LuminUltra GeneCount COVID-19 RT-qPCR Assay Kit

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LuminUltra GeneCount® COVID-19 RT-qPCR

Assay Kit

Instructions for Use

For In vitro Diagnostic (IVD) Use

LuminUltra GeneCount® COVID-19 RT-qPCR Assay Kit has completed

the Section IV.C notification process under FDA’s “Policy for

Coronavirus Disease-2019 Tests During the Public Health Emergency

(Revised)” and has not been reviewed by FDA.

LuminUltra® Technologies Ltd.

Version 1.02

Related to S406.2

Date: January 12, 2021

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Contents

Intended Use ................................................................................................................................................ 3

Product Description .................................................................................................................................... 3

Principle of Procedure ................................................................................................................................ 4

GeneCount® COVID-19 RT-qPCR Test Kit Components ...................................................................... 4

Warnings & Precautions ............................................................................................................................ 6

Reagent Storage, Handling and Stability .................................................................................................. 7

Sample Collection, Handling and Storage ................................................................................................ 8

Control Materials ........................................................................................................................................ 9

Workflow and Procedure ........................................................................................................................... 9

GeneCount® Clinical RNA Isolation Kits Procedure ........................................................................ 10

GeneCount® COVID-19 Assay Kit Procedure ................................................................................... 16

GeneCount® Q-16 Instrument Setup (Windows Software) ................................................................... 18

GeneCount® Q-16 Instrument Setup (Android, Touchscreen Device) ................................................. 21

GeneCount® Q-96 Instrument Setup (Windows Software) ................................................................... 22

Interpretation of Results .......................................................................................................................... 26

Quality Control ......................................................................................................................................... 27

Performance Evaluation ........................................................................................................................... 29

Limit of Detection (LoD) ...................................................................................................................... 29

Inclusivity Analysis ............................................................................................................................... 33

Cross-Reactivity Analysis ..................................................................................................................... 33

Clinical Evaluation ................................................................................................................................ 36

Contact Information ................................................................................................................................. 37

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Intended Use

The GeneCount® COVID-19 RT-qPCR Assay Kit is a RT-qPCR test intended for the qualitative

detection of RNA from SARS-CoV-2 in nasopharyngeal/oropharyngeal swab specimens from

individuals suspected of COVID-19 by their healthcare provider. Testing is limited to qualified

medical laboratories certified to perform high complexity tests. The GeneCount® COVID-19 RT-

qPCR Assay Kit has been validated but FDA’s independent review of this validation is pending.

Laboratories should include a statement such as, “the test has been validated but FDA’s

independent review of this validation is pending” with patient test results to healthcare providers.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally

detectable in upper respiratory tract specimens during the acute phase of infection. Positive results

are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and

other diagnostic information is necessary to determine patient infection status. Positive results do

not rule out bacterial infection or co-infection with other viruses. The agent detected may not be

the definite cause of disease. Laboratories within the United States and its territories are required

to report all positive results to the appropriate public health authorities.

The GeneCount® COVID-19 RT-qPCR Assay Kit is intended for use by qualified and trained

clinical laboratory personnel specifically instructed and trained in the techniques of RT-qPCR and

in vitro diagnostic procedures.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis

for patient management decisions. Negative results must be combined with clinical observations,

patient history, and epidemiological information. Positive results may be due to past or present

infection with non-SARS-CoV-2 coronavirus strains, such as coronavirus HKU1, NL63, OC43,

or 229E.

Product Description

The GeneCount® COVID-19 RT-qPCR Assay Kit is a real-time reverse transcription polymerase

chain reaction (RT-qPCR) test. The primer and probe sets in the kit are designed to detect SARS-

CoV-2 RNA in nasopharyngeal/oropharyngeal specimens from patients suspected of COVID-19

by their healthcare provider.

The oligonucleotide primer and probes used for detection of SARS-CoV-2 in the

GeneCount® COVID-19 RT-qPCR Assay Kit target the N2 gene of SARS-CoV-2 and E gene of

SARS-coronaviruses. An additional primer/probe set is multiplexed into each assay to detect MS2

bacteriophage as an extraction control. The test is designed to detected fluorescence signals on

three different channels. The target gene and corresponding fluorescent dye of the probe are shown

in Table 1.

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Table 1: Corresponding Channels and Targets for GeneCount® COVID-19 RT-qPCR Assay Kit

Channel Target

FAM N2 gene

HEX E gene

Cy5 MS2 extraction control

Principle of Procedure

Nucleic acids are isolated and purified from nasopharyngeal/oropharyngeal specimens using either

the LuminUltra GeneCount® Clinical Advanced RNA Isolation Kit (manual spin column and

centrifuge) or the LuminUltra GeneCount® Clinical Auto RNA Isolation Kit (automated magnetic

bead extraction with LuminUltra GeneCount® E-32 or E-96 Auto Purification instrument). Both

extraction kits are manufactured and distributed by LuminUltra Technologies Ltd. and affiliated

entities.

The purified nucleic acid is reverse transcribed into cDNA and subsequently amplified in a

LuminUltra GeneCount® Q-16 or Q-96 qPCR instrument using LuminUltra GeneCount® COVID-

19 RT-qPCR Master Mix. In the process, the probe anneals to a specific target sequence located

between the forward and reverse primers. During the extension phase of the PCR cycle, the 5’

nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from

the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye

molecules are cleaved from their respective probes, increasing the fluorescence intensity.

Fluorescence intensity is monitored at each PCR cycle by a LuminUltra GeneCount® Q-16 or Q-

96 qPCR instrument.

GeneCount® COVID-19 RT-qPCR Test Kit Components

The following section summarizes all materials required for the GeneCount® COVID-19 RT-

qPCR Assay Kit as well as the GeneCount® Clinical Auto and Advanced RNA Isolation Kits.

Components Included with GeneCount® COVID-19 RT-qPCR Assay Kit

The following test components are manufactured by LuminUltra Technologies Ltd. (FDA

registration number:10075226) or LuminUltra Technologies Inc. and supplied with the

GeneCount COVID-19 RT-qPCR Assay kit. In addition to the components listed in Table 2, all

required consumables can be included with the assay kit.

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Table 2: Components Included with the GeneCount® COVID-19 RT-qPCR Assay Kit

Component Catalog Number Description

COVID-19 Negative

Control

NFW-US-55mL Nuclease-Free Water

(Store from -20°C to 25°C)

COVID-19 Positive

Control

COVPC-US-50uL Plasmid DNA

(Store from -20°C to 25°C)

COVID-19 RT-qPCR

Master Mix

COVMM-US-1.65mL

Includes primers and probe sets

for assay E and N2 target genes

and MS2 bacteriophage.

(Store at -20°C if frozen, ambient

if lyophilized)

PCR Strip Tubes For use with LuminUltra

GeneCount® Q-16 instrument

Components Included with GeneCount® RNA Isolation Kits

The following components listed in Table 3 are included in the GeneCount® Clinical Auto RNA

Isolation Kit and/or GeneCount® Clinical Advanced RNA Isolation Kit.

