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.

A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants

“…For example, clade distribution of A(H3N2) viruses circulating between 1997 and 2006 was studied using the RT-PCR/electrospray ionization mass spectrometry (ESI MS), an approach based on the identification of unique nucleotide compositions of less variable internal genes (25). Although powerful, this approach is limited by access to costly equipment and requires an external database (Abbott Molecular, Carlsbad, CA) for data analysis and interpretation.…”

Microbiota Evaluation of Patients With a Boston Type I Keratoprosthesis Treated With Topical 0.5% Moxifloxacin and 5% Povidone–Iodine

“…The PCR/ESI-MS method has been used to simultaneously identify and type multiple clinically relevant pathogens including viruses, bacteria, and fungi from clinical and environmental samples. [19][20][21]29,30 Molecular methods are very sensitive, and we avoided false positives by using a calibration standard of 500 copies for each primer pair targeted to ribosomal DNA and 100 genome copies for each primer pair targeted to genes encoding housekeeping proteins, which roughly matched the relative abundance of ribosomal operons to housekeeping genes. Because the calibration standard competes with the target organism DNA for amplification, this effectively established a threshold for detection, whereby low levels of endogenous DNA were not detected as a false positive.…”

Influenza in the US Military: An Overview

“…Among adult patients with ARI in one study using this type of testing in the United States in 2012-2013, 5% to 8% were found to sustain viral coinfections, to include influenza, HCoVs, RSV and HRV [35]. One influenza typing kit based on the RT-PCR electrospray ionization mass spectrometry (PCR-ESI-MS) platforms allows detection of all 16 hemagglutinin and 9 neuraminidase subtypes, [36] as well as detection of drift of specific genes over time [36][37][38][39]. Because of its ability to detect newly emerging recombinant, drifted, or shifted influenza viruses, the PCR-ESI-MS typing analysis can be useful in detecting newly emerging influenza strains [40].…”