A novel method for detection of H9N2 influenza viruses by an aptamer-real time-PCR.
H9N2 Influenza subtype has emerged in Tunisia causing epidemics in poultry and resulting in major economic losses. New mutations in their hemagglutinin and neuraminidase proteins were acquired, suggesting their potential to directly infect humans. Effective surveillance tools should be implemented to help prevent potential spillover of the virus across species. We have developed a highly sensitive real time immuno-polymerase chain reaction (RT-I-PCR) method for detecting H9N2 virus. The assay applies aptamers as ligands to capture and detect the virus. First, a panel of specific ssDNA aptamers was selected via a one step high stringency protocol. Next, the panel of selected aptamers was characterized for their affinities and their specificity to H9N2 virus. The aptamer showing the highest binding affinity to the virus was used as ligand to develop a highly sensitive sandwich Aptamer I-PCR. A 3-log increase in analytical sensitivity was achieved as compared to a routinely used ELISA antigen test, highlighting the potential of this approach to detect very low levels of virus particles. The test was validated using clinical samples and constitutes a rapid and a label-free platform, opening a new venue for the development of aptamer -based viability sensing for a variety of microorganisms of economic importance in Tunisia and surrounding regions.
University Tunis El Manar, Laboratory of Epidemiology and Veterinary Microbiology, Institut Pasteur of Tunis,13 Place Pasteur, 1002 Tunis Belvedere, Tunisia. Electronic address: issam.hmila@pasteur.tn.
Issam
I
Hmila
Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota,St. Paul, MN, USA; Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota,St. Paul, MN, USA; Department of Soil, Water, & Climate, and BioTechnology Institute, University of Minnesota, St. Paul, MN 55108, USA.
Manoosak
M
Wongphatcharachai
University Tunis El Manar, Laboratory of Epidemiology and Veterinary Microbiology, Institut Pasteur of Tunis,13 Place Pasteur, 1002 Tunis Belvedere, Tunisia; University of Carthage, Faculty of Sciences Bizerte, 7021 Zarzouna Bizerte, Tunisia.
Nacira
N
Laamiri
University Tunis El Manar, Laboratory of Epidemiology and Veterinary Microbiology, Institut Pasteur of Tunis,13 Place Pasteur, 1002 Tunis Belvedere, Tunisia; University of Carthage, Faculty of Sciences Bizerte, 7021 Zarzouna Bizerte, Tunisia.
Rim
R
Aouini
University Tunis El Manar, Laboratory of Epidemiology and Veterinary Microbiology, Institut Pasteur of Tunis,13 Place Pasteur, 1002 Tunis Belvedere, Tunisia.
Boutheina
B
Marnissi
University Tunis El Manar, Laboratory of Epidemiology and Veterinary Microbiology, Institut Pasteur of Tunis,13 Place Pasteur, 1002 Tunis Belvedere, Tunisia.
Marwa
M
Arbi
Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota,St. Paul, MN, USA; Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota,St. Paul, MN, USA.
Srinand
S
Sreevatsan
University Tunis El Manar, Laboratory of Epidemiology and Veterinary Microbiology, Institut Pasteur of Tunis,13 Place Pasteur, 1002 Tunis Belvedere, Tunisia.
Abdeljelil
A
Ghram
D000818:Animals; D052157:Aptamers, Nucleotide; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011200:Poultry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014416:Tunisia
D052157:Aptamers, Nucleotide
Aptamer; Diagnostic; H9N2; I-PCR; Influenza; SELEX