Table 3: Components Included with GeneCount® Clinical RNA Isolation Kits

Component Catalog # Description

Magnetic Binding Beads

(GeneCount® Clinical Auto RNA

Isolation Kit only)

MB-US-22mL Magnetic binding beads for

the isolation of RNA

Lysis/Binding Buffer Concentrate LBC-US-40mL Solution of Guanidinium

thiocyanate and Triton X-

100

Lysis Supplement 1A LS1A-US-6.6mL

Solution of Proteinase K and

other binding supplements

Wash Solution 1 Concentrate WS1C-US-75mL Solution of Guanidinium

thiocyanate

Wash Solution 2A Concentrate WS2AC-US-150mL Nuclease-Free Water

Nuclease-Free Water NFW-55mL Nuclease-Free Water

Spin Columns with Collection

Tubes (GeneCount® Clinical

Advanced RNA Isolation Kit only)

N/A Plastic spin column with

silica resin

All components are prepared in glass or plastic containers and placed in plastic or foil bags with

absorbent pads if necessary.

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Components Not Included with GeneCount® COVID-19 RT-qPCR Detection Kit

Components required for the GeneCount® COVID-19 RT-qPCR Assay Kit but are not included

in the kit are summarized in Table 4.

Table 4: List of Additional Components not Provided with Test Kits

Consumables and

Equipment • Ethanol, 95-100% (no specific manufacturer required)

• Isopropanol, 95-100% (no specific manufacturer required)

• Disposable powder free exam gloves, safety glasses

• Tube Racks for 1.5/2.0 mL and PCR Strip tube

• Permanent marker

• Adjustable volume 1000 µl and 200 µl pipets with filtered

pipet tips

• Adjustable volume 20 µl Pipet with filtered pipet tips

• Sterile 15 mL or 50 mL Tubes

Warnings & Precautions

• For In Vitro Diagnostic use only

• LuminUltra GeneCount® COVID-19 RT-qPCR Assay Kit has completed the Section

IV.C notification process under FDA’s “Policy for Coronavirus Disease-2019 Tests

During the Public Health Emergency (Revised)” and has not been reviewed by FDA.

• Laboratories are required to report all positive results to the appropriate public health

authorities.

• Laboratories should include a statement such as, “the test has been validated but FDA’s

independent review of this validation is pending” with patient test results to healthcare

providers.

• Follow standard precautions. All patient specimens should be considered infectious

and/or biohazardous and handled accordingly with safe laboratory procedures.

• If infection with COVID-19 is suspected based on current clinical and epidemiological

screening criteria recommended by public health authorities, specimens should be

collected with appropriate infection control precautions.

• Perform all manipulations of live virus samples within a Class II (or higher) biological

safety cabinet (BSC).

• Use personal protective equipment (PPE) consistent with current guidelines for the

handling of potentially infectious samples including but not limited to laboratory gloves,

lab coat and eye protection while performing this assay and handling materials.

• Do not allow Lysis/Binding Buffer Concentrate or mixtures containing Lysis/Binding

Buffer Concentrate to come in contact with sodium hypochlorite (bleach) solution as

Lysis/Binding Buffer Concentrate contains guanidine thiocyanate. This mixture can

produce a highly toxic gas.

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• Do not eat, drink, smoke, apply cosmetics or handle contact lenses in areas where

reagents and specimens are handled.

• Do not use products beyond the expiration date.

• Store kit at 25℃

• If a positive amplification signal appears in the negative control, entire run should be

repeated from residual extracted RNA as the result is considered invalid.

• Amplification technologies such as PCR are sensitive to accidental introduction of PCR

product from previous amplification reactions. Incorrect results could occur if either the

clinical specimen or the reagents used in the amplification step become contaminated by

accidental introduction of amplification product. Workflow in the laboratory should

proceed in a unidirectional manner.

• Maintain separate areas for assay setup and handling of nucleic acids.

• During preparation of samples, compliance with good laboratory techniques is

essential to minimize the risk of cross-contamination between samples and the

inadvertent introduction of nucleases into samples during and after the extraction

procedure. Proper aseptic technique should always be used when working with

nucleic acids.

• Maintain separate, dedicated equipment (e.g. pipettes, microcentrifuges) and

supplies (e.g. microcentrifuge tubes, pipette tips) for assay setup and handling of

extracted nucleic acids.

• Always wear a clean lab coat, powder-free laboratory gloves (not previously

worn) and eye protection when handling kit components.

• Change gloves between samples and whenever contamination is suspected.

• Keep reagent and reaction tubes capped or covered as much as possible.

• Dispose of unused kit reagents and human specimens according to local, state and federal

regulations.

• Work surfaces, pipettes and centrifuges should be clean and decontaminated with

cleaning products such as 70% ethanol to minimize risk of nucleic acid contamination.

• RNA should be maintained on a cold block or on ice during preparation and use to ensure

stability.

• All pipette tips and plastic ware in the assay should be discarded in waste bin with bleach

and discarded after decontamination.

• Avoid exposure of the COVID-19 RT-qPCR Master Mix to light.

Reagent Storage, Handling and Stability

• If Lysis Supplement 1A arrives frozen, the reagents are to be stored at -20℃.

• Store Magnetic Binding Beads at 4℃.

• All other components of the GeneCount® RNA Isolation kits are to be stored at ambient

temperature (15-25℃). Under these conditions, components of the kit are stable until

their expiry date stated on the label.

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• If Lysis Supplement 1A arrives lyophilized, store rehydrated Lysis Supplement 1A at

-20℃ for up to 1 month in single-use aliquots to minimize freeze-thaws. Thaw

completely before use.

• Lysis Buffer (in GeneCount® RNA Isolation Kits) should be used immediately or stored

up to 24 hours at 4℃. Bring to room temperature before use.

• If Master Mix arrives frozen, store at -20℃ for up to 12 months in single aliquots to

minimize free-thaws. Thaw completely before use.

• If Master Mix arrives lyophilized, store at ambient temperature until first use. Under

these conditions, the reagents are stable until their expiry date stated on the label.

• Thawed or freshly rehydrated RT-qPCR Master Mix should be used immediately or

stored frozen at -20 for up to 12 months in single aliquots to minimize free-thaws. Thaw


• Nuclease-Free Water can be stored from -20℃ to 25℃. Under these conditions, the

reagents are stable until their expiry date stated on the label.

• Lyophilized Positive Control is to be stored at ambient temperature until first use. Under

these conditions, the reagents are stable until their expiry date stated on the label.

• Freshly rehydrated Positive Control should be used immediately or stored frozen at –

20°C for up to 12 months in single use aliquots to minimize freeze-thaws. Thaw


• Always check the expiration date prior to use. Do not use expired reagents.

Sample Collection, Handling and Storage

Inadequate or inappropriate specimen collection, storage, and transport are likely to yield false

test results. Sample handlings and storage should be consistent with CDC guidelines.

• Sample Collection

• Sample Collection device and/or sample preservation buffer is not included as

part of the kit.

• Refer to the CDC Interim Guidelines for Collecting, Handling and Testing

Clinical Specimens from Persons Under Investigation (PUI) for COVID-19

https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-

specimens.html

• Follow specimen collection device manufacturer instructions for proper collection

methods.

