title: In vitro transcribed RNA molecules for the diagnosis of pandemic 2009 influenza A(H1N1) virus by real-time RT-PCR.
abstract: The 2009 influenza A(H1N1) outbreak allowed the implementation of new epidemiologic surveillance tools in several countries around the world. A new molecular protocol with appropriate sensitivity and specificity using real-time RT-PCR was developed by the Centers for Disease Control and Prevention (CDC) to identify the pandemic 2009 influenza A (H1N1) virus in human specimens. In the CDC protocol, positive controls are available only upon request and they are taken from cell cultures infected with 2009 influenza A(H1N1) virus, representing a handling risk for laboratory technicians. The poor availability of positive control materials in diagnostic laboratories may limit the public health response. The aim of the work presented in this paper was to develop positive controls for the diagnostic testing of influenza A(H1N1) virus that could be used in the CDC real-time RT-PCR protocol. A series of plasmid constructs bearing partial sequences of the viral genes were created and each construct was used as a template for in vitro transcription. RNA molecules were obtained successfully at high yield, i.e., 2×10(7) assays per microliter. Thus, the inclusion of these molecules in the influenza panel as positive controls is proposed. The in vitro transcribed RNA could also be used as quality standards in the design of international proficiency studies.
authors.affiliation: Department of Molecular Biology, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey 64720, Nuevo León, Mexico; Molecular Diagnostic Laboratory, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey 64720, Nuevo León, Mexico. Electronic address: mario.bermudez@imss.gob.mx.
authors.forename: Mario
authors.initials: M
authors.lastname: Bermúdez de León
authors.forename: Katia
authors.initials: K
authors.lastname: Peñuelas-Urquides
authors.forename: Miguel E
authors.initials: ME
authors.lastname: Aguado-Barrera
authors.forename: María José
authors.initials: MJ
authors.lastname: Currás-Tuala
authors.forename: Brenda L
authors.initials: BL
authors.lastname: Escobedo-Guajardo
authors.forename: Rosa Nelly
authors.initials: RN
authors.lastname: González-Ríos
authors.forename: Viviana L
authors.initials: VL
authors.lastname: Mata-Tijerina
authors.forename: Ofelia E
authors.initials: OE
authors.lastname: Vázquez-Monsiváis
mesh_terms: D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000592:standards*; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
chemical_list: D012367:RNA, Viral
keywords: Influenza A(H1N1) virus; Molecular diagnosis; Positive controls; RNA molecules; Real-time RT-PCR