Genedrive 96 SARS-CoV-2 Kit - Beckman

ID-COV2-IFU-USA V3

Genedrive® 96 SARS-CoV-2 KitID-CoV2-01-LPU (Low Profile Version) ID-CoV2-01-FPU (Fast Plate Version)ID-CoV2-01-BPU (Bio Plate Version)

Store at temperature 2˚C to 30˚C

For the most up to date version of this document, please visit www.genedrive.com

Validation of this test has not been reviewed by FDA. Review under the EUA program is pending.For In Vitro Diagnostic (IVD) UseRx Only

IMPORTANT: Please read these instructions prior to using this kit.

Table of contents 2 Intended use

2 Summary and explanation

3 Principles of the procedure

3 Compatible RNA extraction methodologies

5 Kit components

5 Materials required but not supplied

6 Specimen collection, handling and storage

6 Storage and shelf life

6 Operating conditions

6 Warnings and precautions

7 Limitations of use

8 Test procedure

16 Result interpretation

19 Genedrive® 96 Exporter – result interpretation

22 Quality controls

23 Troubleshooting

24 Performance characteristics

24 Analytical sensitivity

25 Analytical specificity

26 Precision

26 Inclusivity

27 Diagnostic accuracy

29 Quick reference guide – manual set up

32 Manufacturer details

Terms & Abbreviations cDNA Complementary DNA

COVID-19 Coronavirus disease 2019

DNA Deoxyribonucleic acid

dNTPs Deoxyribonucleotide triphosphates

LoD Limit of Detection

PCR Polymerase Chain Reaction

Real-Time PCR Real-Time Polymerase Chain Reaction

RNA Ribonucleic acid

RNase P Ribonuclease P

RT-PCR Reverse Transcription Polymerase Chain Reaction

SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2

WHO World Health Organization

Summary and explanationUntil recently there were six known members of the

Orthocoronavirinae sub-family of viruses (more commonly

referred to as Coronaviruses) that were capable of

infecting humans. Of these, four are known to cause

common cold symptoms (HKU1, NL63, 229E and OC43).

Whilst two are zoonotic and are associated with more

severe respiratory symptoms (SARS-CoV and MERS-CoV)1.

SARS-CoV-2 emerged from China in late 2019. WHO

declared a global pandemic on 11th March 2020.

Since emergence, it has been demonstrated that the

virus can transfer from person to person and through

environmental contamination. Due to the high infectivity

of the virus it is essential that positive individuals are

identified and quarantined to limit onwards transmission

of the disease.

Intended useGenedrive® 96 SARS-CoV-2 Kit is an end point melt

analysis RT-PCR test intended for the qualitative

detection of RNA from the SARS-CoV-2 in upper

respiratory specimens from individuals suspected of

COVID-19 by their healthcare provider. Testing is limited

to laboratories - certified under the Clinical Laboratory

Improvement Amendments of 1988 (CLIA), 42 U.S.C.

§263a, to perform high complexity tests, or by similarly

qualified non-U.S. laboratories.

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

The SARS-CoV-2 RNA is generally detectable in upper

respiratory 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.

SARS-CoV-2 is a novel Coronavirus currently thought

to have been of zoonotic origin that originally infected

humans from close contact with animals, at open markets

in China. Once infected, the virus causes disease

symptoms known as COVID-19. The symptoms may

include a dry cough, fever, shortness of breath, fatigue,

respiratory failure and death.

The Genedrive 96 SARS-CoV-2 Kit is an in vitro

diagnostic (IVD) molecular assay for the detection of

species-specific genetic sequences within the E and N

genes of the SARS-CoV-2 genome2. The kit is indicated for

use by a laboratory professional for the identification of

SARS-CoV-2 in RNA specimens that have been derived

from upper respiratory specimens.

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.

The Genedrive 96 SARS-CoV-2 Kit is intended for use

by qualified and trained clinical laboratory personnel

specifically instructed and trained in the techniques

of Real-Time PCR and in vitro diagnostic procedures.

Validation of this test has not been reviewed by FDA.

Review under the EUA program is pending.

The kit is intended to be run on the following real-time

thermocycler platform:

• Roche LightCycler® 480 II

• Applied Biosystems® 7500 Fast Real-Time PCR instrument

• Bio-Rad CFX96 Real-Time PCR Detection System

References

1. Chu, D., Pan, Y., Cheng, S., Hui, K., Krishnan, P., Liu, Y., Ng, D., Wan, C., Yang, P., Wang, Q., Peiris, M. and Poon, L., 2020. Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia. Clinical Chemistry, 66(4),

pp.549-555

2. WHO.int. 2020. Laboratory Testing For 2019 Novel Coronavirus (2019-Ncov) In Suspected Human Cases. [online] Available at: https://www.who.int/publications-detail/laboratory-testing-for-2019-novel-coronavirus-in-suspected-human-cases-20200117

ID-COV2-IFU-USA V3 2

Compatible RNA extraction methodologiesThe specimen input into the Genedrive 96 SARS-CoV-2 Kit

reaction wells is RNA that has been extracted from upper

respiratory specimens using an extraction instrument and kit.

The following extraction methodologies have been validated

Principles of the procedureGenedrive 96 SARS-CoV-2 Kit is a simplified one-step

test designed for the diagnosis of SARS-CoV-2 from RNA

extracts derived from upper respiratory swabs. All the

reagents required for the test are supplied as a lyophilized

bead, to which the specimen is added prior to running

the test.

The genetic targets are species-specific sequences which

are located in the E and N genes of the virus. The target

is amplified via a one-step RT-PCR reaction, whereby

RNA conversion to cDNA and subsequent asymmetric

amplification of cDNA occurs within the same well without

the need of user intervention. The detection is conducted

via light emission of fluorophores during end-point melt

curve analysis.

The fluorescent probe for each target is designed to melt

from the template within discrete temperature ranges. The

instrument measures the resulting change in fluorescence

and displays the changes as peaks on a temperature

profile, or melt curve, for analysis.

The Genedrive 96 SARS-CoV-2 Kit targets and amplifies

endogenous human RNase P, intended to ensure that

human sample is present in the amplification reaction.

However, levels of endogenous human RNA present

may be donor or extraction kit dependent. Users have

the option to “spike” the patient specimen with the

recommended human RNA control material to normalize

RNase P levels prior to the RNA extraction process.

The chemistry also contains primers and probes for a

synthetic RNA amplification control (IPC). The presence

of the associated peak indicates that amplification has

occurred as expected, therefore the test has been set up

correctly and there are no inhibitory substances within the

added specimen. This provides increased confidence that

a negative result is due to absence of CoV-2 target RNA,

rather than failure of amplification.

Each individual Genedrive 96 SARS-CoV-2 Kit contains

all the components necessary to conduct 96 reactions.

The reagents are lyophilized in reaction wells ready for

PCR and end-point melt analysis (dNTPs, buffers, reverse

transcriptase, DNA polymerase, specific primers/probes).

The lyophilized reagents are rehydrated within the wells,

using the specimen or control material. The plate is then

briefly centrifuged and placed into the appropriate

Real-Time PCR instrument.

The test consists of a target DNA amplification step

followed by an end-point melt curve analysis. The data

requires interpretation after the run is completed.

