: A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes
Session III – Thanh, Tran Tan – YI Applicant
Title of Contribution: A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes
Author(s): Tran Tan Thanh a, Hana Apsari Pawestri b, Nghiem My Ngoc a, c, Vo Minh Hien a, c, Syahrial Harun b, Nguyen Vu Trung d, Rogier van Doorn a, f, Heiman Wertheim e, f, Do Quang Haa, Endang Rahayu Sedyaningsih b, Jeremy J. Farrar a, f, Menno D. de Jong a, f, g,
Affiliation(s):
a. Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam
b. National Institute of Health Research and Development, Jakarta, Indonesia
c. Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam
d. National Institute of Infectious and Tropical Diseases, Ha Noi, Viet Nam
e. Oxford University Clinical Research Unit – The National Institute of Infectious and Tropical Diseases, Ha Noi, Viet Nam
f. Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, Oxford, UK
g. Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Abstract:
The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 Influenza viruses warrant the need for a diagnostic assay that can detect both genetic variants. We developed a real-time RT-PCR assay for generic detection of both clades of H5 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 H5 gene. The analytical sensitivity of the assay for detecting the H5 gene was <0.5 PFU and 10 - 100 ssDNA plasmid copies. A total of 106 human 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%, indicating that this assay is a useful stool for diagnosis of H5 virus infections in regions where different genetic clades may cocirculate.
