RESUMEN
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.
Asunto(s)
Virus de la Influenza A/genética , Gripe Aviar/virología , Gripe Humana/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Aves/virología , China , Humanos , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/transmisión , Gripe Humana/diagnósticoRESUMEN
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.
Asunto(s)
Infecciones por Coronavirus/diagnóstico , Coronavirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Coronavirus/clasificación , Coronavirus/genética , Infecciones por Coronavirus/virología , Técnica del Anticuerpo Fluorescente , Alemania , Humanos , Laboratorios/normas , Polimorfismo de Longitud del Fragmento de Restricción , ARN Viral/sangre , ARN Viral/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Virología/métodosRESUMEN
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.