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.