Diagnostic Approach for the Differentiation of the Pandemic Influenza A(H1N1)v Virus from Recent Human Influenza Viruses by Real-Time PCR

Abstract

BackgroundThe current spread of pandemic influenza A(H1N1)v virus necessitates an intensified surveillance of influenza virus infections worldwide. So far, in many laboratories routine diagnostics were limited to generic influenza virus detection only. To provide interested laboratories with real-time PCR assays for type and subtype identification, we present a bundle of PCR assays with which any human influenza A and B virus can be easily identified, including assays for the detection of the pandemic A(H1N1)v virus.Principal FindingsThe assays show optimal performance characteristics in their validation on plasmids containing the respective assay target sequences. All assays have furthermore been applied to several thousand clinical samples since 2007 (assays for seasonal influenza) and April 2009 (pandemic influenza assays), respectively, and showed excellent results also on clinical material.ConclusionsWe consider the presented assays to be well suited for the detection and subtyping of circulating influenza viruses.

Human to animal transmission of influenza A(H1N1)pdm09 in a turkey breeder flock in Norway

“…Antibodies from egg yolk were extracted with chloroform, as described by Mohammed [12]. Turkey cloacal and oropharyngeal swabs and tissue samples were tested with a pan-influenza A real-time RT-PCR (rRT-PCR) [13], and subtype specific rRT-PCRs for H5, H7 and H1N1pdm09 [14–16]. HA gene sequences from positive samples were based on PCR fragments generated as previously described [17].…”

“…Nucleic acid from human nasopharyngeal swabs were typed [18] and H1 subtyped [14] by rRT-PCR. The HA gene was sequenced by dye-terminator Sanger sequencing (PCR and sequencing primers available upon request).…”

Nested PCR in magnetically actuated circular closed-loop PCR microchip system

“…Each microchip is made of acrylic, which can be cheaply mass fabricated compared to glass, silicon or quartz. With the implementation of the microfluidic devices, molecular diagnostics approaches can be more responsive in identification of pathogens, compare to enzymelinked immunosorbent assay or traditional cell culture, during seasonal flu or infectious outbreak [23,24]. The integrated nested PCR microchip system may be used for personalized medicine in order to provide point-of-care test.…”

Validation and Diagnostic Application of NS and HA Gene-Specific Real-Time Reverse Transcription-PCR Assays for Detection of 2009 Pandemic Influenza A (H1N1) Viruses in Clinical Specimens

“…Soon after the emergence of the 2009 pandemic influenza A (H1N1) virus (1,2,6,7), the WHO provided diagnostic kits (http://www.who.int/csr/resources/publications /swineflu/CDCrealtimeRTPCRprotocol_20090428.pdf) and the information necessary to develop PCRs (8,(12)(13)(14) for the identification of this novel virus.…”