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

• Published in: Journal of virological methods 2013-11, Vol.193 (2), p.487-491
• Main Authors: Bermúdez de León, Mario, Peñuelas-Urquides, Katia, Aguado-Barrera, Miguel E., Currás-Tuala, María José, Escobedo-Guajardo, Brenda L., González-Ríos, Rosa Nelly, Mata-Tijerina, Viviana L., Vázquez-Monsiváis, Ofelia E.
• Format: Article
• Language: English
• Subjects: cell culture; Centers for Disease Control and Prevention; genes; Humans; influenza; Influenza A Virus, H1N1 Subtype - genetics; Influenza A Virus, H1N1 Subtype - isolation & purification; Influenza A(H1N1) virus; Influenza, Human - diagnosis; Influenza, Human - virology; Molecular diagnosis; Molecular Diagnostic Techniques - methods; Molecular Diagnostic Techniques - standards; monitoring; pandemic; Plasmids; Positive controls; public health; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Real-time RT-PCR; Reference Standards; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; risk; RNA; RNA molecules; RNA, Viral - genetics; Sensitivity and Specificity; technicians; Transcription, Genetic; vertebrate viruses; viruses
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2013.07.016

•Plasmid constructs were developed and viral RNA molecules in vitro were synthesized.•In vitro transcribed RNA molecules were specific, stable and useful as positive controls in molecular diagnosis of pandemic 2009 influenza A(H1N1) virus.•These new positive controls substantially reduce the exposure of technicians to highly infectious controls, such as virus-infected cell cultures. 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.