tetano
Editor, Senior Moderator
J Clin Microbiol. 2010 Nov 17. [Epub ahead of print]
Development and characterization of a highly specific and sensitive SYBR Green RT-PCR assay for detection of the 2009 pandemic H1N1 influenza virus based on sequence signatures.
Medina RA, Rojas M, Tuin A, Huff S, Ferres M, Martinez-Valdebenito C, Godoy P, Garc?a-Sastre A, Fofanov Y, Santalucia J Jr.
Department of Microbiology; Global Health and Emerging Pathogens Institute; Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, NY, USA; DNA Software, Inc., Ann Arbor, MI, USA; Department of Computer Science, University of Houston, TX, USA; Department of Biology and Biochemistry, University of Houston, TX, USA; Laboratorio de Infectolog?a y Virolog?a Molecular, Centro de Investigaciones M?dicas y Departamento de Pediatr?a, Facultad de Medicina, Pontificia Universidad Cat?lica de Chile, Marcoleta 391, Santiago, Chile.
Abstract
The emergence and rapid spread of the 2009 H1N1 pandemic influenza left many diagnostic tests unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the United States Centers for Disease Control and Prevention was used for diagnostics. 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 2 mismatches as 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 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 RT-PCR assay with no loss of specificity and sensitivity. We validated the diagnostic accuracy of the NA SYBR Green assay with 125 clinical specimens obtained between May and August of 2009 in Chile, which showed comparable diagnostic efficacy as the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.
PMID: 21084522 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/21084522
Development and characterization of a highly specific and sensitive SYBR Green RT-PCR assay for detection of the 2009 pandemic H1N1 influenza virus based on sequence signatures.
Medina RA, Rojas M, Tuin A, Huff S, Ferres M, Martinez-Valdebenito C, Godoy P, Garc?a-Sastre A, Fofanov Y, Santalucia J Jr.
Department of Microbiology; Global Health and Emerging Pathogens Institute; Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, NY, USA; DNA Software, Inc., Ann Arbor, MI, USA; Department of Computer Science, University of Houston, TX, USA; Department of Biology and Biochemistry, University of Houston, TX, USA; Laboratorio de Infectolog?a y Virolog?a Molecular, Centro de Investigaciones M?dicas y Departamento de Pediatr?a, Facultad de Medicina, Pontificia Universidad Cat?lica de Chile, Marcoleta 391, Santiago, Chile.
Abstract
The emergence and rapid spread of the 2009 H1N1 pandemic influenza left many diagnostic tests unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the United States Centers for Disease Control and Prevention was used for diagnostics. 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 2 mismatches as 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 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 RT-PCR assay with no loss of specificity and sensitivity. We validated the diagnostic accuracy of the NA SYBR Green assay with 125 clinical specimens obtained between May and August of 2009 in Chile, which showed comparable diagnostic efficacy as the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.
PMID: 21084522 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/21084522