• Specimen Transport

• Specimens must be packaged, shipped and transported according to the current

edition of the International Air Transport Association (IATA) Dangerous Goods

Regulation.

https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-specimens.html

https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-specimens.html

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• Storing Specimens

• Specimens can be stored at 2-8 ℃ for up to 48 hours after collection.

• If delivery and processing of sample exceeds 48 hours, specimens should be

transported in dry ice and stored at -70℃ or colder once in laboratory.

• Extracted nucleic acid should be stored at -70℃ or lower.

Control Materials

All controls provided with the GeneCount® COVID-19 RT-qPCR Assay Kit include:

• Negative Control

• A negative template control is needed to check for contamination during assay

preparation and is used for every batch of samples analyzed. Nuclease-free

water is used as the negative control.

• Positive Control

• A positive template control is needed to verify the functionality of the assay and

is used for every batch of samples analyzed. It is comprised of plasmid DNA.

• Extraction control

• An extraction control of MS2 Bacteriophage RNA is needed to validate the

success of the extraction process and assay performance and is used for every

sample and control. It is multiplexed into each assay.

Note: Using the provided positive control is not necessary if a known positive sample was

processed and will be analyzed.

Workflow and Procedure

Using a unidirectional workflow with separate preparation areas for RNA isolation, assay setup

and amplification is recommended to minimize the risk of contamination while working with the

assay kits and clinical samples. Workflow for the GeneCount® COVID-19 RT-qPCR Assay Kit is

shown in Figure 1.

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Figure 1: Workflow with GeneCount® COVID-19 RT-qPCR Assay Kit

GeneCount® Clinical RNA Isolation Kits Procedure

The LuminUltra GeneCount® Clinical Auto RNA Isolation Kit or the LuminUltra GeneCount®

Clinical Advanced RNA Isolation Kit is required for RNA Isolation with the GeneCount COVID-

19 RT-qPCR Assay Kit. Following the GeneCount® Clinical RNA Isolation Kit, the sample is

ready for the GeneCount® COVID-19 RT-qPCR Assay Kit.

Reagent Preparation for GeneCount® Clinical RNA Isolation Kits

Steps for preparing the reagents are identical for both the GeneCount® Clinical Auto and

Advanced RNA Isolation Kits. The reagent preparation steps are as follows:

• Add 25 mL of Ethanol to Wash Solution 1 Concentrate (before first-time use)

• Add 120 mL of Ethanol to Wash Solution 2A Concentrate (before first-time use)

• If Lysis Supplement 1A Arrives Frozen: Gently thaw vial of Lysis Supplement 1A. Mix

intermittently for 1 minute by swirling. Do not invert bottle.

• If Lysis Supplement 1A Arrives Lyophilized: Rehydrate vial of Lysis Supplement 1A

with 6.6 mL of Nuclease-Free Water. Mix intermittently for 1 minute by swirling. Do

not invert bottle.

• Note: Solution may not fully dissolve.

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• Prepare Lysis Buffer by combining required volume of Lysis/Binding Buffer

Concentrate and Lysis Supplement 1A (Refer to Table 5) in a sterile 15 mL or 50 mL

tube. Mix by inverting 5-10 times.

• Note: Lysis Buffer should be used immediately or stored up to 24 hours at 4℃.

Bring to room temperature before use.

• Note: Store unused Lysis Supplement 1A frozen at -20℃ for up to 1 month in

single-use aliquots to minimize freeze-thaws. Thaw completely before use.

Table 5: Required volumes of Lysis Supplement 1A and Lysis/Binding Concentrate for Lysis Buffer

Preparation

Number of

Samples

Lysis Supplement

1A

Lysis/Binding Buffer

Concentrate

16 1 mL 6 mL

32 2 mL 12 mL

48 3 mL 18 mL

64 4 mL 24 mL

80 5 mL 30 mL

96 6 mL 36 mL

GeneCount® Clinical Advanced RNA Isolation Kit Procedure

• Label a spin column, as well as two 1.5 mL microcentrifuge tubes – one each for lysis

and elution – with identifying information for each sample.

• Add 400 µl of Lysis Buffer, followed by 300 µl of Sample stored in transport media

(e.g. VTM, UTM, PBS, etc.) to labeled lysis tube. Gently invert tubes 5-10 times.

• Note: Work in appropriate biosafety cabinet and take appropriate precautions

when handling potentially infectious clinical samples.

• Incubate at room temperature for 5 – 10 minutes.

• Add 400 µl of Isopropanol to each lysis tube and gently invert 5-10 times to mix.

• Place spin column into a collection tube.

• Transfer 600 µl of the Sample from the appropriate lysis tube into the corresponding spin

column and close cap.

• Note: If the sample contains solid materials, first centrifuge at max speed for 2

minutes. Then use supernatant for subsequent processing while avoiding pelleted

materials.

• Centrifuge the column at max speed for 1 minute. Always be sure to balance centrifuge.

• Discard flow-through and transfer the remainder of the sample from the lysis tube into

the spin column.

• Centrifuge the column at max speed for 1 minute and discard flow-through.

• Add 700 µl of Wash Solution 1 and centrifuge the column at max speed for 1 minute.

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• Note: Make sure ethanol has been added to Wash Solution 1 before first use.

• Transfer column into a new collection tube.

• Add 700 µl of Wash Solution 2A, centrifuge the column at max speed for 1 minute and

discard the flow-through. Repeat this step.

• Note: Make sure ethanol has been added to Wash Solution 2A before first use.

• Centrifuge the empty column at max speed for 1 minute to dry.

• Note: Failure to dry the column can leave residual ethanol which can inhibit

downstream analysis.

• Carefully remove the column from the collection tube after centrifugation, making sure

that the column does not contact any of the flow-through liquid. Place the column in a

new labeled 1.5 mL elution tube.

• Note: If the column contacts the flow-through liquid, empty the collection tube,

place the column back into the collection tube and centrifuge for 1 minute before

proceeding. It is important that the column is dry.

• Add 60 µl of Nuclease-Free Water directly to the spin column membrane being careful

not to touch pipette tip to the membrane. Let sit for 1 minute.

• Centrifuge column at maximum speed for 1 minute. Always be sure to balance

centrifuge.

• Remove and discard spin column. The purified RNA is in the Elution Tube and is ready

for immediate RT-qPCR analysis. The eluted RNA may be stored for up to 1 month at -20℃.

LuminUltra GeneCount® Clinical Auto RNA Isolation Kit Procedure Using the

GeneCount® E-32 Auto Purification Instrument

Each plate can process up to 16 samples and two plates can be processed at one time on a

GeneCount® E-32 Auto Purification Instrument for a total of 32 samples. The below instructions

are based on running complete plates, but fewer samples can be run by adjusting protocol

appropriately.

• Orient the Extraction Plate so that the flat corner is facing the back left as seen in Figure

2 to aliquot reagents properly.

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• Add 400 µl of Lysis Buffer to each well in columns 1 and 7 (labeled Lysis/Bind in

Figure 2).

• Add 300 µl of Sample stored in transport media (e.g. VTM, UTM, PBS, etc.) into the

appropriate well in columns 1 or 7. Mix by pipetting up and down 10 times.

• Note: Work in an appropriate biosafety cabinet and take appropriate precautions


• Incubate plate at room temperature for 5-10 minutes.

