Influenza A, B and respiratory syncytial virus simultaneous detection by a new multiplex reverse transcription nested-PCR assay

Filippo Ansaldi, G. Icardi, D. Amicizia, G. Ferro, F. Banfi, P. Morelli, L. Valle, E. Bergatto, O. Friard, O. Righello, R. Gasparini, C. Campello, P. Crovari

Research output: Contribution to journalArticlepeer-review


A multiplex nested reverse-transcription (RT)-PCR was developed for simultaneous detection and typing of influenza A and B and detection of Respiratory Syncytial Virus (RSV). Primers for influenza A detection were designed to amplify specific sequence of A/H1N1, A/H3N2, A/H5N1, A/H7N7 and A/H9N2. Specificity, analytical and clinical sensitivity of the new assay were evaluated. Primers showed no reactivity to specimens collected from patients with Influenza like-illness (ILI) and resulted influenza and RSV negative using a highly sensitive PCR-based test. Dilution series of cell culture-grown virus tested by uniplex nested RT-PCR and multiplex nested RT-PCR showed an identical sensitivity of the two assays for A/H1N1 and A/H3N2 influenza viruses and comparable for influenza B. The detection limit for influenza A subtypes H1N1 and H3N2, influenza B and RSV ranged between 2.5 and 5 target gene copies per reaction. A total of 75 specimens, PCR and culture positive for influenza, collected between 1999 and 2003 were chosen as representative of the cirdilating strains. To evaluate the clinical sensitivity in specimens with a probable low viral load, 20 PCR-positive and culture-negative samples were also tested, while 25 specimens, PCR-positive for RSV, collected during the four seasons were also analysed by multiplex. Influenza A or B virus or RSV were detected by multiplex nested PCR in a total of 109 (90.8%) out of 120 specimens initially uniplex nested PCR positive. Eleven uniplex/multiplex discordant samples were re-tested by both assays. Surprisingly, all samples resulted negative by both assays probably due to RNA degradation. Furthermore, to determine the clinical sensitivity of multiplex nested RT-PCR in detecting avion influenza viruses isolated in humans, 3 specimens, culture positive for A/H5N2, A/H7N7 and A/H9N2, were tested and resulted positive by multiplex. Multiplex nested RT-PCR provides accurate and sensitive diagnosis of influenza virus and RSV infection and represents a sensitive tool for virological surveillance and management of patient with ILL.

Original languageEnglish
Pages (from-to)57-62
Number of pages6
JournalJournal of Preventive Medicine and Hygiene
Issue number3
Publication statusPublished - Sep 2004


  • Influenza
  • Multiplex RT-PCR assay
  • Respiratory Viruses

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health


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