Please see analysis instructions and Table 7 for a result

interpretation matrix. The time from specimen input to

result is 100-119 minutes dependent on the platform

the test is run on.

for use, with verification of analytical LoD across all Real-

Time PCR instruments listed (See Table 1) and validated with

contrived specimens (Qiagen Viral mini, Table 1) and clinical

RNA specimens (Qiagen Viral Mini, Table 1).

Manufacturer RNA Extraction Kit Product code

Real-Time PCR instrument used for Diagnostic Accuracy

Real-Time PCR instrument used for Analytical Sensivity

(LoD)

Qiagen

Virus/Pathogen Kit with QIAamp® MinElute

Virus Spin Kit57704 Roche LightCycler 480 II

QIAamp Viral RNA Mini Kit

52904/52906 Roche LightCycler 480 II Roche LightCycler 480 II

Beckman CoulterRNAdvance Viral RNA

purification KitC63510

ABI 7500 Fast

Bio-Rad CFX96

Roche LightCycler 480 II

CytivaSera-Xtracta Virus/

Pathogen Kit29514201/ 29506009

Bio-Rad CFX96


Table 1

Product name Manufacturer Product code

LightCycler 480 II Roche Diagnostics05 015 278

001

ABI 7500 FastThermo Fisher

Scientific4351106/7

CFX96 Bio-Rad 1845097-IVD

For all RNA extraction methodologies, ensure optional or

additional steps suggested in the extraction kit instructions

for use are taken to reduce ethanol or wash buffer

carryover as much as possible to reduce the chance of

reaction inhibition, which could impact test sensitivity.

Compatible Real-Time PCR instrumentation

The Genedrive 96 SARS-CoV-2 Kit is available as low

profile, bio or fast 96-well plates and is validated for use

with the following Real-Time PCR instrument.

All instruments used must be installed, calibrated,

checked and maintained according to the

manufacturer’s instructions for best results.


Materials required but not supplied:Reagents

• RNA extraction kit• Negative control• Positive control• Molecular grade Nuclease Free Water (e.g. Sigma W4502)• Human Lymphocyte RNA (AMSBIO, ATR1254148-10)

(Extraction control) This accessory can be obtained from Genedrive

Kit components

Catalogue Number Description

ID-CoV2-01-LPU Genedrive 96 SARS-CoV-2 Kit – Low Profile Plate (96 reactions per kit)

ID-CoV2-01-FPU Genedrive 96 SARS-CoV-2 Kit - Fast Plate (96 reactions per kit)

ID-CoV2-01-BPU Genedrive 96 SARS-CoV-2 Kit - Bio Plate (96 reactions per kit)

Equipment

• RNA extraction

instrument (optional)

• Real-Time PCR instrument

• Powder free

disposable gloves

• Calibrated pipettes (p20,

p200)

• Plate centrifuge

• Class 2 safety cabinet

• Plate vortex

Part

Description

96 well low profile, Bio or fast bar-coded plate containing lyophilized reagent mix in each well. Each plate is supplied foil-sealed. Lyophilized contents (each bead):• DNA polymerase enzyme• Reverse transcriptase enzyme• Oligonucleotide primer pairs;

FAM labelled Oligonucleotide probes• dNTPS (dATP, dCTP, dATP, dTTP)• Stabilisers, including Bovine serum

albumin (<1% (w/w))• Synthetic RNA

Optical film seal Desiccant pouch

Quantity 1 per kit 1 per kit 1 per kit

Information Non-hazardous Non-hazardous Non-hazardous

Low profile block Require GDL Product Code: ID-CoV2-01-LPU

Company Model

Roche LightCycler 480 II Real-Time PCR System

Bio plate block Require GDL Product Code: ID-CoV2-01-BPU

Company Model

Bio-Rad CFX96 Real-Time PCR Detection System

Fast plate block Require GDL Product Code: ID-CoV2-01-FPU

Company Model

Applied Biosystems 7500 Fast Real-Time PCR System

Ordering details: Ensure the correct plate type is used for the instrument

• Single-use RNase, DNase-

free aerosol resistant

pipette tips (p20, p200)

• Any PPE required by local

safety guidelines

• 96 well cool block

• Genedrive 96 Exporter

software (product code:

GE-01) (optional)


For suggested controls please see the

Quality Controls section of this document

*WHO Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases Guidance March 2020


Specimen collection, handling and storageThe Genedrive 96 SARS-CoV-2 Kit is intended for use on

RNA extracts stored in viral transport media and derived

from upper respiratory specimens.

Due to the sensitivity of RNA toward shearing by freeze

thaw, do not use specimens that have undergone more

than one freeze-thaw cycle either pre- or post-extraction.

Table 2 indicates the World Health Organization (WHO)

recommendations for handling of specimen types.*

Note: Local regulations for specimen handling must take

priority over these recommendations.

Operating conditionsFor the operating conditions of third party instrumentation

please refer to the associated manufacturer’s user manual.

Nasopharyngeal swabs

Oropharyngeal swabs

Collection

Dacron or

polyester flocked

swabs in viral

transport medium

Dacron or

polyester flocked

swabs in viral

transport medium

Transport temperature

4˚C 4˚C

Initial storage 4˚C for 3≤ days 4˚C for 3≤ days

Long term storage

-70˚C for one

month

-70˚C for one

month

Table 2 WHO recommendations for handling of swab types

Storage and shelf life• Genedrive 96 SARS-CoV-2 Kit should be stored

between 2˚C – 30˚C

• The expiry date for each kit is identified on the kit label

• Expired kits must not be used

Warnings and precautions• RNA is extremely susceptible to shearing by freeze-

thaw cycles. Ensure freeze and thaw of all fresh

specimens and extracted RNA from specimens is

kept to an absolute minimum to prevent failed tests.

• RNA samples are susceptible to degradation by

contaminating RNases. Ensure all specimens are

handled with appropriate laboratory RNA handling

procedures.

• For in vitro diagnostic (IVD) use only

• For prescription use only

• Do not use Genedrive 96 SARS-CoV-2 Kits that have

exceeded the expiry date shown on the outer kit label.

• Each reaction well is designed to perform one test.

Do not reuse any reaction wells.

• The test should be performed in a clean environment,

on a flat surface.

• Each kit is single use, regardless of how many test

wells have been used.

• Immediately discard the kit if the protective foil pouch

has been pierced/broken in transit.

• Visually check for any missing beads in the 96 well

plate. Do not use any wells that do not contain a

lyophilized reagent bead.

• Protect the kits against humidity, prolonged exposure

to humidity may affect the performance of the product.

• Ensure the correct volume of specimen is used

(a change in volume may result in test failure).

• Do not disturb the Real-Time PCR instrument whilst

running a test.

• Do not attempt to remove the optical film seal from

the plate following the completion of the test. This

will result in high risk of local contamination.

• Dispose of plates immediately following run

completion. Prolonged storage following run

completion may increase risk of loss of seal integrity.

• Used plates should be treated as potentially infectious

materials and should be disposed of accordingly as

clinical waste, in accordance with local guidelines.

• Specimens must be treated as potentially infectious.

Take the necessary precautions during collection,

storage, treatment and disposal of specimens.

• PCR must always be carried out using good

laboratory practices.


Limitations of useThe Genedrive 96 SARS-CoV-2 Kit has been validated for

use with upper respiratory specimens run on the Roche

LightCycler 480 II, Applied Biosystems 7500 Fast

Real-Time PCR systems and Bio-Rad CFX96.