• Add 400 µl of Isopropanol to each sample in columns 1 and 7. Mix by pipetting up and

down 10 times.

• Add 700 µl of Wash Solution 1 to each well in columns 2 and 8 of the plate.

• Note: Make sure that ethanol has been added to Wash Solution 1 before first use.

• Add 700 µl of Wash Solution 2A to each well in columns 3, 4, 9 and 10 of the plate.

• Note: Make sure that ethanol has been added to Wash Solution 2A before first

use.

• Add 60 µl of Nuclease-Free Water to each well in columns 5 and 11 of the plate

(labeled Elution in Figure 2).

• Add 200 µl of Magnetic Bind Beads to each well in columns 6 and 12 (ensure beads are

evenly suspended by vortex mixing moderately for 1-2 minutes initially and then

periodically during dispensing).

• Program automated instrument as specified in Table 6 and save the run file for future use.

Figure 2: Extraction Plate Layout

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Table 6: Programming Parameters for GeneCount E-32 Auto Purification Instrument

Column Name Mix

Time

Mix

Speed

Wait

Time

Magnet

Time

Volume

(µL)

6 Bead

Drop

0:30 Slow 0:00 1:00 200

1 Binding 10:00 Slow 0:00 1:00 1100

2 Wash 1 1:00 Fast 0:00 1:00 700

3 Wash 2A 1.00 Fast 0:00 1:00 700

4 Wash 2A 1.00 Fast 0:00 1:00 700

5 Elution* 3:00 Fast 5:00 1:00 60

6 Bead

Drop

0:30 Slow 0:00 0:00 200

*Program the Elution row to heat to 65℃. Elution starts heating at Step 4.

• Carefully insert Extraction Plate into GeneCount® E-32 Auto Purification Instrument,

making sure the plate fits snugly into the holding brackets.

• Place 8-rod covers into the slots on the upper deck for the magnetic rods, making sure the

plastic clip on the handle is locked in place.

• Run the automated extraction protocol.

• When finished, the eluted RNA from rows 5 and 11 can be used immediately for RT-

qPCR analysis. Alternatively, transfer eluted RNA into new nuclease-free

microcentrifuge tubes and store for up to 1 month at -20℃.

LuminUltra GeneCount® Clinical Auto RNA Isolation Kit Procedure Using the

GeneCount® E-96 Auto Purification Instrument

Each E-96 instrument can process up to 96 samples at one time and operates with a max capacity

of 6 extraction plates. Contrary to the E-32 instrument, the E-96 separates the samples and reagents

by full extraction plates rather than columns of a single plate. The following instructions are based

on running complete plates, but fewer samples can be run by adjusting protocol appropriately.

• Set out 6 extraction plates and label according to the layout in Figure 3:

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Figure 3: Plate Layout for E-96 Auto Purification Instrument

• Add 400 µl of Lysis Buffer to each well in plate 1.

• Add 700 µl of Wash Solution 1 to each well in plate 2.

• Note: Make sure that ethanol has been added to Wash Solution 1 before first

use.

• Add 700 µl of Wash Solution 2A to each well in plates 3 and 4.

• Note: Make sure that ethanol has been added to Wash Solution 2A before first

use.

• Add 60 µl of Nuclease-Free Water to each well in plate 6.

• Add 200 µl of Magnetic Bind Beads to each well in plate 5.

• Note: Close bottle and resuspend beads frequently while loading plate to ensure

a consistent concentration of beads across all wells.

• Add 300 µl of Sample stored in transport media (e.g. VTM, UTM, PBS, etc.) into each

well in plate 1. Mix by pipetting up and down 10 times.

• Note: Work in an appropriate biosafety cabinet and take appropriate precautions


• Incubate plate at room temperature for 5-10 minutes.

• Add 400 µl of Isopropanol to each well in plate 1. Mix by pipetting up and down 10

times.

• Program the instrument as specified in Table 7 and save the run file for future use.

• Note: Be sure to use the tip mix option for the lysis and elution steps rather than

the shaking mix method.

Table 7: Programming parameters for GeneCount® E-96 Auto Purification Unit

Plate Name Mix

Time

Mix

Speed

Wait

Time

Magnet (Collect)

Time

Volume (µL)

5 Bead Pickup 0:30 Medium 0:00 1:00 200

1 Lysis/ Binding 10:00 Slow 0:00 1:00 1100

2 Wash 1 1:00 Fast 0:00 1:00 700

3 Wash 2A 1.00 Fast 0:00 1:00 700

4 Wash 2A 1.00 Fast 0:00 1:00 700

6 Elution* 3:00 Fast 5:00 1:00 60

5 Bead Discard 0:30 Slow 0:00 0:00 200

*Program the Elution plate to heat to 65℃ and to start heating at step 5.

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• Carefully insert all 6 Extraction Plates into GeneCount® E-96 Auto

Purification Instrument in the order as they appear in Figure 3, with position 1 being

furthest to the left. Make sure each plate fits snugly into the holding brackets.

• Place the 96-well rod cover into the slot on the upper deck above the 1st plate position,

making sure the plastic clip on the handle is locked in place.

• Run the automated extraction protocol.

• When finished, the eluted RNA from plate 5 can be used immediately for RT-qPCR

analysis. Alternatively, transfer eluted RNA into new nuclease-free microcentrifuge tubes

and store for up to 1 month at -70℃.

GeneCount® COVID-19 Assay Kit Procedure

Initial Setup

• If Master Mix Arrives Frozen: Gently thaw RT-qPCR Master Mix bottle on ice. Invert

tube several times to mix and then centrifuge for 5 to 10 seconds to collect contents to the

bottom of the tube.

• If Master Mix Arrives Lyophilized: Remove and discard rubber stopper and

transfer 1650 µL of Nuclease-Free Water into the RT-qPCR Master Mix bottle. Recap

and let RT-qPCR Master Mix rehydrate for 3 minutes. Mix occasionally by

swirling. Do not invert bottle.

• Note: Each bottle of RT-qPCR Master Mix contains enough for 96 samples.

• Note: Thawed or freshly rehydrated RT-qPCR Master Mix should be used

immediately or stored frozen at -20℃ for up to 12 months in single aliquots to

minimize freeze-thaws. Thaw completely before use.

• Transfer 50 µL of Nuclease Free Water into the Positive Control DNA tube. Recap

tube tightly. Allow Positive Control to rehydrate for 5 minutes. Mix tube occasionally

by inverting. Centrifuge for 5-10 seconds before use.

• Note: Each tube of Positive Control contains enough for 10 reactions.

• Note: Using the provided positive control is not necessary if a known positive

sample was processed and will be analyzed.

• Note: Freshly rehydrated Positive Control should be used immediately

or stored frozen at – 20°C for up to 12 months in single use aliquots to minimize

freeze-thaws. Thaw completely before use.

Assay Setup

• Assemble reactions (as outlined in Table 8) in the provided PCR strip tubes or optionally

in a 96-well PCR plate:

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Table 8: Reaction Assemblies for GeneCount® COVID-19 RT-qPCR Assay Kit

Component Sample Positive

Control

Negative

Control

RT-qPCR

Master Mix

15 µL 15 µL 15 µL

Nuclease-

Free Water

- - 5 µL

Positive

Control

- 5 µL -

Sample

RNA

5 µL - -

• Close caps or seal plate.

