title: Detection of influenza A viruses with a portable real-time PCR instrument.
abstract: Timely identification of respiratory pathogens is essential for appropriate patient care and cohorting. In order to do rapid identification-technology near the patient we utilized the field-deployable RAZOR EX-thermocycler with a reverse transcription real-time PCR assay that detects all subtypes of influenza A virus. In addition, we developed a RT PCR assay for specific detection of influenza A(H1N1)pdm09 virus. These assays amplified segments of the matrix (M)- and the hemagglutinin (HA)-gene, respectively. Detection limits of the M-gene and the influenza A(H1N1)pdm09-specific HA-gene assays were 0.15 PFU and 8.8 PFU per reaction, respectively. With 18 influenza A viruses of different subtypes and influenza B, C, and 7 other respiratory viruses the RAZOR EX and standard real-time PCR assay results were in total agreement. From 104 clinical samples identical results were obtained by both PCR methods. Additional 21 clinical samples were tested under field conditions with the RAZOR EX instrument. Results were achieved in 90 min, including 45 min for sample preparation and they were in complete agreement with those obtained by standard real-time PCR under laboratory conditions. These methods enable highly sensitive and rapid on-site diagnostics to reliably identify patients infected with influenza A, including the influenza A(H1N1)pdm09-virus.
authors.affiliation: Centres for Military Medicine and for Biological Threat Preparedness, Tukholmankatu 8A, FI-00290 Helsinki, Finland.
authors.forename: Markos
authors.initials: M
authors.lastname: Mölsä
authors.forename: Katja A
authors.initials: KA
authors.lastname: Koskela
authors.forename: Esa
authors.initials: E
authors.lastname: Rönkkö
authors.forename: Niina
authors.initials: N
authors.lastname: Ikonen
authors.forename: Thedi
authors.initials: T
authors.lastname: Ziegler
authors.forename: Simo
authors.initials: S
authors.lastname: Nikkari
mesh_terms: D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D019095:Point-of-Care Systems; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000235:genetics; D014773:Virology / Q000295:instrumentation / Q000379:methods*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; C488936:M1 protein, Influenza A virus; D014763:Viral Matrix Proteins; C483468:hemagglutinin, human influenza A virus