A FRET based melting curve analysis to detect nucleotide variations in HA receptor-binding site of H5N1 virus.
Outbreaks of highly pathogenic H5N1 influenza A virus represent a major public health problem because of the possibility of direct transmission of these viruses from avian species to humans. For influenza H5N1 hemagglutinin, a switch from SA-a-2, 3-Gal to SA-a-2, 6-Gal receptor specificity is a critical step that could lead to inter-human transmission. The monitoring of the receptor-binding preference of H5N1 viruses represents an instrument to detect a potential pandemic virus. The aim of this study was to develop a method based on the fluorescence resonance energy transfer (FRET) technology and melting peaks analysis for rapid screening of pandemic H5N1 influenza A virus. Three selected probes corresponding to a 23bp nucleotide sequence of the avian receptor-binding site were used in a real-time RT-PCR to detect nucleotide variations. Five strains of avian influenza A viruses isolated from avian species and two synthesized HA gene were tested. The results showed that the melting peaks analysis is a reliable screening method for detecting the variability of the H5N1 receptor-binding site.
De Santis
R
R
Histology and Molecular Biology Section, Army Medical and Veterinary Research Center, Via S. Stefano Rotondo 4, 00184 Rome, Italy. riccardo.desantis@gmail.com
Faggioni
G
G
Ciammaruconi
A
A
Fillo
S
S
Clausi
V
V
Corcioli
F
F
Lista
F
F
Azzi
A
A
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001717:Birds; D015342:DNA Probes / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D014644:Genetic Variation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005585:Influenza in Birds / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D007251:Influenza, Human / Q000453:epidemiology / Q000517:prevention & control / Q000821:virology; D058873:Pandemics; D012367:RNA, Viral / Q000235:genetics*; D011991:Receptors, Virus / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
D015342:DNA Probes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; D011991:Receptors, Virus