A Real-Time TaqMan RT-PCR Method for Neuraminidase Type 1 (N1) Gene Detection of H5N1 Eurasian Strains of Avian Influenza Virus

• Published in: Avian diseases 2007-03, Vol.51 (s1), p.378-381
• Main Authors: Agüero, Montserrat, Sánchez, Azucena, San Miguel, Elena, Gómez-Tejedor, Concepción, Angel Jiménez-Clavero, Miguel
• Format: Article
• Language: English
• Subjects: accuracy; Animals; Avian influenza virus; Base Sequence; Birds - virology; diagnosis; diagnostic techniques; disease diagnosis; genes; genetic variation; H5N1 subtype influenza A virus; Influenza A virus; Influenza A Virus, H5N1 Subtype - enzymology; Influenza A Virus, H5N1 Subtype - genetics; Influenza A Virus, H5N1 Subtype - isolation & purification; Influenza in Birds - diagnosis; Influenza in Birds - virology; matrix protein; microbial genetics; molecular sequence data; Nervous system diseases; Neuraminidase - genetics; new methods; Newcastle disease virus; nucleotide sequences; Orthomyxoviridae; pathogen identification; real-time reverse transcription polymerase chain reaction; Research Note; Reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA; RNA, Viral; Sensitivity and Specificity; sequence alignment; sialidase; strain differences; strains; TaqMan reverse transcriptase polymerase chain reaction; test sensitivity; test specificity; Tissue samples; vertebrate viruses; Viral diseases; viral proteins; Viruses
• ISSN: 0005-2086; 1938-4351
• DOI: 10.1637/7642-050306r.1

This work describes the development of a real-time RT-PCR (RRT-PCR) procedure for detection of the N1 gene from avian influenza virus (AIV), based on the use of specific primers and a TaqMan-MGB (minor groove binder) probe. Nucleotide sequences of the neuraminidase type 1 gene from a collection of H5N1 Eurasian strains of AIV were aligned using ClustalW software. Conserved regions were located and used to design specific primers and a TaqMan-MGB probe using Primer Express software. A one-step RRT-PCR method was optimized using RNA from the Turkey 2005 H5N1 strain of AIV and can be completed in about 2 hr once the RNA is extracted from the sample. The specificity of the assay was assessed with non-N1 AIV strains, another related avian virus, and different avian tissue samples from healthy animals. Sensitivity was determined using 10-fold serial dilutions of the H5N1 Turkey 2005 strain and was compared with the generic RRT-PCR detection method, targeted at the matrix protein gene of AIV, commonly used at the Spanish AIV National Reference Laboratory. The N1 detection method proved to be even more sensitive than the generic (matrix-based) method, allowing a very quick confirmation (or discarding) of any Eurasian N1 strain when a positive result was obtained with the matrix RRT-PCR assay. Combined with RRT-PCR tests for general detection of AIV and H5 typing in use at the NRL, the procedure here described allows characterizing of any H5N1 Eurasian AIV strain in a field sample within a working day.