title: A real-time RT-PCR for detection of clade 1 and 2 H5N1 influenza A virus using locked nucleic acid (LNA) TaqMan probes.
abstract: BACKGROUND
  The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants.
  
  
  RESULTS
  We developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10-100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%.
  
  
  CONCLUSIONS
  This assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.
authors.affiliation: Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, Dist 05, Ho Chi Minh City, Viet Nam. thanhtt@oucru.org
authors.forename: Tran Tan
authors.initials: TT
authors.lastname: Thanh
authors.forename: Hana Apsari
authors.initials: HA
authors.lastname: Pawestri
authors.forename: Nghiem My
authors.initials: NM
authors.lastname: Ngoc
authors.forename: Vo Minh
authors.initials: VM
authors.lastname: Hien
authors.forename: Harun
authors.initials: H
authors.lastname: Syahrial
authors.forename: Nguyen Vu
authors.initials: NV
authors.lastname: Trung
authors.forename: Rogier H
authors.initials: RH
authors.lastname: van Doorn
authors.forename: Heiman F L
authors.initials: HF
authors.lastname: Wertheim
authors.forename: Nguyen Van Vinh
authors.initials: NV
authors.lastname: Chau
authors.forename: Quang
authors.initials: Q
authors.lastname: Ha do
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Farrar
authors.forename: Tran Tinh
authors.initials: TT
authors.lastname: Hien
authors.forename: Endang R
authors.initials: ER
authors.lastname: Sedyaningsih
authors.forename: Menno D
authors.initials: MD
authors.lastname: de Jong
mesh_terms: D017124:Conserved Sequence; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D015345:Oligonucleotide Probes / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014744:Vietnam
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C109963:hemagglutinin, avian influenza A virus; C477371:locked nucleic acid