Results
Before public release of virus sequences from cases of 2019-nCoV, we relied on social media reports announcing detection of a SARS-like virus. We thus assumed that a SARS-related CoV is involved in the outbreak. We downloaded all complete and partial (if > 400 nt) SARS-related virus sequences available in GenBank by 1 January 2020. The list (n = 729 entries) was manually checked and artificial sequences (laboratory-derived, synthetic, etc), as well as sequence duplicates were removed, resulting in a final list of 375 sequences. These sequences were aligned and the alignment was used for assay design (Supplementary Figure S1). Upon release of the first 2019-nCoV sequence at virological.org, three assays were selected based on how well they matched to the 2019-nCoV genome (Figure 1). The alignment was complemented by additional sequences released independently on GISAID (https://www.gisaid.org), confirming the good matching of selected primers to all sequences. Alignments of primer binding domains with 2019-nCoV, SARS-CoV as well as selected bat-associated SARS-related CoV are shown in Figure 2.
Figure 1  Relative positions of amplicon targets on the SARS coronavirus and the 2019 novel coronavirus genome
E: envelope protein gene; M: membrane protein gene; N: nucleocapsid protein gene; ORF: open reading frame; RdRp: RNA-dependent RNA polymerase gene; S: spike protein gene.
Numbers below amplicons are genome positions according to SARS-CoV, GenBank NC_004718.
Figure 2  Partial alignments of oligonucleotide binding regions, SARS-related coronaviruses (n = 9)
The panels show six available sequences of 2019-nCoV, aligned to the corresponding partial sequences of SARS-CoV strain Frankfurt 1, which can be used as a positive control for all three RT-PCR assays. The alignment also contains a closely related bat virus (Bat SARS-related CoV isolate bat-SL-CoVZC45, GenBank accession number MG772933) as well as the most distant member within the SARS-related bat CoV clade, detected in Bulgaria (GenBank accession number NC_014470). Dots represent identical nucleotides compared with the WH_Human_1 sequence. Nucleotide substitutions are specified. Blue arrows: oligonucleotides as specified in Table 1. More comprehensive alignments can be found in the Supplement.

Assay sensitivity based on SARS coronavirus virions
To obtain a preliminary assessment of analytical sensitivity, we used purified cell culture supernatant containing SARS-CoV strain Frankfurt-1 virions grown on Vero cells. The supernatant was ultrafiltered and thereby concentrated from a ca 20-fold volume of cell culture supernatant. The concentration step simultaneously reduces the relative concentration of background nucleic acids such as not virion-packaged viral RNA. The virion preparation was quantified by real-time RT-PCR using a specific in vitro-transcribed RNA quantification standard as described in Drosten et al. [8]. All assays were subjected to replicate testing in order to determine stochastic detection frequencies at each assay’s sensitivity end point (Figure 3A and B). All assays were highly sensitive, with best results obtained for the E gene and RdRp gene assays (5.2 and 3.8 copies per reaction at 95% detection probability, respectively). These two assays were chosen for further evaluation. One of the laboratories participating in the external evaluation used other basic RT-PCR reagents (TaqMan Fast Virus 1-Step Master Mix) and repeated the sensitivity study, with equivalent results (E gene: 3.2 RNA copies/reaction (95% CI: 2.2–6.8); RdRP: 3.7 RNA copies/reaction (95% CI: 2.8–8.0). Of note, the N gene assay also performed well but was not subjected to intensive further validation because it was slightly less sensitive (Supplementary Figure S2)
Figure 3  Determination of limits of detection based on SARS coronavirus genomic RNA and 2019 novel coronavirus-specific in vitro transcribed RNA
CI: confidence intervals; c/r: copies per reaction; IVT: in vitro-transcribed RNA.
A: E gene assay, evaluated with SARS-CoV genomic RNA. B: RdRp gene assay evaluated with SARS-CoV genomic RNA. C: E-gene assay, evaluated with 2019-nCoV-specific in vitro-transcribed RNA standard. D: RdRp gene assay evaluated with 2019-nCoV-specific in vitro-transcribed RNA standard.
The x-axis shows input RNA copies per reaction. The y-axis shows positive results in all parallel reactions performed, squares are experimental data points resulting from replicate testing of given concentrations (x-axis) in parallels assays (eight replicate reactions per point).
Technical limits of detection are given in the panels headings. The inner line is a probit curve (dose-response rule). The outer dotted lines are 95% CI.

Sensitivity based on in vitro-transcribed RNA identical to 2019 novel coronavirus target sequences
Although both assays detected 2019-nCoV without polymorphisms at oligonucleotide binding sites (Figure 2), we additionally generated in vitro-transcribed RNA standards that exactly matched the sequence of 2019-nCoV for absolute quantification and studying the limit of detection (LOD). Replicate reactions were done at concentrations around the detection end point determined in preliminary dilution experiments. The resulting LOD from replicate tests was 3.9 copies per reaction for the E gene assay and 3.6 copies per reaction for the RdRp assay (Figure 3C and D). These figures were close to the 95% hit rate of 2.9 copies per reaction, according to the Poisson distribution, expected when one RNA molecule is detected.

