https://dx.doi.org/10.17504/protocols.io.bb5piq5n | 2021-04-15 09:16:55 |
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