The FilmArray RP is a multiplexed nucleic acid test intended for use with the FilmArray Instrument for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections. The following organism types and subtypes are identified using the FilmArray RP:
- Coronavirus 229E
- Coronavirus HKU1
- Coronavirus NL63
- Coronavirus OC43
- Human Metapneumovirus
- Influenza A
- Influenza A subtype H1
- Influenza A subtype H3
- Influenza A subtype H1-2009
- Parainfluenza Virus 1
- Parainfluenza Virus 2
- Parainfluenza Virus 3
- Parainfluenza Virus 4
- Human Rhinovirus/Enterovirus
- Respiratory Syncytial Virus
- Bordetella pertussis
- Chlamydophila pneumonia
- Mycoplasma pneumonia
Principle of the Procedure
The FilmArray RP pouch is a closed system disposable that houses all the chemistry required to isolate, amplify and detect nucleic acid from multiple respiratory pathogens within a single NPS specimen. The rigid plastic component (fitment) of the FilmArray RP pouch contains reagents in freeze-dried form. The flexible plastic portion of the pouch is divided into discrete segments (blisters) which, through interactions with actuators and sensors in the FilmArray instrument, are where the required chemical processes are carried out. The user of the FilmArray RP loads the sample into the FilmArray RP pouch, places the pouch into the FilmArray instrument, and starts the run. All other operations are automated.
The following is an overview of the operations and processes that occur during a FilmArray run:
- Nucleic Acid Purification – Nucleic acid purification occurs in the first three blisters of the pouch. The sample is lysed by agitation (bead-beating) and the liberated nucleic acid is captured, washed and eluted using magnetic bead technology. These steps require about ten minutes and the bead-beater apparatus can be heard as a high-pitched whine during the first minute of operation.
- Reverse Transcription and 1st Stage Multiplex PCR – Since many pathogens identified by the FilmArray RP pouch are RNA viruses, a reverse transcription (RT) step is performed to convert the viral RNA into cDNA prior to amplification. The purified nucleic acid solution is combined with a preheated master mix to initiate the RT step and subsequent thermocycling for multiplex PCR. The effect of 1st stage PCR is to enrich for the target nucleic acids present in the sample.
- 2nd Stage PCR – The products of 1st stage PCR are diluted and mixed with fresh PCR reagents containing an intercalating fluorescent DNA dye (LCGreen® Plus, BioFire Diagnostics, LLC). This solution is distributed over the 2nd stage PCR array. The individual wells of the array contain primers for different assays (each present in triplicate) that target specific nucleic acid sequences from each of the pathogens detected, as well as control template material. These primers are ‘nested’ or internal to the specific products of the 1st stage multiplex reaction, which enhances both the sensitivity and specificity of the reactions.
- DNA Melting Analysis – After 2nd stage PCR, the temperature is slowly increased and fluorescence in each well of the array is monitored and analyzed to generate a melting curve. The temperature at which a specific PCR product melts (melting temperature or Tm) is consistent and predictable and the FilmArray software automatically evaluates the data from replicate wells for each assay to report results.
Human nasopharyngeal swabs (NPS).
- NPS specimens should be collected according to standard technique and immediately placed in viral transport media (VTM).
- 300 µL of sample is required for testing.
- Specimens in VTM should be processed and tested as soon as possible. If storage is required, specimens in VTM can be held at room temperature (18–30 ºC) for up to 4 hours, at refrigerator temperature (2-8 °C) for up to 3 days, or at freezer temperature (<-15 °C) for up to 30 days.