A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes | Tran Tan et al. Virology Journal 2010 7 46 http content 7 1 46 VIROLOGY JOURNAL METHODOLOGY Open Access A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid LNA TaqMan probes Thanh Tran Tan1 Hana Apsari Pawestri2 Ngoc Nghiem My3 Hien Vo Minh3 Harun Syahrial2 Trung Nguyen Vu4 H Rogier van Doorn1 6 Heiman FL Wertheim5 6 Chau Nguyen Van Vinh3 Ha Do Quang1 Jeremy J Farrar1 6 Hien Tran Tinh3 Endang R Sedyaningsih2 Menno D de Jong1 6 7 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 PFU and 10 - 100 ssDNA plasmid copies. A total of 106 clinical samples 58 from patients infected with clade 1 or 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. Background Highly pathogenic avian influenza A H5N1 viruses cause sporadic infections in humans and are associated with severe respiratory disease with a mortality of about 60 1 . Since the re-emergence of human H5N1 influenza virus infections in January 2003 2 436 human cases have been documented in 15 countries in Asia Africa and Europe 1 . Genetic studies have revealed that most of the viruses isolated from humans and poultry belong to genotype Z 3 4 .
