BioFire® Respiratory Panel 2.1 (RP2.1) Testing

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BioFire® Respiratory Panel 2.1 (RP2.1) Testing

Purpose

This procedure provides instructions for testing nasopharyngeal swabs (NPS)

collected in transport media or saline, using the BioFire® Respiratory Panel 2.1

(RP2.1) Kit (423742).

Background

The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid

test intended for use with the BioFire® 2.0 or BioFire® Torch Systems 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, including COVID-19.

The following organism types and subtypes are identified using the BioFire RP2.1:

• Adenovirus

• Coronavirus 229E

• Coronavirus HKU1

• Coronavirus NL63

• Coronavirus OC43

• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

• Human Metapneumovirus

• Human Rhinovirus/Enterovirus

• Influenza A, including subtypes H1, H1-2009, and H3

• Influenza B

• Parainfluenza Virus 1

• Parainfluenza Virus 2

• Parainfluenza Virus 3

• Parainfluenza Virus 4

• Respiratory Syncytial Virus

• Bordetella parapertussis (IS1001)

• Bordetella pertussis (ptxP)

• Chlamydia pneumoniae

• Mycoplasma pneumoniae

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Principle of the Procedure

The BioFire RP2.1 pouch is a closed system disposable that stores all the necessary

reagents for sample preparation, reverse transcription, polymerase chain reaction

(PCR), and detection in order to isolate, amplify, and detect nucleic acid from

multiple respiratory pathogens within a single NPS specimen. After sample

collection, the user injects hydration solution and sample combined with BioFire®

Sample Buffer into the pouch, places the pouch into a BioFire® System instrument

module, and starts a run. The entire run process takes about 45 minutes. Additional

detail can be found in the appropriate BioFire System Operator’s Manual.

During a run, the BioFire system:

• Lyses the sample by agitation (bead beading) in addition to chemical lysis

mediated by the Sample Buffer.

• Extracts and purifies all nucleic acids from the sample using magnetic bead

technology.

• Performs nested multiplex PCR by:

o First performing reverse transcription, followed by a multiplexed first

stage PCR reaction (PCR1).

o Then performing multiple simultaneous second-stage PCR reactions

(PCR2) in the array to amplify sequences within the PCR1 products

• Uses endpoint melting curve data to detect target-specific amplicons and

analyses the data to generate a result for each analyte.

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Sample Requirements

The following table describes the requirements for specimen collection, preparation,

and handling that will help ensure accurate test results.

Specimen Type

Nasopharyngeal Swab (NPS) collected according to standard technique and

immediately placed in up to 3 mL of transport media or saline.

Minimum Sample Volume

0.3 mL (300 µL)

Transport and Storage

Specimens should be tested with the BioFire RP2.1 as soon as possible.

If storage is required, specimens can be held:

• At room temperature for up to 4 hours (15-25 °C)

• Refrigerated for up to 3 days (2-8 °C)

• Frozen (≤-15 °C or ≤-70°C) (for up to 30 days)

a Frozen storage for up to 30 days was evaluated for this sample type. However, longer frozen storage at -70°C or lower may be acceptable. Please follow your

institutions rules and protocols regarding sample storage validation.

NOTE: NPS specimens should not be centrifuged before testing.

NOTE: Bleach can damage organisms/nucleic acids within the specimen, potentially causing false negative

results. Contact between bleach and specimens during collection, disinfection, and testing procedures should be

avoided.

Materials

Materials Provided

Materials Required But Not Provided

Each kit contains sufficient reagents to test 30

samples:

• Individually packaged BioFire RP2.1 pouches

• Single-use (1.0 mL) Sample Buffer ampoules

• Single-use pre-filled (1.5 mL) BioFire® Hydration

Injection Vials (blue)

• Single-use Sample Injection Vials (red)

• Individually packaged Transfer Pipettes

• BioFire RP2.1 Pouch Module Software

This software is required to run the BioFire RP2.1

and can be downloaded at

https://www.biofiredx.com/e-labeling/ITIFA20RP2110 if

not already installed on the BioFire 2.0 or BioFire

Torch Systems.

BioFire system including:

• BioFire2.0 or BioFire

Torch Systems and

accompanying system specific core software

• BioFire® Pouch Loading Station’

• 10% bleach solution or a similar disinfectant

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Procedure

Use clean gloves and other Personal Protective Equipment (PPE) when handling

pouches and samples. Only prepare one BioFire RP2.1 pouch at a time and change

gloves between samples and pouches. Once sample is added to the pouch, promptly

transfer to the instrument to start the run. After the run is complete, discard the

pouch in a biohazard container.

