Figure 1.
(A) A FilmArray pouch was injected with mock sample (here colored blue for illustrative purposes) in the left side injection port and hydration solution (colored red) in the right side injection port. (B) The blisters of a FilmArray pouch were filled with different coloring (and the channels between the blisters heat sealed shut). In this pouch the plunger tree was made from plastic dyed blue. The fitment and film are normally at right angles to each other; for clarity the pouch has been flattened. (C) A schematic of the pouch showing a trace of the blisters, channels and array wells (black) and the functional areas of the pouch (red).
Figure 2.
Schematic of second stage PCR mix entering the array.
The layers of film and adhesive attaching the array to the pouch are separated to show the flow of liquid into the cells of the array (figure is not to scale). From the top the layers are: 2nd pouch film, 1st pouch film, array adhesive layer (orange), pricked cover film, array (black, with wells), and array cover film. All of the actual layers are transparent except for the array itself. Second stage PCR primers are spotted into the cells during manufacture and air-dried (Methods). Arrows show the flow of PCR master mix (without primers) entering the array through a hole cut in the 1st pouch film.
Figure 3.
FilmArray instrument with pouch being loaded.
Table 1.
FilmArray RP Pouch Pathogens, Gene Targets and LOD95.
Table 2.
Comparison of FilmArray RP to DFA.
Figure 4.
Real-time amplification and melt curves from the array.
Respiratory Pathogen pouches were injected with viral transport medium spiked with 200 TCID50 FluA H1-seasonal (panels A and B), 4×106 cfu B. per and 200 TCID50 FluA-H1 (panels C and D), or 4×106 cfu B. per (panels E and F) and run on the FilmArray instrument. Real time amplification curves (panels A and C and E) and post-amplification melt curves (panels B and D and F) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red), FluA-H3 (black), B. per (Green), Yeast RNA process control (dark blue), Second stage PCR control (light blue). For clarity the controls are shown in panels E and F only.
Figure 5.
Detection rates of the FilmArray RP pouch compared to DFA.
Pediatric NPA samples (N = 328) were tested either by DFA at PCMC (yellow bars) or on the FilmArray (Blue bars). The percent of samples in which no virus (Negative) or one of the indicated viruses was detected is shown. The viruses are grouped into those in which both DFA and FilmArray assays are available or only the FilmArray assay is available.
Figure 6.
Amplification and melt curves at low target levels.
Respiratory Pathogen pouches were injected with viral transport medium spiked with 1 TCID50 of the FluA- H1 seasonal virus used in Figure 4, and run on the FilmArray instrument. Real-time amplification curves (A) and post-amplification melt curves (B) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red). The ordinate scales are the same as in Figure 4.
Figure 7.
Tm data used to establish assay specific melt windows.
Histograms of the theoretical or observed Tms of the hMPV assay are shown. Tm data for the FilmArray runs includes each of the three replicates of the second stage PCR. A: Tms calculated from 13 sequence variants published in the NCBI databases. B: Tm data generated during the system beta-testing with 37 banked hMPV-positive patient samples. C: Tm data generated during the inclusivity testing with 10 hMPV strains representing subtypes A1, A2, B1 and B2. Multiple FilmArray runs of these strains are included in this data set. D: Tm data from 74 hMPV-positive patient samples collected during the clinical evaluation.
Table 3.
Performance of the FilmArray RP melt curve detection algorithm compared to expert interpretation.
Table 4.
Performance of the FilmArray RP system automated analysis as compared to expert interpretation.