Detection and quantitation of gallid herpesvirus 1 in avian samples by 5′ Taq nuclease assay utilizing Minor Groove Binder technology

• Published in: Avian pathology 2010-02, Vol.39 (1), p.47-52
• Main Authors: Corney, B.G, Diallo, I.S, Wright, L.L, De Jong, A.J, Hewitson, G.R, Tolosa, M.X, Rodwell, B.J, Ossedryver, S.M, Pritchard, L.I, Boyle, D.B
• Format: Article
• Language: English
• Subjects: Alphaherpesvirus; Animals; Biological Assay - methods; Birds; Cells; Chickens; Clinical Laboratory Techniques; Cytopathogenic Effect, Viral; cytopathogenicity; detection limit; diagnostic techniques; disease diagnosis; DNA, Viral; Gallid herpesvirus 1; Genes; Herpesviridae Infections - diagnosis; Herpesviridae Infections - veterinary; Herpesviridae Infections - virology; Herpesvirus 1, Gallid - genetics; Herpesvirus 1, Gallid - isolation & purification; Kidney - cytology; Kidney - virology; kidney cells; Kidneys; Kinases; Laryngitis - veterinary; Laryngitis - virology; methodology; microbial detection; microbial genetics; Minor Groove Binder technology; molecular sequence data; nucleases; nucleotide sequences; optimization; Pathogens; polymerase chain reaction; poultry; poultry diseases; Poultry Diseases - diagnosis; Poultry Diseases - virology; quantitative analysis; rapid methods; Reverse Transcriptase Polymerase Chain Reaction - methods; thymidine kinase; Thymidine Kinase - genetics; Trachea - virology; Tracheitis - veterinary; Tracheitis - virology; vertebrate viruses
• ISSN: 0307-9457; 1465-3338
• DOI: 10.1080/03079450903473582

A 5' Taq nuclease assay utilizing Minor Groove Binder technology and targeting the thymidine kinase gene of gallid herpesvirus 1 (GaHV-1) was designed and optimized for use in diagnosing avian infectious laryngotracheitis. The assay was specific for GaHV-1 in that it did not react with other avian viral or bacterial pathogens. The detection limit was 1.0x10⁻² median tissue culture infectious dose per reaction or 90 target copies per reaction. Fifteen out of 41 diagnostic samples from sick birds reacted in the assay, five of which produced a typical alphaherpesvirus cytopathic effect (CPE) on chicken kidney (CK) cells. Sequencing, using amplicons generated by a polymerase chain reaction with primers flanking the 5' Taq nuclease amplicon, confirmed the presence of GaHV-1 in six samples (two producing alphaherpesvirus CPE on CK cells, three not producing alphaherpesvirus CPE, and one that was not inoculated onto CK cells). Tracheal swabs taken from 18 healthy broilers did not react in the assay. The ability of the assay to determine viral load in samples was demonstrated. Overall the assay is suitable for the rapid diagnosis of infectious laryngotracheitis.