• Gently mix and then using a robust downward motion, shake the contents of qPCR tubes

to the bottom of tube or centrifuge tubes for 5-10 second.

• Note: Be careful to note the correct orientation of the tubes to prevent accidentally

reversing the tubes when inserting them into the qPCR device. A small mark with

a permanent marker on the side of the first tube can help prevent mis-orientating.

GeneCount® Q-16/Q-96 qPCR Analysis

• Set up qPCR instrument according to the manufacturer’s instructions.

• Note: GeneCount® Q-16 RT-qPCR device is pre-programmed with the correct

fluorescent and cycling parameters.

• Data acquisition should be set to use the following channels:

Table 9: Corresponding Channels and Targets for GeneCount® COVID-19 RT-qPCR Assay

Channel Target

FAM N2 gene

HEX E gene

Cy5 MS2 extraction control

• Temperature cycling should be set to the following parameters:

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Table 10: Temperature cycling parameters for GeneCount® Q-16/Q-96 qPCR Analysis

GeneCount® Q-16 GeneCount® Q-96

Step Temperature Time Cycles Temperature Time Cycles

Reverse

Transcription 50℃ 15 min 1 50℃ 15 min 1

Initial Denaturation 95℃ 2 min 1 95℃ 2 min 1

Denaturation 95℃ 10 sec 45 95℃ 10 sec 45

Annealing/Extension 55℃ 60 sec 55℃ 45 sec

• Load PCR strip tubes (or plate) into the qPCR instrument and start the run.

GeneCount® Q-16 Instrument Setup (Windows Software)

• Connect GeneCount® device via USB to desktop or laptop computer.

• Switch on GeneCount® Q-16 device via the switch on the back of the device – ensure

green power symbol on the front of the instrument is on.

• Start GeneCount® Software program on computer and wait for the program to initialize

device (this should only take a few seconds).

• Click Continue as a guest.

o To sign in at any time, click the button in the top right corner.

• On the software dashboard, ensure the HID Device Connected indicator is green.

Under the Setup tab, select the Sample Setup page (Figure 4) to:

1. Name the experiment in the Experiment Name field.

2. Open a previous experiment or a saved template file (if applicable).

3. Update the assay parameters from the LuminUltra™ cloud.

4. Enter sample information.

5. Save a new template.

19

Figure 4: Setup Tab on GeneCount® Q-16 Instrument Software

To Enter Sample Information:

• Select box in the grid that corresponds to the location of the sample in the GeneCount®

Q-16 qPCR instrument.

• Fill out the dialogue box.

• Click Add/Modify.

• Once sample information has been entered for all samples, click Continue setup to take

you to the Program Setup page.

• The Program Setup page displays all the cycling parameters for the chosen assay. The

parameters for each assay are preloaded on the GeneCount® software and do not need to

be modified by the user.

To Start an Experiment Run:

• Click the Start button.

• Experiment will begin immediately, and real-time data will be displayed on the Run tab.

• If for any reason the run needs to be terminated, click the Force Stop button on the Run

tab.

• Once run is complete, view results on the Analysis tab.

20

• Click on the Analysis tab (Figure 5) to:

1. View Amplification Graph

2. View Results Table

3. Normalize graph curves

Figure 5: Analysis Tab on GeneCount® Q-16 Instrument Software

• Once run is complete, click on the Report tab to:

• Print report as a spreadsheet (.csv).

• Save report as a spreadsheet (.csv), experiment file (.json), image (.jpg) or PDF

report.

• Upload report to LuminUltraTM cloud account.

21

GeneCount® Q-16 Instrument Setup (Android, Touchscreen Device)

• Switch on GeneCount® Q-16 device via the switch on the back of the device – ensure

green power symbol on the front of the instrument is on.

• Click Continue as guest.

On the Test Setup page:

• Start a new experiment.

• Open a saved template file (if applicable).

• Update the assay parameters from the LuminUltraTM cloud.

Enter sample data:

• Select the box in the grid that corresponds to the location of the sample in the

GeneCount® Q-16 qPCR instrument.

• Fill out the dialogue box

• Click OK.

Check cycling parameters:

• Select Cycling Program to go to the Cycling Program Setup page.

• The Cycling Program Setup page displays all the cycling parameters for the chosen

assay. The parameters for each assay are preloaded on the GeneCount® software and do

not need to be modified by the user.

To begin experiment run:

• Click Start to begin the experiment run.

• Select Run tab to view real-time data.

• If for any reason the run needs to be terminated, click the Manual Stop button.

Use the Amplification analysis tab to:

1. View Amplification Graph

2. View Results Table

3. Normalize the graph curves

Use the Results tab to:

• Export report to USB drive as a spreadsheet (.csv), experiment file (.json), image

(.jpg), or PDF report.

• Upload report to LuminUltra cloud account.

22

GeneCount® Q-96 Instrument Setup (Windows Software)

To start a new experiment file: Select New → Absolute

To start from a previously saved template: Click Open and select desired template.

To enter assay targets: Select Setup tab → Detector tab → Add Detector.

Use the Detector tab (Figure 6) to:

1. Enter experiment name into Experiment Name field.

2. Add new Detectors by selecting Add detector.

3. Enter target name into the Detector field. Add a separate detector for the three following

targets:

a. N2 gene

b. E gene

c. MS2 extraction control

4. Change fluorescent channel by selecting from options in the drop-down menu in the

Reporter field. Use the following Detector and Reporter combinations:

Table 11: Detector and Reporter combinations on GeneCount Q-96 Software for GeneCount® COVID-19

RT-qPCR Assay

Detector Reporter

N2 Gene FAM

E Gene HEX

MS2 Extraction Control Cy5

23

Figure 6:Setup Tab on GeneCount® Q-96 Instrument Software

Select the Sample tab to:

• Individually input sample names into the Sample ID field

• Press enter.

Select the Plate tab (Figure 7) to assign each cell on the grid:

1. Assay Item (N2 Gene, E Gene and MS2 Extraction Control)

2. Property (Unknown, Standard, Negative or Positive)

3. Sample ID

24

Figure 7: Plate Tab on GeneCount® Q-96 Instrument Software

To load samples on GeneCount® Q-96 Instrument:

• Press Open/Close Rack button

• Place tubes in grid – ensure tubes are in the identical order as shown on Plate Setup tab.

Select Program tab to input Cycling Conditions:

• Required cycling parameters for the GeneCount® COVID-19 RT-qPCR Assay Kit are as

shown in Table 10.

• The Reverse Transcription step must be added before the Initial Denaturation step as follows:

o Select first section on Run Programs Setup page (Figure 8)

o Click Add Step

o Select Before from drop-down menu

o Input appropriate Cycling Conditions

25

Figure 8: Run Programs Setup Page on GeneCount® Q-96 Instrument Software

• Select the Run tab

• Save experiment parameters prior to the experimental run.

• Click the Start Run. The experiment will start immediately.

o All gain and image settings are pre-calibrated and do not need to be changed.

o Fluorescence data can be viewed in real-time on the Fluorescence Curve page

under the Run tab.

