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.