A real-time RT-PCR for detection of clade 1 and 2 H5N1 influenza A virus using locked nucleic acid (LNA) TaqMan probes

• Published in: Virology journal 2010-02, Vol.7 (1), p.46-46, Article 46
• Main Authors: Thanh, Tran Tan, Pawestri, Hana Apsari, Ngoc, Nghiem My, Hien, Vo Minh, Syahrial, Harun, Trung, Nguyen Vu, van Doorn, Rogier H, Wertheim, Heiman F L, Chau, Nguyen Van Vinh, Ha do, Quang, Farrar, Jeremy J, Hien, Tran Tinh, Sedyaningsih, Endang R, de Jong, Menno D
• Format: Article
• Language: English
• Subjects: Avian influenza; Causes of; Conserved Sequence; Diagnosis; Genes; Genetic aspects; Health aspects; Hemagglutinin Glycoproteins, Influenza Virus - genetics; Humans; Influenza A virus; Influenza A Virus, H5N1 Subtype - classification; Influenza A Virus, H5N1 Subtype - genetics; Influenza A Virus, H5N1 Subtype - isolation & purification; Influenza viruses; Influenza, Human - diagnosis; Influenza, Human - virology; Laboratories; Medical research; Methodology; Oligonucleotide Probes - genetics; Oligonucleotides - genetics; Plasmids; Polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity; Vietnam
• ISSN: 1743-422X; 1743-422X
• DOI: 10.1186/1743-422x-7-46

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. 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%. This assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.