PMC:6988269 / 15919-20325 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6988269","sourcedb":"PMC","sourceid":"6988269","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6988269","text":"Specificity testing\n\nChemical stability\nTo 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.\n\nCross-reactivity with other coronaviruses\nCell 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.\nTable 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)\nClinical samples with known viruses Clinical samplesa Virus isolatesb\nHCoV-HKU1 14 1c\nHCoV-OC43 16 2d\nHCoV-NL63 14 1e\nHCoV-229E 18 2f\nMERS-CoV 5 1g\nInfluenza A(H1N1)pdm09 17 1\nInfluenza A(H3N2) 16 1\nInfluenza A (untyped) 11 NA\nInfluenza A(H5N1) 1 1\nInfluenza A(H7N9) 0 1\nInfluenza B (Victoria or Yamagata) 31 1\nRhinovirus/enterovirus 31 NA\nRespiratory syncytial virus (A/B) 33 NA\nParainfluenza 1 virus 12 NA\nParainfluenza 2 virus 11 NA\nParainfluenza 3 virus 14 NA\nParainfluenza 4 virus 11 NA\nHuman metapneumovirus 16 NA\nAdenovirus 13 1\nHuman bocavirus 6 NA\nLegionella spp. 3 NA\nMycoplasma spp. 4 NA\nTotal clinical samples 297 NA\na For samples with multiple viruses detected, the virus with highest concentration is listed, as indicated by real-time PCR Ct value.\nb Directly quantified or spiked in human negative-testing sputum.\nc 1 × 105 RNA copies/mL, determined by specific real-time RT-PCR. Isolated from human airway epithelial culture.\nd 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.\ne 4 × 109 RNA copies/mL, determined by specific real-time RT-PCR.\nf 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.\ng 1 × 108 RNA copies/mL, determined by specific real-time RT-PCR.\n\nExclusivity of 2019 novel coronavirus based on clinical samples pre-tested positive for other respiratory viruses\nUsing 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 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