Any deviations from the procedures detailed within this

IFU may result in erroneous results or failure.

Samples should be handled and treated as if they are

infectious and all biosafety precautions should be followed.

All results should be interpreted by a trained

healthcare professional.

A negative result does not conclusively rule out the

possibility of SARS-CoV-2 infection. Patient medical history

and clinical symptoms must also be taken into account.

Whilst the test is fully exclusive to non-CoV-2 human

pathogens described in Table 11, a negative SARS-CoV-2

test does not rule out disease caused by other pathogens.

Laboratories should include a statement such as

‘the test has been validated but FDA’s independent

review of this validation is pending’ in test reports to

healthcare providers.

False positive results may be caused by:

• Cross-contamination of wells

• Local contamination caused by unsuitable handling of

positive SARS-CoV-2 samples or other positive materials

• Local contamination caused by unsuitable handling of

PCR amplicons.

False negative results may be caused by:

• Unsuitable collection, handling and/or storage of

samples (review RNase P control result)

• Sample taken from patient whilst outside of viraemic

phase of SARS-CoV-2

• IFU has not been followed

• Poor laboratory practice for handling RNA samples.

Test failed results may be caused by:

• Carryover of ethanol from RNA extraction wash buffers

• RNA extraction failure

• IFU has not been followed

• Incorrect automated RNA extraction system

input and eluate volumes

• Not adding specimen or run control to reaction well

• Not including the RNA process extraction control

Use of the Extraction Control - Human Lymphocyte RNA

Human Lymphocyte RNA may be used as an optional

extraction control to assess RNA extraction performance.

When included in the RNA extraction procedure,

successful RNA extraction and test performance will be

indicated by positive detection of RNase P. The Extraction

Control is supplied in an anhydrous 10 µg format. Each vial

may be used to perform between sixty-six and 100 kits

depending on RNA isolation methodology – equivalent to

between 6,336 to 9,600 reactions.

The Extraction Control must first be reconstituted using

Nuclease-Free Water, then dispensed into single use

aliquots, each sufficient to set up one full Genedrive 96

SARS-CoV-2 Kit (1 x 96 well plate). Single use aliquots

must be stored frozen at -80°C. When performing the RNA

extraction procedure for specimens to be analysed using

the Genedrive 96 SARS-CoV-2 Kit, a single aliquot of the

RNA extraction control should be thawed on ice and the

appropriate amount added directly to the RNA extraction

kit lysis/binding buffer before dispensation of the lysis/

binding buffer master mix to each of the specimens.

For specific instructions for each validated RNA extraction

kit see below.

If you require support using the Extraction control

with an extraction kit not listed, please contact

[email protected] for guidance.

1. The Human Lymphocyte RNA is shipped at ambient

temperature. On receipt, store at ambient temperature

(15-30°C) until required.

2. On first use, reconstitute RNA by addition of

200 μL room-temperature NFW, and incubate

at room temperature for 10 minutes. Do not use

ice-cold water for reconstitution. Mix by brief vortex

and centrifugation.

3. Transfer 200 μL reconstituted RNA to a nuclease-free

2 mL microfuge tube and add 1800 μL nuclease-free

water to a final concentration of 5 ng/mL. Mix by

pipetting several times, followed by brief vortex and

centrifugation.

• RNA at 5 ng/μL is stable on ice for up to 8 hours.

• Check Table 3 and the specific instructions below for

the required aliquot volume for the RNA extraction kit

being utilised.

• Prepare aliquots in nuclease-free microfuge tubes and

store as single-use aliquots at -80°C.

• Thaw on ice when required for use.

• Aliquots are intended for single use only, do not

re-freeze thawed aliquots. Repeated freeze-thaw will

impact RNA integrity and risk Extraction Control failure.

• As a guide, each single-use aliquot is sufficient for

1 X 96-well kit with 15% overage.

Calculation of the quantity of extraction control

required per sample (A) is performed according to the

following formula:

Where:

A = Quantity of extraction control to spike per sample

extraction (ng)

B = Quantity of extraction control required per 20 μL

PCR assay (ng)

C = The elution volume of the RNA isolation kit (μL)

20 = Genedrive 96 SARS-CoV-2 Kit volume (20 μL)


Test procedure

1. RNA Extraction The Genedrive 96 SARS-CoV-2 Kit is intended for use with commercially available RNA extraction kits and Universal

Human Lymphocyte RNA (AMSBIO, ATR1254148-10) and is validated for use with the RNA extraction kits listed in Table 3.

Extraction Process Control

RNA Extraction Kit Manufacturer Product

CodeSpecimen Input (µL)

Lysis Buffer Volume/

Specimen (µL)

(A) Quantity of

Extraction Control required per

extraction (ng)

(B) Quantity of Extraction

Control required per 20 µL assay

(ng)

(C) RNA Kit Elution

Volume (µL)

RNAdvance Viral RNA

purification Kit

Beckman Coulter

C63510 200 150 1.25 0.25 100

Sera-Xtracta Virus/Pathogen

KitCytiva

29514201/ 29506009

200 570 1.25 0.25 100

QIAamp Viral RNA Mini Kit

Qiagen52904 or

52906140 560 0.3 0.1 60

Virus/Pathogen Kit with QIAamp MiniElute Virus

Spin Kit

Qiagen 57704 200 200 0.4 0.1 80

Table 3 RNA extraction kits and extraction control requirements

Reagent volume produced will allow for at least 96 extractions (with 15% overage)

Reagent volume produced will allow for at least 96 extractions

1. Remove a single use aliquot (30 μL of 5 ng/μL) of the

RNA extraction control from the freezer (-80°C) and

allow to thaw on ice or a chilled cool block (2-8°C).

2. Mix by laboratory vortex followed by brief centrifugation.

3. Immediately prior to commencing RNA isolation, add

27.5 μL of the RNA extraction control (5 ng/μL) to 16.5

mL RNAdvance Viral Lysis Buffer (LBF) and mix by

laboratory vortex.

4. Use 150 μL of extraction control/LBF mix per

extraction (1.25 ng/extraction – final assay

concentration of 0.25 ng).

5. Perform the extraction according to the RNAdvance

Viral RNA Purification Kit IFU.

1. Remove a single use aliquot (20 μL of 5 ng/μL) of the

RNA extraction control from the freezer (-80°C) and

allow to thaw on ice or a chilled cool block (2-8°C).

2. Mix by laboratory vortex followed by brief

centrifugation.

3. Add carrier RNA provided to AVL buffer (if not

already added) according to the QIAamp Viral

RNA Mini Kit IFU.

4. Add 15 μL of extraction control to 1235 μL Nuclease

Free Water and mix by laboratory vortex, briefly

centrifuge (0.06 ng/μL).

5. Immediately prior to commencing RNA isolation, add

550 μL of diluted RNA extraction control (0.06 ng/μL)

to 61.6 mL AVL buffer and mix by laboratory vortex.

6. Use 560 μL of extraction control/AVL mix per

extraction (0.3 ng per extraction – final assay

concentration of 0.1 ng).

7. Perform the extraction according to the QIAamp Viral

RNA Mini Kit IFU.

RNAdvance Viral RNA Purification Kit (Beckman Coulter):

QIAamp Viral RNA Mini Kit (Qiagen):



The use of carrier RNA is crucial for extraction efficiency and stability of the extracted nucleic acid. Follow RNA extraction kit instructions for processing.CAUTION

The procedure must be conducted in accordance

with the manufacturer’s instructions for use. Ensure

that specimen swabs have been handled according

to the conditions set out in the Specimen collection,

handling and storage section of this IFU.

