Application of three duplex real-time PCR assays for simultaneous detection of human seasonal and avian influenza viruses

• Published in: Archives of virology Vol. 158; no. 8; pp. 1743 - 1753
• Main Authors: Stefańska, Ilona, Dzieciatkowski, Tomasz, Brydak, Lidia B., Romanowska, Magdalena
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01-08-2013; Springer Nature B.V
• Subjects: Avian flu; Biomedical and Life Sciences; Biomedicine; DNA Primers - genetics; Epidemics; Genes; Humans; Infectious Diseases; Influenza A virus - classification; Influenza A virus - genetics; Influenza A virus - isolation & purification; Influenza B virus; Influenza virus; Influenza, Human - diagnosis; Influenza, Human - virology; Laboratories; Medical Microbiology; Molecular Diagnostic Techniques - methods; Multiplex Polymerase Chain Reaction - methods; Oligonucleotide Probes - genetics; Original; Original Article; Pandemics; Pathogens; Quality control; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity; Streptococcus infections; Surveillance; Virology; Virology - methods; Viruses; Poland; California; Florida; United States > US; Influenza Virus; Avian Influenza Virus; Respiratory Specimen; Influenza; qPCR Assay
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s00705-013-1648-0

This study was performed to develop real-time PCR (qPCR) for detection of human seasonal and avian influenza viruses in duplex format. First duplex qPCR detects haemagglutinin (HA) gene of influenza virus A(H1N1)pdm09 and HA gene of influenza virus A(H3N2), the second reaction detects neuraminidase (NA) gene of influenza virus A(H3N2) and NA gene of influenza virus A(H1N1)pdm09 and A(H5N1), and the third reaction detects HA gene of influenza A(H5N1) and nonstructural protein gene of influenza B virus. Primers and probes were designed using multiple alignments of target gene sequences of different reference strains. Assays were optimised for identical thermocycling conditions. Their specificity was confirmed by conventional PCR and monoplex qPCR with nucleic acids isolated from different influenza viruses and other respiratory pathogens. Plasmid constructs with a fragment of specific gene were used to assess sensitivity of the assay. The limit of detection ranged from 27 to 96 cDNA copies/reaction. Clinical specimens (n = 107) have been tested using new assays, immunofluorescence and monoplex qRT-PCR. It has been shown that developed assays have been capable of rapid and accurate simultaneous detection and differentiation of influenza viruses. They are more sensitive than immunofluorescence and at least as sensitive as monoplex qRT-PCR.