Once the run is completed, view final data by selecting Analysis tab → Amplification Plot

(Figure 9).

Figure 9: Amplification plot tab on GeneCount® Q-96 Instrument Software

26

To export results, click Export Experiment on the Report tab and choose to export the data as a

text file (.txt) or spreadsheet (.csv).

Interpretation of Results

Upon completion of qPCR run, analyze data according to manufacturer’s instructions to

determine Ct values.

All test controls should be examined prior to interpretation of patient results. If any control group

does not meet the expected criteria, it is investigated for a root cause, the entire run and/or

extraction may have to be repeated and results cannot be interpreted or reported. The criteria for

the controls used in the process is summarized in Table 12.

Table 12: Expected Performance of GeneCount® COVID-19 RT-qPCR Assay Kit Controls

(Positive result when Ct <40)

Control Type N2 gene E gene MS2

Positive + + -

Negative - - -

Assessment of clinical specimen test results should only be performed after the positive and

negative controls have been examined and determined to be valid and acceptable. If the controls

are not valid, the patient results cannot be interpreted. The interpretation and reporting of clinical

specimens in summarized in Table 13.

27

Table 13: Interpreting Sample Results using the GeneCount® COVID-19 RT-qPCR Assay Kit

E

(Ct < 40)

N2

(Ct < 40)

MS2

(Ct range = 25 -35)

Control

status

Result Action

+/- + +/-

Valid SARS-CoV-

2 detected.

Positive for

COVID-19

by PCR.

Report results to

appropriate public

health authorities

and submitter as

positive for

COVID-19.

+ - +/-

Valid Inconclusive

for COVID-

19.

Repeat extraction

and RT-qPCR

test. Submit new

specimen if

clinically

indicated.

- - +

Valid SARS-CoV-

2 not

detected.

Negative for

COVID-19

by PCR.

Report results to

appropriate public

health authorities

and submitter as

negative for

COVID-19.

-

-

-

Invalid Invalid test. Repeat extraction

and RT-qPCR

test. If repeat

results remain

invalid, suggest

collecting new

specimen sample.

Note: Laboratories should include a statement such as, “the test has been validated but FDA’s

independent review of this validation is pending” with patient test results to healthcare providers.

Quality Control

• Quality control requirements should be performed in conformance with local state, and/or

federal regulations or accreditations requirements and user’s laboratory’s standard quality

control procedures. For further guidance on appropriate quality control practices, refer to

42 CFR 493.1200.

• Quality control procedures are intended to monitor reagent and assay performance.

• Test all positive and negative extraction controls when running diagnostic samples and

with each new test lot to ensure all test reagents and components are working properly.

• Good laboratory practice recommends including a positive extraction control and

negative extraction control in each nucleic acid isolation batch.

• All samples and controls include and external control to validation the success of the

extraction process and assay performance.

28

Kit Limitations

1. The use of this assay as an in vitro diagnostic. LuminUltra GeneCount® COVID-19 RT-

qPCR Assay Kit has completed the Section IV.C notification process under FDA’s

“Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency

(Revised)” and has not been reviewed by FDA.

2. This kit is used for the qualitative detection of SARS-CoV-2 RNA from human

nasopharyngeal swabs.

3. The GeneCount® COVID-19 RT-qPCR Assay Kit has been validated but FDA’s

independent review of this validation is pending.

4. Laboratories should include a statement such as, “the test has been validated but FDA’s

independent review of this validation is pending” with patient test results to healthcare

providers.

5. The GeneCount® COVID-19 RT-qPCR Assay Kit performance has only been established

with the specimen types described in the Intended Use section. Testing other types of

specimen may cause inaccurate results.

6. The specimens to be tested shall be collected, processed, stored and transported in

accordance with the conditions specified in the instructions. Inappropriate specimen

preparation and operation may lead to inaccurate results.

7. Extraction and amplification of nucleic acid from clinical samples must be performed

according to the specific methods listed in this procedure. Other extraction approached

and processing systems have not been evaluated.

8. Amplification and detection of SARS-CoV-2 with the GeneCount® COVID-19 RT-qPCR

Assay Kit has only been validated with the GeneCount® Q-16 and Q-96 qPCR

instruments. Use of other instrument systems may cause inaccurate results.

9. The limit of detection (LoD) is determined based on a 95% confidence of detection.

When SARS-CoV-2 presents at or above the LoD concentration in a test specimen, there

will be a low probability that SARS-CoV-2 is not detected. When SARS-CoV-2 presents

below the LoD concentration in the test specimen, there will also be certain probability

that SARS-CoV-2 can be detected.

10. Negative results do not preclude SARS-CoV-2 infections and should not be used as the

sole basis for treatment or other management systems.

11. Positive results may be due to past or present infection with non-SARS-CoV-2

coronavirus strains, such as coronavirus HKU1, NL63, OC43, or 229E.

12. Laboratories are required to report all positive results to the appropriate public health

authorities.

29

Performance Evaluation

Limit of Detection (LoD)

Studies were performed to determine the analytical limit of detection (LoD) of the

GeneCount® COVID-19 RT-qPCR Test Kit. LoD is defined as the lowest concentration at which

95% of samples are positive for each assay target.

The LoD was determined using contrived clinical nasopharyngeal swab samples. Positive

contrived samples were prepared by spiking negative nasopharyngeal swab specimens obtained

from Boca Biolistics and BioIVT. The negative swabs were spiked with known concentrations of

AccuplexTM SARS-CoV-2 RNA reference material (SeraCare, Cat. # 0505-0159).

In LoD studies, a preliminary LoD was determined by testing a range of concentrations with 5

sample replicates at each concentration. The preliminary LoD was then confirmed by testing the

preliminary LoD at 20 sample replicates each as well as one concentration level below the

preliminary LoD to confirm the true LoD value.

The preliminary LoD was estimated using the GeneCount Clinical Auto RNA Isolation Kit with

the GeneCount® Q-96 qPCR instrument and included 5 sample replicates at each of the 3 dilution

concentrations: 1000, 500 and 170 copies/mL. The preliminary LoD study estimated the LoD for

the GeneCount Clinical Auto RNA Isolation Kit and Q-96 instrument at 500 copies/mL. Results

of the preliminary LoD study are summarized in Table 14.

Table 14: Preliminary LoD Range Results Using GeneCount Q-96 Instrument

Target E Gene N2 Gene MS2 Internal

control

Concentration

(Copies/mL)

170 500 1000 170 500 1000 170 500 1000

Positives/total 4/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

Mean Ct 34.5 33.3 32.5 36.3 34.8 33.4 28.5 28.5 28.4

Standard

deviation

0.43 0.62 0.89 1.10 0.47 0.24 0.13 0.40 0.23

The MS2 bacteriophage internal control was valid for all LoD experiments. Negative controls of

nuclease-free water were also run and confirmed to pass for every batch of samples analyzed.

Positive controls were not included in the LoD experiment as it was not needed to validate the

expected outcome as only positive samples were tested.