If using an automated extraction system, perform the

extraction using specimen input to eluate volume

ratio of 5:1 - for example, use a 200 μL specimen

input volume with a 40 μL elution volume.

The quality of the extracted RNA has a profound impact on the performance of the entire test system. It is recommended to ensure that the system used for nucleic acid extraction is compatible with Real-Time PCR technology.

If using a spin column based sample preparation procedure including washing buffers containing ethanol, it is highly recommended to perform an additional centrifugation step for 1 min at approximately 17000 x g (~ 13000 rpm), using a new collection tube, prior to the elution of the nucleic acid.

CAUTION


2.9 Being careful to contact only the end of the optical

film seal and not the adhesive surface, place the

seal on the plate evenly, and ensure a tight seal is

obtained for each well. A microtitre sealing brayer

(or similar) may be used to ensure an even seal.

Carefully place seal on the plate and then rub the

FLAT edge of the applicator back and forth along the

long edge of the plate, sealing from the middle of

the plate outwards in all directions. Next rub the flat

edge of the applicator back and forth along the short

edge (width) of the plate. Finally, rub the end of the

applicator horizontally and vertically between all wells

and around all the outside edges of the plate, using

small back and forth motions to form a complete seal

around all the outside wells.

2.10 Carefully drop from a 1cm height 3 times onto the

work surface, to promote liquid movement to the

bottom of the well.

2.11 Using a tube vortex with the flat attachment, or plate

vortex, pulse mix cool block/plate for 3 x 15 seconds

at full power.

CAUTION

It is crucial to ensure sufficient mixing at this stage

of the procedure.

2.12 Drop from 1cm height 3 times onto the work

surface, to promote liquid movement to the

bottom of the well.

2.13 Incubate on cool block for 5 minutes.

2.14 Using a tube vortex with the flat attachment, or

plate vortex, pulse mix cool block/plate for 3 x 15

seconds at full power (total 45 seconds).

(Proceed to step 2.18)

CAUTION

It is crucial to ensure sufficient mixing at this stage

of the procedure.

Alternatively, pipette based reagent mixing can be

pursued to prepare the reactions following set up steps

on the next page:

2. Reaction setup and run protocol – amplification and detection on RT-PCR instrument

To ensure high test sensitivity and specificity, keep

the 96 well plate cool during specimen addition,

mixing and incubation steps. We recommend using

a 96 well plate cool block that has been cooled

down to 2-8 °C.

• Follow good laboratory practice for RNA

handling and PCR.

• It is important to ensure the set up area is free

of RNase and sources of PCR contamination.

• Perform all steps in a clean PCR hood.

2.1 Open the kit pouch using the tear seal.

2.2 Remove the 96-well plate and the optical film seal

provided. Once removed from the pouch, the sealed

assay kit is stable for 5 days.

2.3 Place the Genedrive 96 SARS-CoV-2 Kit 96 well plate

in a 96 well cold block.

2.4 Carefully peel back the plate foil seal. It is

recommended to peel back in sections so as to

provide further protection against contamination.

Once foil seal is removed, the assay kit is stable for

4 hours.

2.5 Inspect each well for the presence of a reagent bead.

2.6 Add 20 μL of specimen (or control) to each well,

using a fresh pipette tip. Ensure you do not touch

the lyophilised reagent bead with the pipette tip. The

specimen will begin to rehydrate the reagent bead.

2.7 Once reconstituted, the assay kit is stable for 1

hour (from reconstitution to initiation of cycling

programme).

If reactions are insufficiently mixed performance of

the kit may be variable.

It is critical that no well is left non-reconstituted. Empty wells containing a reagent bead will cause substantial interference that results in incorrect readings on LightCycler and CFX96 systems, and with ABI 7500 Fast may result in overall plate failure.

CAUTION

Manual reaction set up

If setting up the reactions manually perform the

following actions:

2.8 Once plate dispensation is complete, prepare to seal

the plate with the optical film seal provided. Separate

the optical film seal from the paper backing by

separation of seal/paper at the ends.


Pipette based reagent mixing

2.15 Once plate dispensation is complete leave to

reconstitute for 5 mins.

2.16 Set multi-channel pipette step volume to 15 μL,

and mix by pipetting in and out 10 times.

2.17 Remove the plate from the automated laboratory

system and apply the optical film seal – being

careful to contact only the end of the optical film

seal and not the adhesive surface. Place the

seal on the plate evenly, and ensure a tight seal

is obtained for each well. A microtitre sealing

brayer (or similar) may be used to ensure an

even seal. Carefully place seal on the plate

and then rub the FLAT edge of the applicator

back and forth along the long edge of the plate,

sealing from the middle of the plate outwards

in all directions. Next rub the flat edge of the

applicator back and forth along the short edge

(width) of the plate. Finally, rub the end of the

applicator horizontally and vertically between

all wells and around all the outside edges of the

plate, using small back and forth motions to form

a complete seal around all the outside wells.

2.18 Centrifuge in a plate centrifuge for 2 minutes

at 2000 rpm.

Ensure that the time between reconstitution of reagent

beads and initiation of cycling programme is no longer

than a maximum of 1 hour.

2.19 If using the Roche LightCycler 480 II, ensure the plate

barcode is on the correct side. The instrument will not

load the plate if the plate is labelled incorrectly. Ensure

the barcode is on the back of the plate. If not, remove

the barcode and re-attach it.

2.20 Load the plate into the Real-Time PCR instrument and

start the PCR cycling immediately.

Following completion of run, remove plate and dispose

of immediately.


Manual set up instructions for Roche LightCycler 480 II

• Input the following cycling parameters into the

instrument according to Table 4.

StageTemperature

(°C)Time

(hh:mm:ss)Ramp rate (°C/s)

Number of cycles

Reverse Transcription 42 00:10:00 4.4 1

Amplification

Denaturation 95 00:00:20 4.4

50Annealing 58 00:00:20 2.2

Extension 72 00:00:20 4.4

Melt

curve analysis

95 00:01:00 4.4

140 00:02:00 2.2

95 Continuous 0.11

Cooling 40 00:00:30 2.2 1

Table 4 Reverse Transcription-PCR cycling and melt analysis parameters

3. Real-Time PCR instrument setup

How to Setup a Genedrive 96 SARS-CoV-2 assay on the

Roche LightCycler 480 II System

To set up instrument parameters using a template file follow

the instructions below:

1. Download the Roche LightCycler 480 II template file

from the genedrive website at:

https://genedrive.com/assays/genedrive-96-exporter.php

2. On the instrument software home screen, click on the

‘Navigator’ icon and click on ‘Import’

3. Locate the Run Template File Filepath and click ‘Open’

4. The Run Template File will now be open

5. Click on the ‘Save’ icon and save in the Template Folder

6. Click on the ‘Close’ icon

7. To Start a run, Open New Experiment from Template

8. Locate the Saved template and open

9. Use the “Sample Editor” tab to enter specimen names

10. Click start run to open the save experiment dialog box,

save the run in a suitable location and click “OK” to start

the run

* Note – The Genedrive 96 SARS-CoV-2 test does not use SYBR green chemistry. This information is only used for the Roche LightCycler 480 II instrument wavelength filter settings.

