Influenza B virus

HBoV DNA positive samples were tested for 16 other potential pathogens, including influenza A virus, influenza B virus, parainfluenza virus (1, 2, 3, 4), respiratory syncytial virus, adenovirus, enterovirus, human metapneumovirus, human coronavirus (229E, OC43, NL63, HKU1), Mycoplasma pneumoniae, and Chlamydia pneumoniae by Taqman real-time PCR, in accordance with the manufacturer's protocol (Guangzhou HuYanSuo Medical Technology Co., Ltd).

The specimens were vortexed, and a 100-µL volume was used for total nucleic acid extraction using the QIAamp Viral RNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. One step qRT-PCR was performed by using the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, Carlsbad, California, USA). NP and OP specimens from each patient were separately tested by singleplex qRT-PCR for eight viral pathogens: adenovirus, influenza A virus, influenza B virus, human metapneumovirus (hMPV), parainfluenza viruses (PIV) 1–3, and respiratory syncytial virus (RSV). The primers, probes and positive controls for all viruses were provided by CDC-Atlanta. Sequences for the primers and probes are shown in Table 2[11] . We tested for influenza A virus with the conserved matrix gene-base qRT-PCR; positive influenza A samples were also subtyped as 2009 pandemic influenza A (H1N1) virus (2009 H1N1), influenza A (H3N2) virus (H3N2), and seasonal influenza A (H1N1) virus (H1N1) [12] . Fluorescence was read at the combined annealing-extension step at 57°C and recorded as threshold cycle (C_t) values. A C_t value ≤39.9 was regarded as positive; C_t values ≥40.0 were regarded as negative. The qRT-PCR test did not discriminate between viral mRNA and genomic RNA. Specimens were not tested if the following conditions existed when the specimen arrived at the lab: there was no swab, the volume was less than 600 µL, the specimen was at room temperature, patient identification was absent or inadequate, or the patient questionnaire was absent. In addition, the test results were discarded for any specimen whose internal control (human ribonuclease P gene) was negative.

To determine the analytical sensitivity of the One-Step LAMP assay, tenfold serial dilutions from 1× 10⁶ to 1 × 10^− 3 copies of the RNA standard strain of SARS-CoV-2 were prepared in 1X HBSS (Gibco, 14,025–092) using the qPCR. The copy numbers of the RNA standard in each dilution were calculated using the qPCR according to Ji and colleagues’ method [38 (link)]. The accuracy of the analytical sensitivity results was confirmed by repeating the tests three times. Also, to ensure the results obtained for the analytical sensitivity test and to avoid possible visual error in the reaction tubes’ color examination, the reaction product was electrophoresed on a 1.5% agarose gel and evaluated under UV in the gel documentation. A set of 15 positive and 10 negative clinical samples previously tested by RT-qPCR were also selected to determine the clinical sensitivity of the One-Step LAMP assay using the optimized One-Step LAMP protocol.
The analytical specificity of the One-Step LAMP assay was examined by detecting the various templates, including Influenza A virus, Influenza B virus, Respiratory syncytial virus, Adenovirus, Parainfluenza virus, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenza, Pseudomonas aeruginosa, Legionella pneumophila, Bordetella Pertussis, Staphylococcus aureus, Mycoplasma pneumoniae, and Chlamydia pneumoniae as well as human positive samples of HIV ، HBV ، HCV ، EBV ، CMV ، HPV, and HSV1, and 2., and synthetic nucleic acid sequences prepared as a gift from Infectious Disease Control Center, Ministry of Health and Medical Education, Tehran, Iran.

We used commercial kits to detect viral genomes. For pooled collections, we used Allplex Seegene (Eurobio, https://www.eurobio-scientific.com) on the sample of collected liquid, according to the manufacturer’s recommendations. The kit detects influenza A virus; influenza A(H1) virus; influenza A(H1N1)pdm09 virus; influenza A(H3) virus; influenza B virus; respiratory syncytial viruses A and B; adenovirus; enterovirus; human metapneumovirus (hMPV); parainfluenza viruses 1–4; bocaviruses 1–4; coronaviruses 229E, NL63, and OC43; and human rhinovirus. For individual testing, and particularly to detect SARS-CoV-2 RNA, we used TaqPath (ThermoFisher Scientific, https://www.thermofisher.com), Alinity (Abbott, https://www.corelaboratory.abbott), GeneXpert (Cepheid, https://info.cepheid.com), and FilmArray (bioMérieux, https://www.biomerieux.com). To detect cytomegalovirus (CMV), Epstein-Barr virus, and human parvovirus B19 genome, as part of our routine daily workflow we used RealStar assays (Altona, https://www.altona-diagnostics.com). All products were used as recommended by the manufacturers. For the kinetic study on SARS-CoV-2–positive samples and the comparison of signals between nasopharyngeal swab samples and tissues, we obtained all Ct values from a SARS-CoV-2 AMP Kit automatized on Alinity. This test amplifies 2 genome target genes (RNA polymerase and nucleocapsid), and the obtained signal is provided as a single Ct. Concordance between the signals obtained by the different commercial techniques routinely used in our laboratory is excellent. Our laboratory is certified according to COFRAC NF EN ISO 15189.