Initial assessment of the ability of published coronavirus primers sets to detect the Wuhan coronavirus
Jan 15, 2020
The Shanghai Public Health Clinical Center & School of Public Health, in collaboration with the Central Hospital of Wuhan, Huazhong University of Science and Technology, the Wuhan Center for Disease Control and Prevention, the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control, and the University of Sydney, Sydney, Australia released a coronavirus genome from a case of a respiratory disease from the Wuhan outbreak of pneumonia cases (Novel 2019 coronavirus genome). In addition, 5 sequences were release on GISAID*. These suggest some diversity, but our current assessment is that that most likely reflects the use of different platforms and software, rather than true differences.
We have run a software algorithm that allows the alignment of large set of genomes against any primerset, to analyse a selected set of available PCR protocols for testing SARS-like viruses for mismatches with the new Wuhan coronavirus**. For this specific analysis, we have selected all available sequences with taxid 694009 (Severe acute respiratory syndrome (SARS)-related coronavirus) and ran the algorithm to check if currently used primer sets are able to detect these viruses, in silico . In addition, we have evaluated the potential of the most promising primer sets specifically against the 6 recently released Wuhan coronavirus sequences.
Primer sets with the least mismatches were specific SARS primers  and two sets of primers developed to detect a broad range of coronaviruses (panCoV) [2, 3]. As expected, none of the primer-sets used in routine respiratory virus diagnostic testing would be suitable. Also, none of the available primer sets have a 100% match to the Wuhan coronavirus sequences (Annex 5). The pan-CoV primer set by Chu et al, which is a nested PCR protocol, is the most promising primer set with only one mismatch in the reverse primer in the first PCR but the nested PCR will be more problematic. A disadvantage of this pan-CoV primer set is that it is not a real-time PCR assay and requires additional sequencing to determine which specific CoV is detected since this PCR is able to detect a broad range of different coronaviruses.
Specific Wuhan coronavirus primer and probe sets have been developed in China and by Charité in Berlin. The primer set developed by Charité is currently tested by laboratories in Berlin, London, Hongkong and Rotterdam. Technical validation based on controls will be developed on the basis of the available sequence information. The protocol describing the specific Wuhan real-time PCR developed by Charité has been released on the WHO website . However, clinical sensitivity validation will require testing of patient samples which may not be immediately available. For preparedness, the use of different primer sets and sharing of findings will be important. More details are expected in the coming 1-2 weeks.
Details can be found in the following files:
A document describing the primer sets analysed (Annex 1).
A document showing the results of the screening of all the primers against all the viruses with taxid 694009 (Annex 2).
A document providing more details of each primer set alignment, showing exact positions of mismatches for all the viruses with taxid 694009 (Annex 3).
A (very long) document showing all the alignments for primers that have mismatches for all the viruses with taxid 694009 (Annex 4).
A document showing all the alignments for primers that have mismatches against the Wuhan coronaviruses (Annex 5).
ErasmusMC, Rotterdam, The Netherlands:
Bas Oude Munnink, Richard Molenkamp, Reina Sikkema, Zsofia Igloi, Mart Lamers, Annemiek Baltissen – van der Eijk, Bart Haagmans, Ron Fouchier, Marion Koopmans
With the help of:
Hong Kong University, Hong Kong:
Daniel Chu, Malik Peiris
- We gratefully acknowledge all contributors to the public repositories for genomic data, as well as the authors, originating and submitting laboratories of the sequences from GISAID’s EpiFlu™ Database  on which this research is based. The list is detailed below.
|Virus name||Accession ID||Passage details/history||Collection date||Host||Originating lab||Submitting lab|
|BetaCoV/Wuhan/IVDC-HB-01/2019||EPI_ISL_402119||Virus Isolate, Passage 1||2019-12-30||Human||National Institute for Viral Disease Control and Prevention, China CDC||National Institute for Viral Disease Control and Prevention, China CDC|
|BetaCoV/Wuhan/IVDC-HB-04/2020||EPI_ISL_402120||Original||2020-01-01||Human||National Institute for Viral Disease Control and Prevention, China CDC||National Institute for Viral Disease Control and Prevention, China CDC|
|BetaCoV/Wuhan/IVDC-HB-05/2019||EPI_ISL_402121||Original||2019-12-30||Human||National Institute for Viral Disease Control and Prevention, China CDC||National Institute for Viral Disease Control and Prevention, China CDC|
|BetaCoV/Wuhan/IPBCAMS-WH-01/2019||EPI_ISL_402123||Original||2019-12-24||Human||Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College||Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College|
|BetaCoV/Wuhan/WIV04/2019||EPI_ISL_402124||Original||2019-12-30||Human||Wuhan Jinyintan Hospital||Wuhan Institute of Virology, Chinese Academy of Sciences|
**The software has been developed through collaboration with colleagues from DTU, in the EU funded project COMPARE (www.compare-europe.eu). This software is freely available at Bitbucket (https://bitbucket.org/jszarvas/oligo/src/master/). It makes use of the KVIT database (https://bitbucket.org/genomicepidemiology/kvit/src/master/) that is automatically updated after new coronavirus genomes appear in the public domain.
Kuiken, T. et al. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362 , 263–270 (2003).
De Souza Luna, L. K. et al. Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and nonfluorescent low-density microarray. J. Clin. Microbiol. 45 , 1049–1052 (2007).
Chu, D. K. W. et al. Avian Coronavirus in Wild Aquatic Birds. J. Virol. 85 , 12815–12820 (2011).
Shu, Y & McCauley, J. GISAID: Global initiative on sharing all influenza data – from vision to reality. Eurosurveillance 22 , (2017).
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