The set up information contained in the template file is

outlined below in case you need to check or troubleshoot

the set up.

Instrument settings

Reaction volume 20 μL

Detection format SYBR green*/HRM dye

Target Reporter Quencher

SARS-CoV-2 E/N gene FAM* None

Internal RNA assay control

FAM None

Human RNase P target control

FAM None

• Input the following settings: • Fluorescent dyes

*FAM - Fluorescein labelled thymine residues

Dispose of plate after cycling complete. DO NOT OPEN as this will result in local PCR contamination.


How to Setup a Genedrive 96 SARS-CoV-2 assay

on the ABI 7500 Fast Real-Time PCR System

These instructions and associated run template file are

for use with the 7500 Software only.

To set up instrument parameters using a template file

follow the instructions below:

1. Download the ABI 7500 Fast template file from the

Genedrive website at:


2. On the instrument software home screen, click on ‘New

Experiment’ drop down tab (top left of screen) and

select ‘From Template’

3. Locate the Template File Filepath and click ‘Open’

4. The ‘Experiment Menu’ will now be open

5. Input an ‘Experiment Name’ within the ‘Experiment

Properties’ tab

6. Click “Start run”. An alert message will appear saying

“Applied Biosystems recommends the SYBR Green

reagents to perform a melt curve experiment”, click

“OK” to start run

7. Save the run file in a suitable location, the reaction will

now start

8. Once the run has started select the “Plate setup” tab.

Insert specimen names into the “Define Samples” box,

then assign to wells using the “Assign Targets and

Samples” tab

Experimental Properties

Instrument Type: 7500 Fast (96 Wells)

Experiment Type: Quantitation - Standard Curve

Reagent Type: Other – Include Melt Curve

Ramp Speed: Fast

Run Method

Reaction volume: 20 μL

Expert mode: Tick, select filters 1, 2 and 3

Stage Step Temp (°C) Time (mm:ss)

Ramp Rate (%)

Holding Stage Step 1 42 10:00 100

Cycling Stage

(50 Cycles)

Step 1 95 00:20 100

Step 2 (Data Collection On) 58 00:20 100

Step 3 72 00:20 100

Melt Curve Stage

Step 1 95 01:00 100

Step 2 40 02:00 100

Step 3 (Data Collection On - Continuous) 95 00:05 3

Step 4 40 00:30 100

Target data

Reporter: JOE

Quencher: None

Passive reference dye: None

Manual set up instructions for ABI 7500 Fast Real-Time

PCR System

The set up information contained in the template file is


the set up

Table 5 Reverse Transcription-PCR cycling and melt analysis parameters


How to Setup a Genedrive 96 SARS-CoV-2 assay on the

Bio-Rad CFX96 Real-Time PCR Detection System

To set up instrument parameters using a template file

follow the instructions below:

1. Download the Bio-Rad CFX96 template zip file

containing the protocol template and the plate template

from the Genedrive website at: https://genedrive.com/

assays/genedrive-96-exporter.php

2. Open the Bio-Rad CFX Maestro software.

3. On the Startup Wizard pop-up, select ‘CFX96’ as the

instrument then select ‘User-defined’ run type.

4. On the Protocol tab of the Run Setup window, click

‘Select Existing’ and locate the Protocol Template

Filepath and click ‘Open’. Click ‘Next’ to move onto

the Plate tab.

5. On the Plate tab, click ‘Select Existing’ and locate the

Plate Template Filepath and click ‘Open’. Click ‘Next’ to

move on to the ‘Start Run’ tab.

6. If running multiple units, ensure the correct block is

selected.

7. Click ‘Start Run’ and save the run when prompted.

8. Sample names and biological groups for each well

may be input whilst the reaction is running. Click the

Real-Time Status tab then select “Plate Setup” then

“View/Edit Plate”. Use the plate editor window to detail

biological groups (i.e. wells assigned to controls and

clinical specimens), and also to enter specimen names

for individual wells.

Note: two templates are required to set up the test, a

protocol template and a plate template. Ensure that

you have used both templates.

Stage Step Temp (°C) Time (hh:mm:ss)

Holding Stage Step 1 42 00:10:00

Cycling Stage

Step 2 95 00:00:20

Step 3 (Plate read) 58 00:00:20

Step 4 72 00:00:20

Step 5 - Go to Step 2 44 times n/a n/a

Melt Curve Stage

Step 6 95 00:01:00

Step 7 40 00:02:00

Step 8 (0.5°C Increment, Plate read) 40-85 00:00:05

Step 9 40 00:00:30

Within ‘Plate Editor’ select the following parameters:

Fluorophore – FAM

Scan Mode – SYBR/FAM only

Plate Type – BR Clear

Manual set up instructions for Bio-Rad CFX96

• Within User-Defined run type and Protocol Editor,

input the following cycling parameters, accoring to

Table 5 using the ‘Protocol’ tab.

The set-up information contained in the template file is


the set up

Table 6 Cycling and melt analysis parameters

Sample volume - 20 μL


4. Result interpretation

Graph showing 6 Tm windows

4.1 Data analysis instructions for use with the Roche LightCycler 480 II• Select “Tm Calling”

• Set Max peaks to “6 or less”, followed by

“Calculate”

• Check the “Manual Tm Method” box, followed by

clicking the corner of the plate map to activate.

• Check all 6 Tm boxes in “Manual Tm”

• Enter the following:

• TM 1 56.1°C

• TM 2 61.5°C

• TM 3 65.0°C

• TM 4 72.7°C

• TM 5 73.5°C

• TM 6 80.1°C

RNase P SARS-CoV-2 IPC

56.1°C – 61.5°C 65.0°C – 72.7°C 73.5°C – 80.1°C

Human RNA control (RNase P) peak

SARS-CoV-2 peak Internal Process Control (IPC) peak

TM1 TM2 TM3 TM4 TM5 TM6

40

40.472

35.972

31.472

26.972

22.472

17.972

13.472

-0.028

4.472

-4.528

8.972

42 44 46 48 50 52 54 56 58 60 62 64 66

Temperature (˚C)

(d/d

T)

Flu

ore

sce

nce

(4

65

-510

)

68 70 72 74 76 78 80 82 84 86 88 90 92 94

Inspect First Order Derivative melt data visually

for presence of peaks in the respective target

Tm ranges. Control wells should be interpreted

first to validate the performance of the run. If

controls have passed, move on to analysing

specimen wells.

SARS-COV-2 Positive melt curve graph

Target Tm ranges


56.1°C – 61.5°C 65.0°C – 72.7°C 73.5°C – 80.1°C

40

33.614

30.114

26.614

23.114

19.614

16.114

12.614

9.114

-1.836

2.114

-4.886

5.614

42 44 46 48 50 52 54 56 58 60 62 64 66

Temperature (˚C)

(d/d

T)

Flu

ore

sce

nce

(4

65

-510

)

68 70 72 74 76 78 80 82 84 86 88 90 92 94



4.2 Manual data analysis instructions for use with the ABI 7500 Fast Real-Time PCR System• Select “Analyse Experiment” if opening a saved

file and/or proceed to select “Melt Curve” in the

Analysis module.

• Select “Derivative Reporter” in the drop down

menu plot settings.

• FODs for each well are displayed.

• Enter the following Tm ranges.

