In vitro transcribed RNA molecules for the diagnosis of pandemic 2009 influenza A(H1N1) virus by real-time RT-PCR

Mario Bermúdez de León, Katia Peñuelas-Urquides, Miguel E. Aguado-Barrera, María José Currás-Tuala, Brenda L. Escobedo-Guajardo, Rosa Nelly González-Ríos, Viviana L. Mata-Tijerina, Ofelia E. Vázquez-Monsiváis

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)487-491
Number of pages5
JournalJournal of Virological Methods
Volume193
Issue number2
DOIs
Publication statusPublished - 1 Nov 2013
Externally publishedYes

Bibliographical note

Funding Information:
The authors thank specially to Ana María Garza Sánchez for her secretarial assistance. This work was supported partially by the Instituto Mexicano del Seguro Social and the Consejo Nacional de Ciencia y Tecnología – México (Grant numbers 78764-M and SALUD-2011-1-162243 ).

Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.

All Science Journal Classification (ASJC) codes

  • Virology

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