Method Description

The BioFire FilmArray Respiratory Panel is a closed system that performs all the chemistry required to isolate, amplify, and detect nucleic acid from multiple viral and bacterial respiratory pathogens within a single nasopharyngeal swab specimen. The panel contains reagents in freeze-dried form and is divided into discrete segments where the required chemical processes are carried out. Patient sample and hydration fluid are drawn by vacuum into the panel and then placed into the FilmArray instrument. The detection process operations are automated (nucleic acid purification, first stage polymerase chain reaction [PCR], second stage PCR, and melt analysis) and complete in about 45 minutes in this closed system.

Nucleic Acid Purification: The sample is lysed by a combination of chemical and mechanical mechanisms, and the liberated nucleic acid is captured, washed, and eluted using magnetic bead technology.

First-Stage PCR: A reverse transcription step is performed to convert viral RNA into complementary DNA prior to amplification. The purified nucleic acid solution is combined with a preheated master mix to initiate the reverse transcription step and subsequent thermocycling for multiplex PCR.

Second-Stage PCR: Products of first stage PCR are diluted and mixed with fresh PCR reagents, which is distributed over the second stage PCR array. The individual wells of the array contain primers for different assays (in triplicate) that target specific nucleic acid sequences from each of the pathogens detected, as well as control template material.

DNA Melting Analysis: Temperature is slowly increased, and fluorescence in each well of the array is monitored and analyzed to generate a melt curve.

Analysis of Melt Curves: The software evaluates the DNA melt curve for each well to determine if a PCR product was present in that well. If the melt profile indicates the presence of a PCR product, then the analysis software calculates the melting temperature of the curve, which is then compared against the expected range for the assay. When the software determines that the melt curve falls inside the assay-specific melt temp range, it is called positive. When it determines that the melt curve is not in the appropriate range, it is called negative.

Analysis of Replicates: Melt curves of each of the 3 replicates for each assay are evaluated to determine the assay result. For an assay to be called positive, at least 2 of the 3 associated melt curves must be called positive, and the melting temperature for at least 2 of the 3 positive melt curves must be similar (within 1 degree C). Assays that do not meet these criteria are called negative.(Instruction manual: FilmArray Respiratory Panel 2.1. BioFire Diagnostics, LLC; BFR0000-8579-01, 03/2021)