Step 1: Prepare Pouch

Thoroughly clean the work area and the BioFire Pouch Loading Station with freshly prepared 10% bleach

(or suitable disinfectant) followed by a water rinse.

Remove the pouch from its vacuum-sealed package by tearing or cutting the notched outer packaging and

opening the protective aluminum canister.

NOTE: The pouch may still be used even if the vacuum seal of the pouch is not intact. Attempt to hydrate

the pouch using the steps in the Hydrate Pouch section. If hydration is successful, continue with the run. If

hydration fails, discard the pouch and use a new pouch to test the sample.

Check the expiration date on the pouch. Do not use expired pouches.

Insert the pouch into the Pouch Loading Station, aligning the red and blue labels on the

pouch with the red and blue arrows on the Pouch Loading Station.

Place a red-capped Sample Injection Vial into the red well of the BioFire Pouch Loading

Station.

Place a blue-capped Hydration Injection Vial into the blue well of the BioFire Pouch Loading Station.

Step 2: Hydrate Pouch

Unscrew the Hydration Injection Vial from the blue cap.

Remove the Hydration Injection Vial, leaving the blue cap in the BioFire Pouch Loading Station.

Insert the Hydration Injection Vial’s cannula tip into the pouch hydration port located directly

below the blue arrow of the BioFire Pouch Loading Station.

Forcefully push down in a firm and quick motion to puncture seal until a faint “pop” is heard and

there is an ease in resistance. Wait as the correct volume of Hydration Solution is pulled into the

pouch by vacuum.

• If the hydration solution is not automatically drawn into the pouch, repeat Step 2 to verify that the

seal of the pouch hydration port was broken. If hydration solution is again not drawn into the

pouch, discard the current pouch, retrieve a new pouch, and repeat from Step 1: Prepare Pouch.

Verify that the pouch has been hydrated.

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• Flip the barcode label down and check to see that fluid has entered the reagent wells (located at

the base of the rigid plastic part of the pouch). Small air bubbles may be seen.

• If the pouch fails to hydrate (dry reagents appear as white pellets), repeat Step 2 to verify that the

seal of the pouch hydration port was broken. If hydration solution is still not drawn into the pouch,

discard the current pouch, retrieve a new pouch, and repeat from Step 1: Prepare Pouch.

Step 3: Prepare Sample Mix

Add Sample Buffer to the Sample Injection Vial.

• Hold the Sample Buffer ampoule with the tip facing up.

NOTE: Avoid touching the ampoule tip during handling, as this may introduce

contamination.

• Firmly pinch at textured plastic tab on the side of the ampoule until the seal snaps.

• Invert the ampoule over the red-capped Sample Injection Vial and dispense Sample Buffer

using a slow, forceful squeeze followed by a second squeeze.

NOTE: Avoid squeezing the ampoule additional times. This will generate foaming, which should be

avoided.

WARNING: The Sample Buffer is harmful if swallowed and can cause serious eye damage

and skin irritation.

Thoroughly mix the NPS specimen by vortex or inversion.

Use the transfer pipette provided in the test kit to draw specimen to the third line

(approximately 0.3 mL) of the transfer pipette.

Add the specimen to the Sample Buffer in the Sample Injection Vial.

Tightly close the lid of the Sample Injection Vial and discard the transfer pipette in a biohazard

waste container.

NOTE: DO NOT use the Transfer Pipette to mix the sample once it is loaded into the Sample

Injection Vial.

Remove the Sample Injection Vial from the Pouch Loading Station and invert the vial at least

3 times to mix.

Return the Sample Injection Vial to the red well of the Pouch Loading Station.

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Step 4: Load Sample Mix

Slowly twist to unscrew the Sample Injection Vial from the red cap and wait for 5 seconds with the vial

resting in the cap.

NOTE: Waiting 5 seconds decreases the risk of dripping and contamination from the sample.

Lift the Sample Injection Vial, leaving red cap in the well of the Pouch Loading Station, and insert the Sample

Injection Vial cannula tip into the pouch sample port located directly below the red arrow of the Pouch

Loading Station.

Forcefully push down in a firm and quick motion to puncture seal (a faint “pop” is heard) and

sample is pulled into the pouch by vacuum.

Verify that the sample has been loaded.

• Flip the barcode label down and check to see that fluid has entered the reagent well

next to the sample loading port.

• If the pouch fails to pull sample from the Sample Injection Vial, the pouch should

be discarded. Retrieve a new pouch and repeat from Step 1: Prepare Pouch.

Discard the Sample Injection Vial and the Hydration Injection Vial in appropriate biohazard sharps container.

Record the Sample ID in the provided area on the pouch label (or affix a barcoded Sample ID) and remove

the pouch from the BioFire Pouch Loading Station.

Step 5: Run Pouch

The BioFire

Software includes step-by-step on-screen instructions that guide the operator through performing a

run. Brief instructions for BioFire 2.0 and BioFire Torch systems are given below. Refer to the appropriate BioFire

System Operator’s Manual for more detailed instructions.

BioFire 2.0

Ensure that the system (instrument and computer) is powered on and the software is launched.

Follow on-screen instructions and procedures described in the Operator’s Manual to place the pouch in a

module, enter pouch, sample, and operator information.

Pouch identification (Lot Number and Serial Number), Pouch Type and Protocol information will be

automatically entered when the barcode is scanned. If it is not possible to scan the barcode, the pouch Lot

Number, Serial Number, Pouch Type and Protocol can be manually entered from the information provided

on the pouch label into the appropriate fields. To reduce data entry errors, it is strongly recommended that

the pouch information be entered by scanning the barcode.

NOTE: When selecting a Pouch Type manually, ensure that the Pouch Type matches the label on the BioFire

RP2.1 pouch.

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Enter the Sample ID. The Sample ID can be entered manually or scanned in by using the barcode scanner

when a barcoded Sample ID is used.

If necessary, select and/or confirm the appropriate protocol for your sample type from the Protocol drop

down list. The BioFire RP2.1 has a single protocol available in the drop down list.

Enter a username and password in the Name and Password fields.

NOTE: The font color of the username is red until the username is recognized by the software.

Review the entered run information on the screen. If correct, select Start Run.

Once the run has started, the screen displays a list of the steps being performed by the instrument and

the number of minutes remaining in the run.

NOTE: The bead-beater apparatus can be heard as a high-pitched noise during the first minute of operation.

When the run is finished, follow the on-screen instructions to remove the pouch, then immediately discard

it in a biohazard waste container.

The run file is automatically saved in the BioFire Software database, and the test report can be viewed,

printed, and/or saved as a PDF file.

BioFire Torch

Ensure that the system is powered on.

Select an available module on the touch screen or scan the barcode on the pouch using the barcode

scanner.

Pouch identification (Lot Number and Serial Number), Pouch Type and Protocol information will be

automatically entered when the barcode is scanned. If it is not possible to scan the barcode, the pouch Lot

Number, Serial Number, Pouch Type and Protocol can be manually entered from the information provided

on the pouch label into the appropriate fields. To reduce data entry errors, it is strongly recommended that

the pouch information be entered by scanning the barcode.

NOTE: When selecting a Pouch Type manually, ensure that the Pouch Type matches the label on the BioFire

RP2.1 pouch.

Enter the Sample ID. The Sample ID can be entered manually or scanned in by using the barcode scanner

when a barcoded Sample ID is used.

Insert the pouch into the available module.

• Ensure that the pouch fitment label is lying flat on top of pouch and not folded over. As the pouch

is inserted, the module will grab onto the pouch and pull it into the chamber.

If necessary, select and/or confirm the appropriate protocol for your sample type from the Protocol drop

down list. The BioFire RP2.1 has a single protocol available in the drop down list.

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Enter operator username and password, then select Next.

NOTE: The font color of the username is red until the username is recognized by the software.

Review the entered run information on the screen. If correct, select Start Run.

Once the run has started, the screen displays a list of the steps being performed by the module and the

number of minutes remaining in the run.

NOTE: The bead-beater apparatus can be heard as a high-pitched noise during the first minute of operation.

At the end of the run, remove the partially ejected pouch, then immediately discard it in a biohazard waste

container.

The run file is automatically saved in the BioFire database, and the test report can be viewed, printed,

and/or saved as a PDF file.

Assay Interpretation

When PCR2 is complete, the instrument performs a high resolution DNA melting

analysis on the PCR products and measures the fluorescence signal generated in

each well (for more information see appropriate BioFire System Operator’s

Manual). The BioFire Software then performs several analyses and assigns a final

assay result. The steps in the analyses are described below.

Analysis of melt curves. The BioFire Software evaluates the DNA melt curve for

each well of the PCR2 array 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 (Tm) of the curve and compares it

against the expected Tm range for the assay. If the software determines that the

Tm falls inside the assay-specific Tm range, the melt curve is called positive. If the

software determines that the melt curve is not in the appropriate Tm range, the

melt curve is called negative.