Target Tm ranges

Graph to show SARS-CoV-2 positive melt curve on ABI 7500 Fast Real-Time PCR System

SARS-CoV-2RNAse P IPC


48.8 - 54.1°C 54.8 - 64.3°C 65.8 - 75.0°C

4.3 Manual data analysis instructions for use with the Bio-Rad CFX96 Real-Time PCR Detection System• Once the run has finished the ‘Data Analysis’

window will open.

• Enter the following Tm ranges.


46.0°C - 56.0°C 59.0 - 66.0°C 67.0 to 75.0°C

Target Tm ranges

Graph to show example of SARS-Cov-2 melt curve on Bio-Rad CFX96 Real-Time PCR Detection System

RNase P IPCSARS-CoV-2


Follow instructions in Troubleshooting section for interpretation of failed results.

Table 7 Interpretation matrix

RNase PSARS-

CoV-2 E/NIPC Status Result Action

NEG NEG NEG INVALID TEST FAIL

Ensure RNA extraction

procedure is set up correctly.

Ensure the test procedure has

been followed correctly. Repeat

test using 50% dilution of

extracted specimen. If repeated

result remains invalid, consider

collecting a new specimen

NEG NEG POS INVALID

TEST FAIL

(No extraction

control detected)

Ensure RNA extraction

procedure is set up correctly.

Repeat extraction step

and re-test

POS or NEG POS POS or NEG VALIDSARS-CoV-2

POSITIVENone

POS NEG POS VALIDSARS-CoV-2

NEGATIVENone

POS NEG NEG VALIDSARS-CoV-2

NEGATIVENone

The absence and/or presence of the RNase P, SARS-CoV-2 and IPC peaks should be noted for each specimen and

the interpretation matrix used to perform result interpretation, see Table 7.


*Genedrive 96 Exporter software has been validated for use on a 64 bit computer running Windows 10.

Roche LightCycler 480 II1. Within the LightCycler 480 software:

2. Use the ‘Melt Curve Genotyping’ analysis settings,

highlight the wells to be analysed and click

‘calculate’

3. Right click on the ‘Melting curve’ graph (NOT the

melt peak first order derivative (FOD) data) and

click ‘export chart’

4. Change the tab to ‘data’ and ensure text file

is selected

5. Name the file and save in a suitable location

ABI 7500 FAST1. Within the 7500 Fast software:

2. Ensure all wells on the plate are selected in the

software

3. Select “Export”

4. Ensure “Results” and “Multicomponent data” check

boxes are selected

5. Choose file name and a suitable location – use .xls

format for export

6. Select “Start Export”

4.4 Genedrive 96 Exporter – result interpretation

Bio-Rad CFX961. Within the CFX Maestro software:

2. Once the run has finished the ‘Data Analysis’

window will open

3. On the toolbar click ‘Export’ then ‘Export all data

sheets’ as ‘CSV’. Choose a file name and save in a

suitable location

4. The “Filename - Melt Curve RFU Results_FAM”

can then be imported into the Genedrive 96

Exporter software

Genedrive 96 Exporter steps1. Open the software

2. Click ‘Input file’ and select export file type in the box

on the bottom right of the input screen. Navigate to

where the text file was saved and import the file

3. The analysis will complete automatically

Inspect run controls

1. Inspect run controls to ensure the validity of the run

before reporting the specimen results

2. When using an extraction control, ensure the

“Options” “Extraction control used” box has been

checked

3. If the run control has been directly added to the

plate without being taken through RNA extraction

– i.e. without the use of the extraction control –

ensure the “Options” “Extraction control used” box

has been not been checked

Result interpretation may be automated using the

optional Genedrive 96 Exporter software*

1. Go to the website address below and download the

Genedrive 96 Exporter software installer


2. Unzip the file

3. Double-click the ‘Genedrive 96 Exporter’ Icon to

install the software

4. The Genedrive 96 Exporter will be installed in

C:\Genedrive96Exporter

5. Double-click the ‘Genedrive 96 Exporter’ icon to open

the software


Borderline specimen result interpretationA “Check Result” flag indicates that whilst Genedrive 96

Exporter classified the specimen as SARS-CoV-2-Positive

or Negative, the result is borderline. The graph for the

specimen should be visually inspected to determine the

final result

1. To perform a manual check - whilst holding down the

‘ctrl’ key on the keyboard - select a well containing

the positive run control, a well containing a negative

run control and the well with either the ‘Check result’

or ‘Test failed’ result

2. Selecting the positive and negative control wells

will allow any potential peaks to be compared to the

baseline (negative run control well) and a known

positive reaction (positive run control well)

NOTE: If a well contains a reagent bead that has not

been reconstituted it will result in interference and

produce an erroneous result. Ensure each plate is

inspected for any such wells and the results from

these wells are discounted.

3. Individual or multiple wells can be selected to display

the raw melt peak data (Tm, peak height, peak width)

and a graphical visualisation of FOD melt data

Colour Symbol ResultRed + SARS-CoV-2 POSITIVE

Hashed Red + CHECK RESULT

Dark Green - SARS-CoV-2 NEGATIVE

Hashed Green - CHECK RESULT

Blue X TEST FAIL

Result reporting

1. To interpret results from specimens ensure the

“Options” “Extraction control used” box has been

checked

2. Final sample result calls will be displayed

automatically on the image of the plate. Each well will

be colour coded and contain a symbol based upon

the result. Coding is as follows:

Export results

Results may be exported as .csv or .xls format

1. Select wells required for export

2. Data may be exported for each well individually, or by

row, column or entire plate

3. Select the wells of interest, then Select ‘Export

Results’ and save to desired location as either a .csv

or .xls format


Image of Genedrive 96 Exporter software screen


Quality controlsEach test in the Genedrive 96 SARS-CoV-2 Kit contains

primers and probes for an extraction control (RNaseP).

The presence of the associated peak indicates a RNA

extraction was performed successfully and gives

increased confidence that negative results were not due

to poor RNA extraction.

Each test also contains primers, probes and target RNA

for an internal PCR control (IPC). This control amplifies

even within negative controls and the presence of the

peak indicates that PCR has functioned as expected and

has not been impacted by inhibition.

It is recommended that a positive and negative run control

are used when running the Genedrive 96 SARS-CoV-2 Kit.

Nuclease Free Water may be taken through the RNA

extraction method alongside clinical specimens to

function as a negative run control that can help monitor

the full process, including RNA extraction.

The Genedrive 96 SARS-CoV-2 Kit has been validated

using the following controls:

* Do not use for LoD verification studies

** If taken through RNA extraction process when using the RNA extraction control.

Positive run control materials

Product name ManufacturerProduct

Code

RNA extraction required?

Instruction Expected result

SARS-CoV-2

Standard

Exact

DiagnosticsCOV019 No

Dilute to 30 copies/

reaction or 1500

copies/mL. Follow test

procedure steps

RNase P peak

SARS-CoV-2 peak

IPC Peak

2019-nCoV_N

Positive Control

Integrated DNA

Technologies

(IDT)

10006625 No

Dilute to 1:2000 and

add 20 μl/well. Follow

test procedure steps*

SARS-CoV-2 peak

IPC peak

AccuPlex™

SARS-CoV-2

Reference

Material Kit

(positive control)

Seracare 0505-0126 Yes

Perform RNA

extraction. Follow test

procedure steps

SARS-CoV-2 Peak

IPC peak

AccuPlex™

SARS-CoV-2

verification panel

Seracare 0505-0168 Yes

Perform RNA

extraction. Follow test

procedure steps

SARS-CoV-2 Peak

IPC peak

Table 8 Positive control material

For the running of instrumentation control tests please refer to the respective instrument manufacturer user manuals.

