title: Detection of a novel avian influenza A (H7N9) virus in humans by multiplex one-step real-time RT-PCR assay.
abstract: BACKGROUND
  A novel avian influenza A (H7N9) virus emerged in eastern China in February 2013. 413 confirmed human cases, including 157 deaths, have been recorded as of July 31, 2014.
  
  
  METHODS
  Clinical specimens, including throat swabs, sputum or tracheal aspirates, etc., were obtained from patients exhibiting influenza-like illness (ILIs), especially from those having pneumonia and a history of occupational exposure to poultry and wild birds. RNA was extracted from these samples and a multiplex one-step real-time RT-PCR assay was developed to specifically detect the influenza A virus (FluA). PCR primers targeted the conserved M and Rnase P (RP) genes, as well as the hemagglutinin and neuraminidase genes of the H7N9 virus.
  
  
  RESULTS
  The multiplex assay specifically detected the avian H7N9 virus, and no cross-reaction with other common respiratory pathogens was observed. The detection limit of the assay was approximately 0.05 50% tissue culture infective doses (TCID50), or 100 copies per reaction. Positive detection of the H7N9 virus in sputum/tracheal aspirates was higher than in throat swabs during the surveillance of patients with ILIs. Additionally, detection of the matrix (M) and Rnase P genes aided in the determination of the novel avian H7N9 virus and ensured the quality of the clinical samples.
  
  
  CONCLUSIONS
  These results demonstrate that the multiplex assay detected the novel avian H7N9 virus with high specificity and sensitivity, which is essential for the early diagnosis and treatment of infected patients.
authors.forename: Jian
authors.initials: J
authors.lastname: Fan
authors.forename: David
authors.initials: D
authors.lastname: Cui
authors.forename: Siuying
authors.initials: S
authors.lastname: Lau
authors.forename: Guoliang
authors.initials: G
authors.lastname: Xie
authors.forename: Xichao
authors.initials: X
authors.lastname: Guo
authors.forename: Shufa
authors.initials: S
authors.lastname: Zheng
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Huang
authors.forename: Shigui
authors.initials: S
authors.lastname: Yang
authors.forename: Xianzhi
authors.initials: X
authors.lastname: Yang
authors.forename: Zhaoxia
authors.initials: Z
authors.lastname: Huo
authors.forename: Fei
authors.initials: F
authors.lastname: Yu
authors.forename: Jianzhou
authors.initials: J
authors.lastname: Lou
authors.forename: Li
authors.initials: L
authors.lastname: Tian
authors.forename: Xuefen
authors.initials: X
authors.lastname: Li
authors.forename: Yuejiao
authors.initials: Y
authors.lastname: Dong
authors.forename: Qiaoyun
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Department of Clinical Laboratory, the First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China. chenyu_zy@163.com.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D002681:China / Q000453:epidemiology; D004285:Dogs; D005260:Female; D006801:Humans; D064766:Influenza A Virus, H7N9 Subtype / Q000235:genetics*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D057230:Limit of Detection; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction