Influenza species and subtypes circulation among hospitalized patients in Laleh hospital during two influenza seasonal (2016-2017 and 2017-2018) using a multiplex Real Time-Polymerase Chain Reaction

Azhar, Iman Rezaee; Mohraz, Minoo; Mardani, Masoud; Tavakoli, Mohammad Ali; Afshar, Amin Ehteshami; Zamani, Mohammad; Sadeghpoor, Simin; Safari, Saeid; Dadashpoor, Roozbeh; Rezaee, Mahsa; Shirvani, Fariba; Azimi, Shohreh; Heydarifard, Zahra; Ranjbar, Hamidreza Hagh; Lotfi, Amir Hossain; Mosadegh, Farhad; Hashemnejad, Farhad; Jazayeri, Seyed Mohammad

Viral nucleic acids from different specimens were extracted using a viral RNA/DNA nucleic acid extraction kit (ROCHE, Mannheim, Germany) according to manufacturer's recommendation. Internal control was added to each specimen before loading the trays. The use of an internal control of each reaction tube excluded false negatives due to nonspecific inhibitors of the PCR enzymes. DNA was eluted using 50 µL of elution buffer. Qualitative Multiplex Real Time PCR was carried out on 5µL of extracted materials using Flu kit, for the detection of flu A, flu A (H1N1) and flu B (Fast-track diagnostics/SIEMENS, Luxembourg), according to manufacturer's instructions. This kit could differentiate between the pandemic influenza A (H1N1)pdm strain and other (untyped) influenza A strains; the results were classified as and flu A (H1N1) if a sample was positive for to both influenza A and H1, and flu A if a sample was positive to influenza A and negative to H1. If a positive laboratory test result was not compatible with the clinical picture of the patients, the assay was repeated by another technician on the same sample. The time period from extraction to the end of result was between 24 and 36 hours (excluding public holidays).