Negative run control materials

Product name Manufacturer Product codeRNA

extraction required?

InstructionExpected

result

Nuclease Free

WaterVarious Various No

Follow test

procedure steps

IPC peak

RNase P peak**

Table 9 Negative control material


Troubleshooting

Sample type

Issue/Error Probable Cause Solution

Positive

control

SARS-CoV-2 peak

absent

Positive control

material not

added

Repeat test with new kit

PCR inhibitors

present

Clean area and repeat test with new kit. Ensure

appropriate PPE worn to reduce contamination risk

Human RNase P peak

present*

Contamination

with Human RNA


appropriate PPE worn to reduce contamination risk

No peaks present PCR fail


appropriate PPE worn to reduce contamination risk.

Ensure instrument parameters are correct

Negative

control

SARS-CoV-2 peak or

Human RNase P peak

present

Local

contamination



Negative control material may be contaminated.

Consider using a fresh tube

Internal RNA control

peak absentPCR fail



Ensure instrument parameters are correct

Test

sample

Human RNase P

peak absent

Inefficient / failed

RNA extraction

or no extraction

control added

Repeat extraction step and re-test

No peaks present PCR fail



Ensure instrument parameters are correct. Check

specimen was added to well. Ensure automated RNA

extraction parameters are correct. - e.g. Ensure input

and eluate volumes are correct (See Table 2). Specimen

input volume: eluate volume ratio is 5:1

*In cases where an RNase P peak is not expected - i.e. when an

extraction control has not been used or the control used does not

contain RNase P RNA.

Table 10 Troubleshooting


Analytical sensitivityThe analytical sensitivity, or Limit of detection (LoD), is

defined as the lowest concentration which >95% of the

tested samples generate a positive result.

LoD of the Genedrive 96 SARS-CoV-2 kit was determined

by spiking of either Seracare Accuplex CoV-2 reference

material or intact heat inactivated SARS-CoV-2 virus into

CoV-2 negative oropharyngeal and nasopharyngeal

samples separately collected in viral transport media.

Isolated RNA was utilized for probit analysis at 24

replicates per dilution level, with the the LoD of the

Genedrive 96 SARS-CoV-2 assay being established as

10 copies/reaction (CI 8.2 – 12.77) or 0.5 copies/μl, and

verified across all platforms listed.

Performance characteristicsAll performance characteristics data were determined using manual result interpretation.

RNA Extraction

Kit

Real-Time PCR

Platform

SARS CoV-2 Standard

Target Titration

pre- or post-extraction

Copies / ml

Sample

Copies / Reaction

Detected Positive

LoD (copies per

reaction (95% CI))

LoD (copies/µL (95% CI))

Qiagen QIAamp

Viral RNA Mini Kit

Roche LightCycler

480 II

*Accuplex (V2)

Post-extraction

NA

20 10 5

2.5 1.25

48/48 45/48 35/48 23/48 14/48

9.82 (8.19 - 12.77)

0.49 (0.41-0.64)

Beckman Coulter

RNAdvance Viral RNA

purification Kit

ABI 7500 Fast

*Accuplex (V2)

Post-extraction

NA

20 10 5

2.5 1.25

24/24 24/24 23/24 20/24 15/24

4.53 (3.33 - 10.59)

0.23 (0.17-0.53)

ABI 7500 Fast

**Intact Heat

Inactivated SARS CoV-2

Pre-extraction

500 250 125 62.5 31.25

20 10 5

2.5 1.25

24/24 21/24 24/24 9/24 6/24

9.55 (7.58 - 13.97)

0.48 (0.38-0.70)

Roche LightCycler

480 II

**Intact Heat


Pre-extraction

500 250 125 62.5 31.25

20 10 5

2.5 1.25

24/24 21/24 22/24 16/24 7/24

10.25 (7.76 - 17.15)

0.51 (0.39-0.86)

Bio-Rad CFX96

**Intact Heat


Pre-extraction

500 250 125 62.5 31.25

20 10 5

2.5 1.25

24/24 24/24 22/24 14/24 10/24

5.7 (4.44 - 9.42)

0.29 (0.22-0.47)

Cytiva Sera-Xtracta

Virus/Pathogen

Kit

Bio-Rad CFX96

*Accuplex (V2)

Post-extraction

NA

20 10 5

2.5 1.25

24/24 22/24 16/24 9/24 4/24

10.27 (8.28 - 14.49)

0.51 (0.41-0.72)

*AccuPlex™ SARS-CoV-2 Verification Panel; V2, partial genome (SeraCare, 0505-0129)

**Heat inactivated SARS CoV-2 virus (ATCC® VR-1986HK™, strain 2019-oV/USA-WA1/2020)

Table 11 LoD across Real-Time PCR platforms


The Genedrive 96 SARS-CoV-2 Kit is exclusive for non-

CoV-2 human pathogens listed in the table below, assessed

firstly by in silico analysis (<80% match of primers and probe,

Analytical specificity

Table 12 Exclusivity

with none resulting in viable amplification products detected

in the detection windows), and also by experimental

assessment at the copies per reaction detailed.

Pathogen In silico

Experimental (Copies per

reaction)

Human coronavirus 229E Yes 4.0 X 105

Human coronavirus OC43 Yes 3.4 X 105

Human coronavirus HKU1 Yes Not tested

Human coronavirus NL63 Yes 1.0 X 105

SARS-Coronavirus Yes 2.8 X 105

MERS coronavirus Yes 3.2 X 105

Parainfluenza virus 1 Yes 3.0 X 105




Rhinovirus Yes 1.0 X 105

Influenza A (H1N1) Yes 2.8 X 105

Influenza A /(H3N2) Yes 3.6 X 105

Influenza B Yes 2.5 X 105

Respiratory syncytial virus

(subtype A)Yes 3.6 X 105

Respiratory syncytial virus

(subtype B)Yes 3.0 X 105

Adenovirus Yes 3.8 X 105

Adenovirus (Ad.41) Yes 2.4 X 105

Haemophilus ducreyi Not tested 3.6 X 105

Haemophilus influenzae Yes 4.0 X 105

Human Metapneumovirus (hMPV) Yes 1.0 X 105

Streptococcus pneumoniae Yes 3.4 X 105

Mycoplasma pneumoniae Yes 3.2 X 105

Streptococcus pyogenes Yes 1.0 X 105

Bordetella pertussis Yes 3.8 X 105

Bordetella holmesii Yes 3.2 X 105

Bortedella Parapertussis Yes 3.4 X 105

Pathogen In silico

Experimental (Copies per

reaction)

Chlamydia trachomatis Yes 3.4 X 105

Candida albicans Yes 2.5 X 105

Candida auris Yes 2.6 X 105

Legionella pneumophila Yes 2.8 X 105

Staphylococcus salivarius Yes 1.0 X 106

Staphylococcus epidermis Yes 1.0 X 106

Pseudomonas aeruginosa Yes 1.0 X 106

Mycobacterium tuberculosis Yes 3.4 X 105

Chlamydia psittaci Yes Not tested

Chlamydia pneumonia Yes Not tested

Influenza C Yes Not tested

Parechovirus Yes Not tested

Corynebacterium diptheriae Yes Not tested

Legionella feeleei Yes Not tested

Legionella micdadei Yes Not tested

Legionella longbeachae Yes Not tested

Bacillus anthracosis (Anthrax) Yes Not tested

Moraxella cararrhalis Yes Not tested

Neisseria elongate and

miningitidisYes Not tested

Leptospirosis Yes Not tested

Coxiella burneti (Q-Fever) Yes Not tested

Staphylococcus aureus Yes Not tested

Enterovirus (type 71) Yes Not tested

Pneumocystis jirovecii (PJP) Yes Not tested

Bacterioides oralis Yes Not tested

Chlamydophila pneumoniae Yes Not tested

Nasal wash N/ANo assay cross-

reactivity (N/A)


InclusivityIn silico analysis concluded that the Genedrive 96 SARS

CoV-2 assay will detect all analyzed SARS-CoV-2 sequences

in the GISAID database by using a dual target design.

