RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses

Abstract

Reverse-transcription PCR (RT-PCR) coupled with electrospray ionization mass spectrometry (ESI-MS) is a high-throughput nucleic acid-based technology that relies on the accurate measurement of the molecular weight of PCR amplicons that can be used to deduce the base counts (number of As, Gs, Cs and Ts) of DNA. These amplicons represent highly variable regions with information-rich sequences, which are flanked by broad-range primers designed based on highly conserved loci. This technology was first introduced in 2005 for microbial identification and subtyping, and was later applied to influenza virus detection and identification. The influenza RT-PCR/ESI-MS assay allows analysis of approximately 300 samples per 24 h, and aids in the characterization of influenza viruses based on their ‘core’ gene signatures. Notably, this assay was used to identify one of the first cases of the 2009 H1N1 pandemic viruses. One of the main advantages of the RT-PCR/ESI-MS technology is its universality and adaptability for pathogen characterization. Efforts are being made to customize the currently used influenza surveillance assay for use in the diagnosis of the H1N1 pandemic virus. In this article, we provide a summary of known applications of the RT-PCR/ESI-MS assay in the field of influenza.

Keywords::

• core genes
• H1N1 pandemic
• mass spectrometry
• PLEX-ID
• reverse-transcription PCR/electrospray ionization mass spectrometry
• seasonal influenza
• T5000

Acknowledgements

The authors thank Alexander Klimov, Michael Shaw and members of the Molecular Epidemiology Team from the Influenza Division, CDC, for fruitful discussions.

Financial & competing interests disclosure

Rangarajan Sampath is an employee of the Ibis/Abbott Company that developed the Influenza reverse-transcription PCR/electrospray ionization mass spectrometry assay. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interest. The study was funded by the Centers for Disease Control and Prevention.The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The manuscript was cleared for submission to the journal following the CDC’s standard operating procedures. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.