Discrimination of 2019 novel coronavirus from SARS coronavirus by RdRp assay
Following the rationale that SARS-CoV RNA can be used as a positive control for the entire laboratory procedure, thus obviating the need to handle 2019-nCoV RNA, we formulated the RdRp assay so that it contains two probes: a broad-range probe reacting with SARS-CoV and 2019-nCoV and an additional probe that reacts only with 2019-nCoV. By limiting dilution experiments, we confirmed that both probes, whether used individually or in combination, provided the same LOD for each target virus. The specific probe RdRP_SARSr-P2 detected only the 2019-nCoV RNA transcript but not the SARS-CoV RNA.

Detection range for SARS-related coronaviruses from bats
At present, the potential exposure to a common environmental source in early reported cases implicates the possibility of independent zoonotic infections with increased sequence variability [5]. To show that the assays can detect other bat-associated SARS-related viruses, we used the E gene assay to test six bat-derived faecal samples available from Drexler et al. [13] und Muth et al. [14]. These virus-positive samples stemmed from European rhinolophid bats. Detection of these phylogenetic outliers within the SARS-related CoV clade suggests that all Asian viruses are likely to be detected. This would, theoretically, ensure broad sensitivity even in case of multiple independent acquisitions of variant viruses from an animal reservoir.

Specificity testing

Chemical stability
To exclude non-specific reactivity of oligonucleotides among each other, causing artificial fluorescent signals, all assays were tested 120 times in parallel with water and no other nucleic acid except the provided oligonucleotides. In none of these reactions was any positive signal detected.

Cross-reactivity with other coronaviruses
Cell culture supernatants containing all endemic human coronaviruses (HCoV)‑229E, ‑NL63, ‑OC43 and ‑HKU1 as well as MERS-CoV were tested in duplicate in all three assays (Table 2). For the non-cultivable HCoV-HKU1, supernatant from human airway culture was used. Viral RNA concentration in all samples was determined by specific real-time RT-PCRs and in vitro-transcribed RNA standards designed for absolute quantification of viral load. Additional undiluted (but not quantified) cell culture supernatants were tested as summarised in Table 2. These were additionally mixed into negative human sputum samples. None of the tested viruses or virus preparations showed reactivity with any assay.
Table 2  Tests of known respiratory viruses and bacteria in clinical samples and cell culture preparations for cross-reactivity in 2019 novel coronavirus E and RdRp gene assays (n = 310)
Clinical samples with known viruses  Clinical samplesa  Virus isolatesb
HCoV-HKU1  14  1c
HCoV-OC43  16  2d
HCoV-NL63  14  1e
HCoV-229E  18  2f
MERS-CoV  5  1g
Influenza A(H1N1)pdm09  17  1
Influenza A(H3N2)  16  1
Influenza A (untyped)  11   NA
Influenza A(H5N1)  1  1
Influenza A(H7N9)  0  1
Influenza B (Victoria or Yamagata)  31  1
Rhinovirus/enterovirus  31  NA
Respiratory syncytial virus (A/B)  33  NA
Parainfluenza 1 virus  12  NA
Parainfluenza 2 virus  11  NA
Parainfluenza 3 virus  14  NA
Parainfluenza 4 virus  11  NA
Human metapneumovirus  16  NA
Adenovirus  13  1
Human bocavirus  6  NA
Legionella spp.  3  NA
Mycoplasma spp.  4  NA
Total clinical samples   297  NA
a For samples with multiple viruses detected, the virus with highest concentration is listed, as indicated by real-time PCR Ct value.
b Directly quantified or spiked in human negative-testing sputum.
c 1 × 105 RNA copies/mL, determined by specific real-time RT-PCR. Isolated from human airway epithelial culture.
d 1 × 1010 RNA copies/mL, determined by specific real-time RT-PCR of one isolate. The other isolate was not quantified but spiked in human negative-testing sputum.
e 4 × 109 RNA copies/mL, determined by specific real-time RT-PCR.
f 3 × 109 RNA copies/mL, determined by specific real-time RT-PCR of one isolate. The other isolate was not quantified spiked in human negative-testing sputum.
g 1 × 108 RNA copies/mL, determined by specific real-time RT-PCR.

Exclusivity of 2019 novel coronavirus based on clinical samples pre-tested positive for other respiratory viruses
Using the E and RdRp gene assays, we tested a total of 297 clinical samples from patients with respiratory disease from the biobanks of five laboratories that provide diagnostic services (one in Germany, two in the Netherlands, one in Hong Kong, one in the UK). We selected 198 samples from three university medical centres where patients from general and intensive care wards as well as mainly paediatric outpatient departments are seen (Germany, the Netherlands, Hong Kong). The remaining samples were contributed by national public health services performing surveillance studies (RIVM, PHE), with samples mainly submitted by practitioners. The samples contained the broadest range of respiratory agents possible and reflected the general spectrum of virus concentrations encountered in diagnostic laboratories in these countries (Table 2). In total, this testing yielded no false positive outcomes. In four individual test reactions, weak initial reactivity was seen but they were negative upon retesting with the same assay. These signals were not associated with any particular virus, and for each virus with which initial positive reactivity occurred, there were other samples that contained the same virus at a higher concentration but did not test positive. Given the results from the extensive technical qualification described above, it was concluded that this initial reactivity was not due to chemical instability of real-time PCR probes but most probably to handling issues caused by the rapid introduction of new diagnostic tests and controls during this evaluation study.