title: Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.
abstract: The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Medina
authors.forename: Mark
authors.initials: M
authors.lastname: Rojas
authors.forename: Astrid
authors.initials: A
authors.lastname: Tuin
authors.forename: Stephen
authors.initials: S
authors.lastname: Huff
authors.forename: Marcela
authors.initials: M
authors.lastname: Ferres
authors.forename: Constanza
authors.initials: C
authors.lastname: Martinez-Valdebenito
authors.forename: Paula
authors.initials: P
authors.lastname: Godoy
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Fofanov
authors.forename: John
authors.initials: J
authors.lastname: SantaLucia
mesh_terms: D052160:Benzothiazoles; D002677:Chile; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis*; D009439:Neuraminidase / Q000235:genetics; D009930:Organic Chemicals / Q000378:metabolism*; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014481:United States; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009930:Organic Chemicals; D011804:Quinolines; D014764:Viral Proteins; C098022:SYBR Green I; C487630:NA protein, influenza A virus; D009439:Neuraminidase