1Zastosowanie reakcji duplex real-time PCR do identyfikacji wirusów grypy podtypu A(H1)pdm09 i A(H3)
[Development of duplex real-time PCR assay for identification of influenza viruses of subtype A(H1)pdm09 and A(H3)]

Streszczenie

Zaprojektowano zestaw starterów i sond oraz zoptymalizowano warunki reakcji real-time PCR w formacie duplex do różnicowania wirusów grypy typu A, obejmujących podtyp A(H1)pdm09 oraz A(H3). Opracowana me­toda może być stosowana w wirusologicznym nadzorze nad grypą, a także diagnostyce laboratoryjnej wykonywanej na potrzeby pacjenta.

Abstract

Introduction: Among different laboratory techniques used in influenza surveillance, methods based on molecular biology, such as real-time PCR, play increasingly important role. They allow detection and identification of viruses currently circulating in human population and responsible for infections and diseases. The aim of the study was to develop duplex real-time PCR assay for detection and differentiation of influenza virus subtype A(H1N1)pdm09 and A(H3N2).Methods: Specific primers targeting a highly conservative region of haemagglutinin gene and a dual-color TaqMan probes, labeled with fluorophore JOE (560 nm) and quencher BHQ-1 or CalFluor Red 610 (610 nm) and BHQ-2 were designed. The specificity of duplex reaction was evaluated using RNA isolated from reference strains of influenza virus A(H1N1)pdm09, A(H3N2), A(H1N1) and A(H5N1), B and other respiratory pathogens. Sensitivity of the assay was tested using serial dilutions of plasmid constructs carrying fragment of specific viral HA gene, in range between 10 and 105 copies/sample. A number of 116 clinical samples weretested using developed duplex qPCR assay in comparison with the CDC monoplex assay. Results: Positive duplex qPCR results were obtained only in samples containing RNA of influenza viruses of a specific subtype. The limit of detection (LOD) of developed method amounted up to 27 copies/sample for A(H1N1)pdm09 and up to 37 copies/sample for A(H3N2). Conclusions: The results show that developed TaqMan-based duplex qPCR assay is a va­luable diagnostic tool in influenza virus surveillance for using in direct study of clinical specimens as well as identification of isolated strains of influenza virus.