Abstract
As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR). Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic), H1N1s (seasonal), and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 104 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.
Highlights
Influenza is a major pathogen of humans and causes significant respiratory infections, resulting in 250,000 to 500,000 deaths worldwide annually [1,2,3]
Droplet PCR formats are useful for amplifying DNA in small volumes in that droplets act as discrete reaction vessels, facilitating faster temperature cycling and reaction times as well as limiting sample carryover and contamination
Rapid subtyping is of high clinical value in that during some flu seasons, the viral susceptibility to antiviral medications like oseltamivir differs based upon subtype when multiple subtypes are in circulation
Summary
Influenza is a major pathogen of humans and causes significant respiratory infections, resulting in 250,000 to 500,000 deaths worldwide annually [1,2,3]. Influenza A viruses are of the most concern to public health and are responsible for the yearly seasonal flu epidemics as well as global pandemic events characterized by high morbidity and mortality [5]. The World Health Organization assigns names to each virus strain with an antigenic description of the hemagglutinin (HA) and neuraminidase (NA) surface proteins such that there are 13 different HA types and 9 NA types among influenza A viruses. ‘‘antigenic drift’’) in the HA and NA proteins is responsible for most of the seasonal variation in influenza A viruses. ‘‘antigenic shift’’) gives rise to influenza virus strains that are essentially unrecognizable to the human immune system, leading to the possibility of global pandemic events [6,7,8,9] The infrequent occurrence of profound alterations in these surface antigens (i.e. ‘‘antigenic shift’’) gives rise to influenza virus strains that are essentially unrecognizable to the human immune system, leading to the possibility of global pandemic events [6,7,8,9]
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