Analysis of replicates. Once melt curves have been identified, the software

evaluates the three replicates for each assay to determine the assay result. For an

assay to be called positive, at least two of the three associated melt curves must be

called positive, and the Tm for at least two of the three positive melt curves must be

similar (within 1°C). Assays that do not meet these criteria are called negative.

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Organism Interpretation

For most organisms detected by the BioFire RP2.1, the organism is reported as

Detected if a single corresponding assay is positive. For example, Human

Metapneumovirus will have a test report result of Human Metapneumovirus

Detected if the hMPV assay is positive (at least two of the three hMPV assay wells

on the array have similar positive melt peaks with Tm values that are within the

assay-specific Tm range). The test results for Adenovirus and Influenza A, and

SARS-CoV-2 depend on the interpretation of results from more than one assay.

Interpretation and actions for the multi-assay results are provided below.

Adenovirus

The BioFire RP2.1 pouch contains five different assays (Adeno2, Adeno3, Adeno6,

Adeno7.1, and Adeno8) for the detection of Adenovirus. The BioFire Software

interprets each of these assays independently (as described above) and the results

are combined as a final test result for the virus. If one or any combination of assays

is positive, the test report result will be Adenovirus Detected. If all assays are

negative, the test report result will be Adenovirus Not Detected.

Influenza A

The assays in the BioFire RP2.1 are designed to both detect Influenza A and to

differentiate common hemagglutinin subtypes. To accomplish this, the BioFire

RP2.1 uses two Influenza A assays, (FluA-pan-1 and FluA-pan-2) and three

subtyping assays directed at the hemagglutinin gene (FluA-H1-2, FluA-H1-2009,

and FluA-H3). Each of the individual assays is interpreted independently (as

described above) and the test result reported for Influenza A is based on the

combined results of the five assays as outlined in Table 1. An Influenza A (or

subtyping) Equivocal result could occur when the titer of the virus in the specimen

is low and not detected by one or more required assays (Error! Reference source

not found.). An Influenza A Equivocal result could also indicate the presence of an

atypical Influenza A subtype (e.g. avian H7N9 or H5N1 types), or a novel Influenza

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A strain. Specimens with an Equivocal result or multiple Influenza A subtypes

detected should be retested once.

Table 1. Possible Assay Results for Influenza A and the Corresponding Interpretation

Assay

Result

FluA-pan

Assays

(n=2)

FluA-H1-2

FluA-H1-

2009

FluA-H3

Action

Influenza A Not Detected

Negative

None

Influenza A H1

≥1 positive

Positive

Negative

Influenza A H3

≥1 positive

Negative

Positive

Influenza A H1-2009

≥1 positive

Any result

Positive

Negative

Influenza A H1

Influenza A H3

≥1 positive

Positive

Negative

Positive

Multiple infections

are possible but

rare

, retest ONCE

to confirm result

Influenza A H1-2009

Influenza A H3

≥1 positive

Any result

Positive

Influenza A (no subtype

detected)

2 positive

Negative

Retest

(see below)

Influenza A Equivocal

1 positive

Negative

Retest once

(see Result Summary

section below for

further instruction).

Influenza A H1

Equivocal

Negative

Positive

Negative

Influenza A H3

Equivocal

Negative

Positive

Influenza A H1-2009

Equivocal

Negative

Any result

Positive

Negative

The BioFire RP2.1 can simultaneously detect multiple influenza viruses contained in multivalent vaccines (see Limitations).

Repeated multiple positives should be further confirmed by other FDA cleared Influenza subtyping tests.

Influenza A (no subtype detected)

If both FluA-pan assays are positive, but none of the hemagglutinin subtyping

assays are positive, then the interpretation is Influenza A (no subtype detected).

This result could occur when the titer of the virus in the specimen is low and not

detected by the subtyping assays. This result could also indicate the presence of a

novel Influenza A strain. In both cases, the sample in question should be retested. If

the retest provides a different result, test the sample a third time to ensure the

accuracy of the result. If the retest provides the same result, then the function of

the BioFire RP2.1 pouches should be verified by testing with appropriate external

control materials (known positive samples for Influenza A H1, Influenza A H3 and

Influenza A H1-2009), and a negative control should also be run to test for PCR-

product contamination. If the BioFire RP2.1 accurately identifies the external and

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negative controls, contact the appropriate public health authorities for confirmatory

testing.

