title: A multiplex PCR assay for the detection of five influenza viruses using a dual priming oligonucleotide system.
abstract: BACKGROUND
  A cost-effective, accurate and rapid simultaneous multiplex assay is required for testing and diagnoses of conventional and emerging viruses in clinical virology laboratories. We developed and optimized a dual priming oligonucleotide (DPO) multiplex PCR assay for detecting influenza viruses including seasonal H1N1, 2009 pandemic H1N1, H3N2, influenza B and H5N1.
  
  
  METHODS
  The optimized multiplex DPO PCR was used to detect 233 clinical human samples. The results were compared to those obtained with RT-qPCR, conventional PCR and immunochromatographic assay.
  
  
  RESULTS
  Specificity analysis revealed that the DPO PCR assay amplified each target virus without any cross-amplification. Statistical analysis demonstrated that the multiplex DPO-PCR sensitivity was higher than for the immunochromatographic assay and lower than for qPCR, while no significant difference was observed compared with conventional PCR, when detecting influenza A and B. Additional experiments using the same sample panel indicated no significant differences between the number of positive samples detected by multiplex DPO PCR and RT-qPCR when applying a Cq with a value lower than 30.
  
  
  CONCLUSIONS
  The five-targeted simultaneous multiplex DPO PCR assay could be easily adopted into routine practice. This approach is cost effective with a short running time, low technical requirements for the detection of influenza virus and early diagnosis in clinical laboratories.
authors.affiliation: Institute of Health and Quarantine, Chinese Academy of Inspection and Quarantine, |No.A3, Gaobeidian North Road, Chaoyang District, Beijing, 100123, China. maxz@caiq.gov.cn.
authors.forename: Xuezheng
authors.initials: X
authors.lastname: Ma
authors.affiliation: Institute of Health and Quarantine, Chinese Academy of Inspection and Quarantine, |No.A3, Gaobeidian North Road, Chaoyang District, Beijing, 100123, China. 2249835269@qq.com.
authors.forename: Huanzhou
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Disease Control and Prevention, Shenzhen International Travel Health Care Center, Shenzhen, Guangdong Province, 518045, China. leishi1116@126.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: Huaian Center for Disease Control and Prevention, No.118, Huaihai North Road, Qinghe District, Huaian, Jiangsu Province, China. biomaster@126.com.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Yang
authors.affiliation: Institute of Health and Quarantine, Chinese Academy of Inspection and Quarantine, |No.A3, Gaobeidian North Road, Chaoyang District, Beijing, 100123, China. madamao2001@163.com.
authors.forename: Liping
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Institute of Health and Quarantine, Chinese Academy of Inspection and Quarantine, |No.A3, Gaobeidian North Road, Chaoyang District, Beijing, 100123, China. 31311386@qq.com.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Sun
authors.affiliation: Institute of Health and Quarantine, Chinese Academy of Inspection and Quarantine, |No.A3, Gaobeidian North Road, Chaoyang District, Beijing, 100123, China. yhu134@gmail.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhen
authors.affiliation: Institute of Health and Quarantine, Chinese Academy of Inspection and Quarantine, |No.A3, Gaobeidian North Road, Chaoyang District, Beijing, 100123, China. hukongxin@caiq.gov.cn.
authors.forename: Kongxin
authors.initials: K
authors.lastname: Hu
mesh_terms: D017931:DNA Primers; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000592:standards*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers