Multiplexed detection of viral infections using rapid in situ RNA analysis on a chip

Abstract

Viral infections are a major cause of human disease, but many require molecular assays for conclusive diagnosis. Current assays typically rely on RT-PCR or ELISA; however, these tests often have limited speed, sensitivity or specificity. Here, we demonstrate that rapid RNA FISH is a viable alternative method that could improve upon these limitations. We describe a platform beginning with software to generate RNA FISH probes both for distinguishing related strains of virus (even those different by a single base) and for capturing large numbers of strains simultaneously. Next, we present a simple fluidic device for reliably performing RNA FISH assays in an automated fashion. Finally, we describe an automated image processing pipeline to robustly identify uninfected and infected samples. Together, our results establish RNA FISH as a methodology with potential for viral point-of-care diagnostics.

Fig. 1

RNA FISH platform rapidly determines whether samples are uninfected or infected with a virus. The pipeline includes probe design software to generate subtype specific probe sets or probe sets that label many viral subtypes. Next, we perform RNA FISH on the microfluidic chip which consists of a 5 minute hybridization and 3 one minute wash steps. Finally, we image the sample directly on the chip and then our image processing pipeline determines whether a sample is uninfected or infected.

Fig. 2

Subtype-specific RNA FISH probes discriminate influenza subtypes and rhinovirus “pan-probe” targets all rhinovirus strains. A) Overview of our subtype-specific probing strategy, in which we design oligonucleotides that only bind to one influenza subtype and do not cross hybridize to other subtypes. B) We used this software to design probes to target A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Brisbane/60/2008. We then infected MDCK cells with A/California/07/2009, A/Victoria/361/2011, and B/Florida/4/2006 and performed RNA FISH with subtype-specific probes. These three strains of influenza that were close enough in sequence similarity to our designs that we would expect our subtype-specific probes to bind. The RNA FISH probes produce bright fluorescent signal in the subtype to which they are designed and do not produce signal in the other subtypes. DAPI stain labels nuclei in blue, and RNA FISH is in white. C) Overview of “pan-probe” design, in which we optimize for the minimum number of oligonucleotides that will bind to all sequences. D) We used our “pan-probe” software to design RNA FISH probes that target 348 rhinovirus sequences with a minimum of 10 oligonucleotides per strain. The results of this design are summarized by the heatmap where each box represents a pair between an RNA FISH oligonucleotide and a viral strain. Light blue boxes indicate that the oligonucleotide is a perfect match somewhere in the virus and thus should bind, while dark blue boxes represent that the oligonucleotide does not bind to that strain. E) We infected HeLa cells with different strains of rhinovirus and performed rapid RNA FISH with the “pan-probe” for rhinovirus. Cells infected with each strain had bright fluorescent signal by RNA FISH and the uninfected cells remained dark. DAPI (nuclear stain) is in blue, and RNA FISH is in white. White scale bar represents 5 μm.

Fig. 3

Automated computational analysis demonstrates high specificity and sensitivity for viral detection. A) Overview of our image analysis software, in which microscopy images are automatically stitched, viral RNA (vRNA) fluorescence intensity is measured in and surrounding each DAPI-stained nucleus, and the percentage of positive cells is calculated using a cutoff that best distinguishes devices with infected cells from uninfected cells. B) Histogram and receiver operating characteristic curves for devices with 1.87% and 0.26% percent infected samples. C) Optimized intensity cutoff from A and applied to an independent dataset of uninfected devices and devices with 1.53% infected samples. White scale bar represents 25 microns.

Fig. 4

Multiplex RNA FISH detects four different viruses in one test. (A) We used an RNA FISH probe cocktail including probe sets for influenza A H1N1, influenza A H3N2, influenza B, and rhinovirus labeled with four differ fluorophores. For each virus, we loaded the chip with infected cells and hybridized on the probe cocktail. (B) We found that the probe sets brightly labeled the correct virus with minimal fluorescence from the probe sets for other viruses. In the analysis, we set the cutoff for calling a cell positive as four standard deviations from the mean intensity of the uninfected control. The numbers in the upper right-hand corner of each image indicate the percentage of cells designated as positive for that virus. DAPI (nuclear stain) is in blue, and RNA FISH is in white. All images are 20X magnification. White scale bar represents 25 μm.

Fig. 5

Viral SNP FISH detects drug resistant viruses with one base pair sequence difference. (A) We use a “mask” oligonucleotide to improve the specificity of a single probe by magnifying the relative energy contribution from a one base mismatch. (B) We use the SNP FISH technique to detect a point mutation in the neuraminidase gene that changes a histidine to a tyrosine, which alters the structure of neuraminidase such that the neuraminidase inhibitor oseltamivir is less effective in treating influenza . (C) Here, we infected MDCK cells with wild-type influenza A H1N1 and mutant influenza A H1N1 containing the oseltamivir resistance mutation. Representative images of SNP FISH on these cells show that most RNA transcripts are correctly labeled by the probes. DAPI (nuclear stain) is in blue, and RNA FISH is in white. Both images are 100X magnification. White scale bar represents 5 μm. (D) A receiver-operator characteristic curve demonstrates that this assay is effective for classifying cells as infected with wild-type or resistant mutant virus.