RESUMO
In response to a recent outbreak in China, detection assays for a novel avian influenza A(H7N9) virus need to be implemented in a large number of public health laboratories. Here we present real-time reverse-transcription polymerase chain reaction (RT-PCR) assays for specific detection of this virus, along with clinical validation data and biologically-safe positive controls.
Assuntos
Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Aves/virologia , China , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/transmissão , Influenza Humana/diagnósticoRESUMO
We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.
Assuntos
Infecções por Coronavirus/diagnóstico , Coronavirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coronavirus/classificação , Coronavirus/genética , Infecções por Coronavirus/virologia , Imunofluorescência , Alemanha , Humanos , Laboratórios/normas , Polimorfismo de Fragmento de Restrição , RNA Viral/sangue , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Virologia/métodosRESUMO
We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.56.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.
