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

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dc.contributor.authorMedina, Rafael A.
dc.contributor.authorRojas, Mark
dc.contributor.authorTuin, Astrid
dc.contributor.authorHuff, Stephen
dc.contributor.authorFerres, Marcela
dc.contributor.authorMartinez Valdebenito, Constanza
dc.contributor.authorGodoy, Paula
dc.contributor.authorGarcia Sastre, Adolfo
dc.contributor.authorFofanov, Yuriy
dc.contributor.authorSantaLucia, John, Jr.
dc.date.accessioned2024-01-10T12:38:19Z
dc.date.available2024-01-10T12:38:19Z
dc.date.issued2011
dc.description.abstractThe 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.
dc.description.funderNIAID Center for Research in Influenza Pathogenesis
dc.description.funderNIAID
dc.description.funderNIH
dc.description.funderDepartment of Homeland Security and Technology Directorate
dc.description.funderDepartment of Homeland Security Science and Technology Directorate
dc.description.funderKeck Center
dc.description.funderNational Science Foundation through the Rice-Houston Alliance
dc.description.funderNATIONAL HUMAN GENOME RESEARCH INSTITUTE
dc.description.funderNATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
dc.description.funderNATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
dc.fechaingreso.objetodigital2024-05-06
dc.format.extent10 páginas
dc.fuente.origenWOS
dc.identifier.doi10.1128/JCM.01142-10
dc.identifier.eissn1098-660X
dc.identifier.issn0095-1137
dc.identifier.pubmedidMEDLINE:21084522
dc.identifier.urihttps://doi.org/10.1128/JCM.01142-10
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/77023
dc.identifier.wosidWOS:000285787100044
dc.information.autorucMedicina;Ferres M;S/I;66180
dc.information.autorucMedicina;Godoy P ;S/I;113511
dc.information.autorucMedicina;Martinez C;S/I;1007539
dc.issue.numero1
dc.language.isoen
dc.nota.accesocontenido completo
dc.pagina.final344
dc.pagina.inicio335
dc.publisherAMER SOC MICROBIOLOGY
dc.revistaJOURNAL OF CLINICAL MICROBIOLOGY
dc.rightsacceso abierto
dc.subjectA H1N1
dc.subject.ods03 Good Health and Well-being
dc.subject.odspa03 Salud y bienestar
dc.titleDevelopment 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
dc.typeartículo
dc.volumen49
sipa.codpersvinculados66180
sipa.codpersvinculados113511
sipa.codpersvinculados1007539
sipa.indexWOS
sipa.indexScopus
sipa.trazabilidadCarga SIPA;09-01-2024
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