(681d) Application of a Novel Tablet PCR Platform for Detection of Influenza Subtypes From Clinical Samples

 

Developing diagnostics which can
quickly and effectively identify viral infections ranging from HIV to hepatitis
is crucially important for addressing the global health impact of these
diseases. This is particularly true in the case of influenza, a disease which
not only spreads quickly, but also mutates rapidly. The gold standard approach for
influenza subtyping is RT-PCR (qRT-PCR) which allows for detection of the
target sequence and quantification the number of viral particles present.
Because RT-PCR and qRT-PCR require thermal cycling in order to amplify the
clinical sample for detection, a typical apparatus for these techniques is
costly, bulky, and requires a large sample volume. Thus, a microfluidic
approach may prove advantageous in addressing these issues. Thus, we present
our work on a droplet platform for the differentiation of influenza strains
using qRT-PCR using a novel microdroplet device. Previous data have
demonstrated this platform's ability to detect both lambda phage DNA and
synthetic H3 influenza RNA in a dose-dependent fashion. We now further
demonstrate that the platform can effectively differentiate between H1 and H3
strains of influenza, as well as between swine-origin and seasonal strains of
H1. Detection using this platform is highly sensitive, amplifying clinical
samples as dilute as 10² virions/mL. The tablet platform is described
and its efficacy demonstrated elsewhere. Primers were designed using consensus
sequences from the online NCBI Influenza Virus Sequence Database to
differentiate H3 and H1 swine and seasonal subtypes. SYBR Green I dye was used
for real-time fluorescence detection. Each RT-PCR reaction consisted of 50ul of
RT-PCR mix from the Superscript III RT-PCR kit. Both reverse transcription and
PCR were carried out in the same tube or on the droplet platform. This included
1X Taq buffer, 0.2mM dNTPs, 1.5mM MgCl₂ and 0.2uM of both the
forward and reverse primers. Cycling consisted of a 30 minute RT step at 50°C,
followed by 40 cycles of 94°C for 15 seconds, the primer-specific annealing
temperature for 15 seconds, and 68°C for 30 seconds. An initial denature was
done following the RT step for 15 minutes for off-chip controls and 2 minutes
for platform droplet amplification. We first determined primer efficacy with
blinded spiked heat inactivated influenza virus particles in transport media as
a serial dilution for the H3N2 and H1N1 (seasonal) subtypes. We effectively
identified the sample subtypes which are differentiated by amplicon length. Samples
A-H (except C) were positive for the H3 subtype, displayed by the amplicon of
233 bp. Sample C is a negative control, while samples J and K are H1 seasonal
positive with an amplicon of 183 bp. The lowest concentration detected was
sample E, at 10² virions/ml. We also tested each sample against the
other primer sets and found no false positives. We subsequently performed
qRT-PCR on the serially diluted samples A-H for the H3 subtype to develop a
standard curve for efficiency utilizing our novel tablet platform. We then
tested our PCR protocol against clinical samples of unknown subtype and
concentration to identify seasonal H1N1, swine H1N1 or seasonal H3N2. The
platform is robust, using a simple apparatus and producing results which are
both replicable for influenza and applicable to other diseases for which
diagnostics currently rely on PCR. Thus, the development of this platform
represents an important step towards improved diagnostic technologies for
influenza and other infectious diseases.