tetano
Editor, Senior Moderator
J Virol Methods. 2013 Jul 24. pii: S0166-0934(13)00270-X. doi: 10.1016/j.jviromet.2013.07.016. [Epub ahead of print]
In vitro transcribed RNA molecules for the diagnosis of pandemic 2009 influenza A(H1N1) virus by real-time RT-PCR.
de Le?n MB, Pe?uelas-Urquides K, Aguado-Barrera ME, Curr?s-Tuala MJ, Escobedo-Guajardo BL, Gonz?lez-R?os RN, Mata-Tijerina VL, V?zquez-Monsiv?is OE.
Source
Department of Molecular Biology, Centro de Investigaci?n Biom?dica del Noreste, Instituto Mexicano del Seguro Social, Monterrey 64720, Nuevo Le?n, Mexico; Molecular Diagnostic Laboratory, Centro de Investigaci?n Biom?dica del Noreste, Instituto Mexicano del Seguro Social, Monterrey 64720, Nuevo Le?n, Mexico. Electronic address: mario.bermudez@imss.gob.mx.
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?107 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.
Copyright ? 2013 Elsevier B.V. All rights reserved.
KEYWORDS:
Influenza A(H1N1) virus, Molecular diagnosis, Positive controls, RNA molecules, Real-time RT-PCR
PMID:
23891871
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/23891871
In vitro transcribed RNA molecules for the diagnosis of pandemic 2009 influenza A(H1N1) virus by real-time RT-PCR.
de Le?n MB, Pe?uelas-Urquides K, Aguado-Barrera ME, Curr?s-Tuala MJ, Escobedo-Guajardo BL, Gonz?lez-R?os RN, Mata-Tijerina VL, V?zquez-Monsiv?is OE.
Source
Department of Molecular Biology, Centro de Investigaci?n Biom?dica del Noreste, Instituto Mexicano del Seguro Social, Monterrey 64720, Nuevo Le?n, Mexico; Molecular Diagnostic Laboratory, Centro de Investigaci?n Biom?dica del Noreste, Instituto Mexicano del Seguro Social, Monterrey 64720, Nuevo Le?n, Mexico. Electronic address: mario.bermudez@imss.gob.mx.
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?107 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.
Copyright ? 2013 Elsevier B.V. All rights reserved.
KEYWORDS:
Influenza A(H1N1) virus, Molecular diagnosis, Positive controls, RNA molecules, Real-time RT-PCR
PMID:
23891871
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/23891871