30

Confirming LoD with GeneCount Clinical Auto RNA Isolation Kit

Based on the preliminary results, a confirmatory study was conducted for the GeneCount COVID-

19 RT-qPCR Assay Kit when using the GeneCount Clinical Auto RNA Isolation kit and both the

GeneCount® Q-16 and Q-96 qPCR instruments. In the study, 20 sample replicates spiked at a

concentration of 500 copies/mL were tested.

The GeneCount® Q-16 showed a 100% detection rate for all targets while the GeneCount® Q-96

had a 95% detection rate for all targets therefore the LoD was confirmed at 500 copies/mL for

both Q-16 and Q-96 instruments.

To further confirm the limit of detection, 20 samples at a concentration of 0.17 copies/ul were

tested with the GeneCount Q-16 instrument. Results showed that only 18/20 samples detected all

targets therefore the limit of detection was again confirmed at 500 copies/ml. Results from the

confirmatory studies are summarized in Table 15.

Table 15: Results of Confirmatory LoD Study using GeneCount® Q-16 and Q-96 Devices

GeneCount® Q-16 GeneCount Q-96

Target E N2 MS2 E N2 MS2

Concentration

(copies/mL)

170 500 170 500 170 500 500 500 500

Positives/total 20/20 20/20 18/20 20/20 20/20 20/20 19/20 19/20 20/20

Mean Ct 33.07 33.51 37.83 37.87 29.26 29.67 33.04 37.43 29.55

Standard

deviation

0.95 1.23 1.09 1.07 0.80 0.95 0.81 1.46 0.45

Confirming LoD with GeneCount Clinical Advanced RNA Isolation Kit

A confirmatory LoD study was also performed for the GeneCount COVID-19 RT-qPCR Assay

Kit when using the GeneCount Clinical Advanced RNA Isolation Kit. These studies were

performed following the LoD studies for the GeneCount Clinical Auto RNA Isolation kit which

found identical LoD values for both the Q-16 and Q-96 instruments. Therefore, for simplicity the

confirmatory LoD study for when using the GeneCount Clinical Advanced RNA Isolation Kit was

conducted only on the GeneCount Q-96 instrument.

For the study, samples were spiked at concentrations of 500, 170 and 60 copies/mL with 20

replicate samples tested at each concentration. Results confirmed the LoD for the GeneCount

COVID-19 RT-qPCR Assay Kit when using the GeneCount Clinical Advanced RNA Isolation kit

at 170 copies/mL which achieved a 100% detection rate for all targets.

31

To further confirm the LoD, 20 samples were also tested at 60 copies/ml where the E gene was

accurately detected in 70% of the samples while the N2 gene was accurately detected in only 50%

of positive samples therefore the LoD was again confirmed at 170 copies/mL. Results of the LoD

study are summarized in Table 16.

Table 16: Results of Confirmatory LoD Study using GeneCount® Clinical Advanced RNA

Isolation kit and Q-96 Device

GeneCount® Q-96

Target E N2 MS2

Concentration

(copies/mL)

60 170 500 60 170 500 60 170 500

Positives/total 14/20 20/20 20/20 10/20 20/20 20/20 20/20 20/20 20/20

Mean Ct 33.60 33.20 31.88 38.42 36.87 34.36 29.15 28.58 28.36

Standard

deviation

1.01 0.92 0.57 1.18 1.16 0.54 0.93 0.65 0.47

Confirming LoD with Lyophilized COVID-19 RT-qPCR Master Mix

All previous LoD studies were performed using a frozen version of the COVID-19 RT-qPCR

Master Mix. Further studies were also performed to determine if using lyophilized COVID-19 RT-

qPCR Master Mix with the GeneCount® COVID-19 RT-qPCR Assay would affect LoD. Since

identical LoDs were found for the Q-96 and Q-16 instruments in previous studies when using the

same GeneCount® Clinical RNA Isolation Kit, only the Q-96 instrument was used in the study for

simplicity.

Samples were spiked at concentrations of 500 and 170 copies/mL with 20 replicate samples tested

at each concentration. Results confirmed the LoD for the GeneCount® COVID-19 RT-qPCR Assay

Kit when using the GeneCount® Clinical Auto RNA Isolation kit and lyophilized Master Mix at

500 copies/mL which achieved a 100% detection rate for all targets.

As seen in Table 17, samples at 170 copies/mL only had accurate detection of the E gene in 75%

of samples while the N2 gene was accurately detected in 85% of positive samples therefore the

LoD was again confirmed at 500 copies/mL. Results of the LoD study are summarized in Table

17.

The LoD of 500 copies/mL is identical to the LoD determined when using a frozen version of the

COVID-19 RT-qPCR Master Mix. Based on these results, lyophilized COVID-19 RT-qPCR

Master Mix does not affect LoD of the assay.

32

Table 17: Results of Confirmatory LoD Study using Lyophilized COVID-19 RT-qPCR Master

Mix

GeneCount® Q-96

Target E N2 MS2

Concentration

(copies/mL)

170 500 170 500 170 500

Positives/total 15/20 20/20 17/20 20/20 20/20 20/20

Mean Ct 33.73 33.51 35.03 37.87 29.97 27.79

Standard deviation 0.65 1.23 0.49 1.07 0.48 0.32

Confirming LoD with GeneCount® E-96 Auto Purification Unit

All previous LoD studies using the GeneCount® Clinical Auto RNA Isolation Kit were performed

using the GeneCount® E-32 Auto Purification Unit therefore a LoD study was also performed

using the GeneCount® E-96 Auto Purification Unit as part of the GeneCount® Clinical Auto RNA

Isolation Kit to determine method LoD. Lyophilized COVID-19 RT-qPCR Master Mix Master

Mix was also used in this study.

Based on results from previous studies using the GeneCount® E-32 Auto Purification Unit,

samples were spiked at concentrations of 500 and 170 copies/mL with 20 replicate samples tested

at each concentration. Results confirmed the LoD for the GeneCount Clinical Auto RNA Isolation

kit when using the GeneCount® E-96 Auto Purification Unit at 500 copies/mL with the E gene

accurately detected in 95% of samples and the N2 gene accurately detected in 100% of samples.

This confirmed LoD is identical to the confirmed LoD when using the GeneCount® E-32 Auto

Purification Unit therefore either of the auto purification units can be used without affecting the

limit of detection.

When testing the method at a spike concentration of 170 copies/mL, the N2 gene was accurately

detected in 100% of samples however the E gene was accurately detected in 85% of the samples

therefore the LoD was again confirmed at 500 copies/mL. Results of the LoD study are

summarized in Table 18.

33

Table 18: Results of Confirmatory LoD Study using GeneCount® E-96 Auto Purification Unit

GeneCount® E-96/GeneCount® Q-96

Target E N2 MS2

Concentration

(copies/mL)

170 500 170 500 170 500

Positives/total 17/20 19/20 20/20 20/20 20/20 20/20

Mean Ct 36.71 32.50 33.14 36.00 28.92 27.95

Standard deviation 1.96 0.79

1.11 1.31 0.24 0.31

Inclusivity Analysis

In silico inclusivity analysis was performed using NCBI Blastn queries to evaluate all assay E and

N2 primer and probe sequences against the 15,372 publicly available, full and partially sequenced

strains of SARS-CoV-2 genomes available in the NCBI Betacoronavirus Genbank database as of

August 11, 2020. All E and primer and probe sequences showed 100% homology to all SARS-

CoV-2 sequences. N2 primers shows 100% homology to all SARS-CoV-2 sequences and the N2

probe had 100% homology to SARS-CoV-2 sequences with the exception of a single mismatch

found with 2 SARS-CoV-2 sequences (96% homology). The N2 gene has been well characterized

and used by many EUA-granted assays including the United States Centers for Disease Control

and Prevention (CDC 2019-nCoV Real-Time RT-qPCR Diagnostic Panel, CDC-006000019, Rev.