SARS-CoV-2

The BioFire RP2.1 pouch contains two different assays for the detection of the SARS-

CoV-2. The target of each assay is shown in Table 2 below. The BioFire Software

interprets each assay independently and if either one or both of the assays is positive,

the test report will show Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-

CoV-2) as Detected. If both assays are negative, the test report result will be Severe

Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Not Detected.

Table 2. Gene Targets for SARS-CoV-2 Assays on the BioFire RP2.1

Assay Name

Gene Target

SARSCoV2-1

Spike protein (S) gene

SARSCoV2-2

Membrane protein (M) gene

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BioFire RP2.1 Test Report

The BioFire RP2.1 test report is automatically displayed upon completion of a run

and can be printed or saved as a PDF file. Each report contains a Run Summary, a

Result Summary, and a Run Details section.

Run Summary

The Run Summary section of the test report provides the Sample ID, time and

date of the run, control results and an overall summary of the test results. Any

organism with a Detected result will be listed in the corresponding field of the

summary. If all of the organism assays were negative then ‘None’ will be displayed

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in the Detected field. Controls are listed as Passed, Failed, or Invalid. Table

provides additional information for each of the possible control field results.

Table 3. Interpretation of Controls Field on the BioFire RP2.1 Test Report

Control

Result

Explanation

Action

Passed

The run was successfully completed

AND

Both pouch controls were successful.

None

Report the results provided on the test report

Failed

The run was successfully completed

BUT

At least one of the pouch controls

(RNA Process Control and/or PCR2

Control) failed.

Repeat the test using a new pouch.

If the error persists, contact Technical Support

for further instruction.

Invalid

The controls are invalid because the

run did not complete.

(Typically this indicates a software or

hardware error).

Note any error codes displayed during the run

and the Run Status field in the Run Details

section of the report. Refer to the appropriate

BioFire Operator’s Manual or contact Technical

Support for further instruction.

Once the error is resolved, repeat the test or

repeat the test using another instrument.

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Results Summary

The Result Summary section of the test report lists the result for each target

tested by the panel. Possible results for each organism are Detected, Not Detected,

or Invalid (Equivocal is also a possible result for Influenza A and its subtypes).

Table 4 provides an explanation for each interpretation and any follow-up necessary

to obtain a final result.

Table 4. Reporting of Results and Required Actions

Result

Explanation

Action

Detected

The run was successfully completed

AND

The pouch controls were successful (Passed)

AND

The assay(s) for the organism were POSITIVE

(i.e., met the requirements for a positive result described in the Assay

Interpretation section above)

Report results.

Not Detected

The run was successfully completed

AND

The pouch controls were successful (Passed)

AND

The assay(s) for the organism were NEGATIVE

(i.e., did not meet the requirements for a positive result described in the Assay

Interpretation section above)

Report results.

Equivocal

The run was successfully completed

AND

The pouch controls were successful (Passed)

AND

The combination of positive and negative assay results for Influenza A were

inconclusive

(seeTable)

Retest the original

sample ONCE and

report the result of

the retest

Invalid

The pouch controls were not successful (Failed)

The run was not successful

(Run Status displayed as: Aborted, Incomplete, Instrument Error or Software

Error)

See Table 4 ,

Interpretation of

Control Field on the

BioFire Test Report

for instruction.

a If four or more organisms are detected in a specimen, retesting is recommended to confirm the polymicrobial result.

b Equivocal results can occur when the titer of the virus in the specimen is low (below LoD). Equivocal results could also indicate the presence

of a novel Influenza A strain or reactivity with non-human influenza A viruses or rare human influenza A viruses that are not H1, H1-2009 or

H3. Such strains generally produce Influenza A Equivocal or Influenza A (no subtype detected) results.

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Run Details

The Run Details section provides additional information about the run including:

pouch information (type, lot number, and serial number), Run Status (Completed,

Incomplete, Aborted, Instrument Error, or Software Error), the protocol that was

used to perform the test, the identity of the operator that performed the test, and

the instrument used to perform the test.

Change Summary

It is possible to edit the Sample ID once a run has completed. If this information

has been changed, an additional section called Change Summary will be added to

the test report. This Change Summary section lists the field that was changed, the

original entry, the revised entry, the operator that made the change, and the date

that the change was made. Sample ID is the only field of the report that can be

changed.

References/Related Documents

BioFire RP2.1 De Novo Instructions for Use, (BFR0000-8579-01) BioFire

Diagnostics, LLC.