title: Detection of human novel influenza A (H1N1) viruses using multi-fluorescent real-time RT-PCR.
abstract: The novel influenza A (H1N1) virus is now rapidly spreading across the world. Early detection is one of the most effective measures to prevent further transmission of the virus. 4 sets of proprietary primers and probes designed for detection of influenza A viruses (InfA), human and swine H1N1 viruses (SH1), the novel H1N1 viruses (NH1) and RNaseP gene (RP) respectively were pooled together in a single tube for a multi-fluorescent real-time RT-PCR assay. The detection limit was found to be one order more sensitive than that employing the WHO recommended protocol. The NH1 probe was negative for all control samples including human seasonal H1N1 virus, other subtypes of human influenza A viruses (H3, H5, H9), human influenza B virus and nasopharyngeal swabs from patients with noninfluenza respiratory diseases, indicating its high specificity, capable of discriminating the novel influenza A virus from the previously identified H1N1 viruses. For confirmation, the PCR amplified fragment of the hemagglutinin gene was sequenced which could provide enough information to identify the novel H1N1 virus as a distinct cluster among all viruses of subtype H1 through average distance clustering analysis. Although these assays should be useful in the current outbreak for rapid detection and discrimination of the novel H1N1 from swine H1N1 and other human seasonal H1N1 viruses, further design improvement is suggested to match possible sequence variations in the detected region along with the course of the epidemic.
authors.affiliation: Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Dong
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hui
authors.initials: H
authors.lastname: Xiong
authors.forename: An
authors.initials: A
authors.lastname: Yan
authors.forename: Guohui
authors.initials: G
authors.lastname: Ding
authors.forename: Yangyi
authors.initials: Y
authors.lastname: Chen
authors.forename: Liqun
authors.initials: L
authors.lastname: Xie
authors.forename: Jiazheng
authors.initials: J
authors.lastname: Chen
authors.forename: Guoqing
authors.initials: G
authors.lastname: Zhang
authors.forename: Pei
authors.initials: P
authors.lastname: Hao
authors.forename: Liming
authors.initials: L
authors.lastname: Cong
authors.forename: Yiyu
authors.initials: Y
authors.lastname: Lu
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Che
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
authors.forename: Yixue
authors.initials: Y
authors.lastname: Li
authors.forename: Kwok-Yung
authors.initials: KY
authors.lastname: Yuen
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhao
authors.forename: Weirong
authors.initials: W
authors.lastname: Jin
mesh_terms: D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D006389:Hemagglutinins, Viral / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
chemical_list: D017931:DNA Primers; D006389:Hemagglutinins, Viral; D012367:RNA, Viral; C458881:hemagglutinin fusogenic peptide, influenza virus