05), therefore these two single mismatches are not expected to negatively impact the use of this

test.

Cross-Reactivity Analysis

Cross-reactivity analysis of the assay primer and probe sequences was performed through in silico

analysis using National Center for Biotechnology Information (NCBI) BLASTn queries against

public domain nucleotide sequences. The database search included GenBank, EMBL, DDBJ, PDB

and RefSeq sequences but excluded EST, STS, GSS, WGS, TSA, patent sequences as well as 0, 1

and 2 HTGS sequences and sequences longer than 100 mb. The database is non-redundant with

identical sequences merged into a single entry, while preserving the accession, GI, title and

taxonomy information for each entry. The database was updated on August 11, 2020.

34

In silico analysis evaluated E and N2 primer and probe sequences to all sequences in NCBI

standard databases (Nucleotide collection) for all organisms listed in Table 19. Organisms with

> 80% homology to the primer and probe sequences are categorized as potentially cross-reactive.

Table 19: In Silico Cross-Reactivity Analysis of E and N2 Oligonucleotide Targets

Pathogen Name Tax ID E Homology N2 Homology

Human coronavirus 229E taxid:11137 Fwd: < 80% similarity

Rev: < 80% similarity

Probe: < 80% similarity

Fwd: < 80% similarity



Human coronavirus OC43 taxid:31631 Fwd: < 80% similarity






Human coronavirus HKU1 taxid:290028 Fwd: < 80% similarity






Human coronavirus NL63 taxid:277944 Fwd: < 80% similarity






SARS-coronavirus taxid:694009 Fwd: 100% similarity

Rev: 100%similarity

Probe: 100% similarity

Fwd: 100% similarity



MERS-coronavirus taxid:1335626 Fwd: < 80% similarity






Adenovirus (e.g. C1 Ad. 71) taxid:1907210 Fwd: < 80% similarity






Human Metapneumovirus

(hMPV)

taxid:162145 Fwd: < 80% similarity






Parainfluenza virus 1-4 taxid:11210

taxid:11214

taxid:11217

taxid:11224

taxid:11226







Influenza A & B taxid:11320

taxid:11520







Enterovirus taxid:42789 Fwd: < 80% similarity






Respiratory syncytial virus taxid:12814 Fwd: < 80% similarity






Rhinovirus taxid:169066 Fwd: < 80% similarity






Chlamydia pneumoniae taxid:83558 Fwd: < 80% similarity






35

Haemophilus influenzae taxid:727 Fwd: < 80% similarity






Legionella pneumophila taxid:446 Fwd: < 80% similarity






Mycobacterium tuberculosis taxid:1773 Fwd: < 80% similarity






Streptococcus pneumoniae taxid:1313 Fwd: < 80% similarity






Streptococcus pyogenes taxid:1314 Fwd: < 80% similarity






Bordetella pertussis taxid:520 Fwd: < 80% similarity






Mycoplasma pneumoniae taxid:2104 Fwd: < 80% similarity






Pneumocystis jirovecii (PJP) taxid:42068 Fwd: < 80% similarity






Candida albicans taxid:5476 Fwd: < 80% similarity






Pseudomonas aeruginosa taxid:287 Fwd: < 80% similarity






Staphylococcus epidermis taxid:1282 Fwd: < 80% similarity






Streptococcus salivarius taxid:1304 Fwd: < 80% similarity






All E primers and probe sequences showed 100% homology with SARS-coronavirus as the

primers/probe are specific to both SARS-CoV-2 and SARS-coronavirus. This is not clinically

significant as the E primers and probes are used as an additional confirmatory indicator of infection

in this assay and alone cannot give a conclusive result for COVID-19 infection, as per the

interpretation of results. There is also a low prevalence of SARS-coronavirus and the virus is not

currently known to be circulating in the human population therefore the cross-reactivity with the

E gene is of minimal clinical concern.

As was found in the in silico analysis results published by CDC (CDC-006-00019, Rev: 05), the

N2 forward primer sequence showed high similarity to SARS-coronavirus. The N2 reverse primer

and probe sequences showed no significant homology with SARS-coronavirus, human genome,

36

other coronaviruses or human microflora. Combining in silico analysis results for all N2 primers

and probes, there is no prediction of potential false positive results.

Based on the in silico analysis results, cross reactivity is not expected with human genome, other

coronaviruses or human microflora resulting in a potential false positive RT-qPCR result. The

homology of the E primer/probe is accounted for in the interpretation of results.

Clinical Evaluation

The clinical performance of the GeneCount® COVID-19 RT-qPCR Test Kit was evaluated using

a total of 60 frozen remnant nasopharyngeal swab specimens (30 positive samples and 30 negative

samples obtained from Boca Biolistics and BioIVT). Samples obtained from Boca Biolistic were

previously confirmed positive or negative in a CLIA lab using the Hologic Panther Fusion SARS-

CoV-2 Assay or the PerkinElmer New Coronavirus Nucleic Acid Detection Kit, both of which

have FDA EUA status. Samples obtained from BioIVT were previously confirmed positive or

negative in a CLIA lab using the Hologic Panther Fusion SARS-CoV-2 Assay which has FDA

EUA status. For the clinical evaluation, RNA was isolated using the GeneCount Clinical Auto

RNA Isolation Kit. All clinical evaluation data is provided in Appendix A.

All 30 confirmed positive specimens tested positive with the GeneCount® COVID-19 RT-qPCR

Test. 29 out of the 30 confirmed negative specimens tested negative with one presumptive negative

specimen testing positive for the N2 gene but negative for the E gene. Table 20 summarizes the

clinical evaluation results between known positive and negative samples confirmed using FDA

EUA granted tests and the GeneCount COVID-19 RT-qPCR Test Kit.

Table 20: Clinical Evaluation Study Results for GeneCount COVID-19 RT-qPCR Assay Kit

EUA Authorized Comparator (Hologic

Panther Fusion SARS-CoV-2 Assay or the

PerkinElmer New Coronavirus Nucleic Acid

Detection Kit)

Positive Inconclusive Negative Total

LuminUltra

GeneCount

SARS-CoV-2

Assay Kit

Positive 30 0 1 31

Inconclusive 0 0 0 0

Negative 0 0 29 29

Total 30 0 30 60

PPA: 30/30 = 100% (95% Confidence Interval = 85.9% - 100%)

NPA: 29/30 = 97% (95% Confidence Interval = 80.9% - 99.8%)

37

Contact Information

LuminUltra Technologies

520 King Street

Fredericton, New Brunswick

Canada, E3B 6G3

Phone: +1 (506) 459-8777

Website: www.luminultra.com

Documents

LuminUltra GeneCount COVID-19 RT-qPCR Assay Kit