The new coronavirus 2019-nCoV recently identified in patients with acute respiratory disease is genetically similar to SARS coronavirus and bat SARS-like coronavirus. Accurate molecular tests are needed for early detection and identification of infected patients. For this purpose, Chu et al. recently designed two 1-step quantitative real-time reverse-transcription PCR assays for the detection of regions ORF1B and N of the viral genome.
Real-time RT-PCR assays targeting the ORF1b and N gene regions of 2019-nCoV were designed based on the first publicly available sequence in Genbank (Accession number: MN908947). Regions (ORF1b and N) that are highly conserved amongst sarbecoviruses were selected for primer and probe designs.
Primers and Probes
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Name
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Label 5’
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Oligonucleotide Sequence (5’>3’)
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Label 3’
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Working Conc.
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ORF1b gene
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ORF1b-F
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TGGGGYTTTACRGGTAACCT
Y = C/T, R = A/G
18816 TGGGGTTTTACAGGTAACCT 18835
Sequence ID: MT093631.1 29911
Present in 2019-nCoVs, SARS, bat-SARS, BetaCov and other coronaviruses.
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0.5 nmol/ml
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ORF1b-R
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AACRCGCTTAACAAAGCACTC
R = A/G
18947 AACACGCTTAACAAAGCACTC 18927
Present in 2019-nCoVs, SARS, bat-SARS, BetaCov and other coronaviruses.
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0.5 nmol/ml
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Probe
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FAM
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TAGTTGTGATGCWATCATGACTAG
W = A/T
18887 TAGTTGTGATGCAATCATGACTAG 18910
Present in 2019-nCoVs, SARS, bat-SARS, BetaCov, Pangolin CoV, and Bat coronavirus.
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ZEN
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0.25 nmol/ml
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Name
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Label 5’
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Oligonucleotide Sequence (5’>3’)
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Label 3’
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Working Conc.
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N gene
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N-F
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29183 TAATCAGACAAGGAACTGATTA 29204
Present in 2019-nCoVs, SARS, bat-SARS, BetaCov and other coronaviruses.
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0.5 nmol/ml
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N-R
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29292 CGAAGGTGTGACTTCCATG 29274
Present in 2019-nCoVs, SARS, bat-SARS, BetaCov and other coronaviruses. |
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0.5 nmol/ml
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Probe
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FAM
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29234 GCAAATTGTGCAATTTGCGG 29215
Present in 2019-nCoVs, SARS, bat-SARS, BetaCov, Pangolin CoV, and Bat coronavirus.
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ZEN
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0.25 nmol/ml
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Typical RT-PCR Condition
Extraction
For RNA and DNA extractions use a viral RNA purification kit (e,g. QIAamp Viral RNA Mini Kit, Qiagen) and a DNA plasmid purification kit (e.g. QIAprep Spin Miniprep Kit, Qiagen). Follow the instructed of the manual from the manufacturer. Typical volumes are: RNA extractions, use ~140 µL of sample and eluted in 60 µL elution buffer containing poly(A) carrier RNA.
PCR
For a total volume of 20 μl reaction use 4 μl of 4X master reaction mixture (e.g. TagMan Fast Virus 1-Step Master Mix, ThermoFisher), 0.5 nmol/ml of forward primer, 0.5 nmol/ml of reverse primer, 0.24 nmol/ml of the probe, and 4 μl of RNA sample.
Use a RT-PCR system for PCR reactions as follows: Reverse transcription at 50 °C for 5 minutes, inactivation at 95 °C for 20 seconds, 30 to 40 cycles of PCR amplification (Denaturing at 95 °C for 5 seconds; Annealing/extension at 60 °C for 30 seconds). The duration of a RT-PCR usually is in the range of 1 hour 15 minutes to 2 hours.
Reference
Daniel K W Chu, Yang Pan, Samuel M S Cheng, Kenrie P Y Hui, Pavithra Krishnan, Yingzhi Liu, Daisy Y M Ng, Carrie K C Wan, Peng Yang, Quanyi Wang, Malik Peiris, Leo L M Poon, Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia, Clinical Chemistry, , hvaa029, https://doi.org/10.1093/clinchem/hvaa029
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