As of 22nd October 2020, 155,276 CoV-2 sequences

were available in the GISAID database for detailed

analysis of Genedrive 96 SARS CoV-2 Assay E & N gene

primer and probe homology. Of these, 137,246 genomes

(88.4%) covered the assay E & N gene target regions fully

to permit sequence alignment analysis of both target

assays together, and demonstrated 100% homology to

all Genedrive 96 SARS CoV-2 assay target primers and

probes. Summary of individual N and E gene assay target

alignments are shown in Table 14.

For the E gene, greater than 99% of sequences had 100%

homology to amplification primers and detection probe.

The majority of those with less than 100% homology contain

non-significant single mismatches distal to 3’ ends, being

PrecisionIntra-assay (within run), Inter-assay (between run),

Inter-instrument and Inter-lot (between reagent lot)

variability end-point melt analysis signal intensity for

SARS-CoV-2 RNase P

%CV SD %CV SD

Intra assay 11.68 0.21 14.64 0.14

Inter assay 5.11 0.09 9.10 0.09

Inter lot 3.00 0.05 6.61 0.06

Inter instrument 11.98 0.11 9.82 0.10

N gene E gene

Amplification primers

Detection Probe

Amplification primers

Detection Probe

Number of sequences 153,587 (100%) 154,234 (100%)

Number of sequences with 100% homology 141,219 (91.94%) 152,868 (99.53%) 152,873 (99.12%) 154,078 (99.9%)

Number of sequences with <100% homology 12,368 (8.05%) 719 (0.47%) 1,361 (0.88%) 156 (0.1%)

Number of sequences with predicted

potential to impact detection

386 (0.25%) 2 (0.001%) 137 (0.09%) 0 (0%)

Number detected by alternative dual target 385 / 386 719 / 719 136 / 137 n/a

Number of sequences remaining potentially

not detected

1 (0.0007%)* 0 (0%) 1 (0.0006%)* n/a

Table 13 Precision data

Table 14 Summary of Individual N and E gene target alignments

predicted not to affect amplification or detection. The

remaining 137 sequences with 2 or more mismatches

(0.09%) were 100% homologous to the alternative N

gene assay (with the exception of 1 sequence*; Egypt/

MASRI-025/2020; 3 consecutive mismatches in the E

gene forward primer at positions -6 to -8 relative to 3’).

Conversely, for the N gene, greater than 92% of

sequences are 100% homologous for amplification

primers (>99% for detection probe). The majority of

the remaining 12,368 sequences (11,982 sequences)

contain non-significant single mismatches distal to

3’ ends, being predicted not to affect amplification.

Those 386 sequences with 2 or more mismatches

(0.25%) were 100% homologous to the alternative

E-gene assay (with the exception of 1 sequence*; Egypt/

MASRI-025/2020; single mismatch in the N gene

reverse primer at position -3 relative to 3’ (estimated to

still result in 80% efficiency).

Genedrive 96 SARS-CoV-2 Kit probes when expressed as

a ratio of the internal PCR control signal intensity. All results

displayed to 2 decimal points.


Diagnostic accuracyContrived Upper Respiratory Tract Specimens

A clinical performance study was performed to evaluate

the performance of the Genedrive 96 SARS-CoV-2 Kit

using nasopharyngeal (NP) and oropharyngeal (OP) swabs.

Sixty contrived upper respiratory tract specimens were

tested:

• Thirty contrived positive NP/OP specimens

• Of which twenty were 2x the Limit of Detection (429

copies/mL, projected assay concentration of 20

copies/reaction)

• Of which ten were 5x the Limit of Detection (1071

copies/mL, projected assay concentration of 50

copies/reaction)

• Thirty contrived negative NP/OP specimens

Specimens were contrived by spiking known

concentrations of viral protein-coated RNA (Seracare

Accuplex Verification Panel), relative to the product

LoD, into pooled NP/OP specimen matrix which was

determined to be negative by the collection site prior

to spiking in the RNA. Specimens were randomized

and blinded. RNA Extraction was performed using the

Qiagen QIAmp Viral RNA mini Kit. RT-PCR analysis was

performed using the Roche LightCycler 480 II across

three reagent lots.

100.00% (30/30, 95% CI 88.65 to 100.00) of positive

specimens returned a positive result, and 100.00% (30/30,

95% CI 88.65 to 100.00) of negative specimens returned a

negative result, see Table 15.

Table 15 Clinical performance of the Genedrive 96 SARS-CoV-2 Kit on contrived upper respiratory

tract specimens performed using the Roche LightCycler 480 II Real-Time PCR Instrument.

Expected result

Positive Negative Total

Genedrive 96

SARS-CoV-2 Kit

Positive 30 0 30

Negative 0 30 30

Total 30 30 60

Positive Specimens Detected 100.00% 95% CI 88.65% to 100.00%

Negative Specimens Not Detected 100.00% 95% CI 88.65% to 100.00%


EUA SARS-CoV-2 Assay

Positive Negative Total

Genedrive 96

SARS-CoV-2 Kit

Positive 100 0 100

Negative 0 50 50

Total 100 50 150

Positive Percent Agreement 100.00% 95% CI 96.30% to 100.00%

Negative Percent Agreement 100.00% 95% CI 92.87% to 100.00%

Table 16: Clinical performance of the Genedrive 96 SARS-CoV-2 Kit on upper respiratory tract

specimens performed using the Roche LightCycler 480 II Real-Time PCR Instrument.

Upper Respiratory Specimens – EUA SARS-CoV-2 Assay

comparator

A clinical performance study was performed to evaluate the

performance of the Genedrive 96 SARS-CoV-2 Kit using

nasopharyngeal (NP) and oropharyngeal (OP) swabs.

150 natural NP/OP specimens were tested using the

Genedrive 96 SARS-CoV-2 Kit on the Roche LightCycler

480 II Real Time PCR Instrument. Specimens were

collected from one site, and each specimen was tested

using an EUA SARS-CoV-2 Assay.

Extraction of specimens was performed using three

different RNA extraction methods: Maxwell® RSC Buccal

Swab DNA Kit (N=120); Roche cobas extraction (N=21); and

Qiagen Virus/Pathogen Kit with QIAamp MinElute Virus

Spin Kit (N=9).

The positive percent agreement was 100.00% (95%

CI 96.30% to 100.00%), whilst the negative percent

agreement was 100.00% (95% CI 92.87% to 100.00%),

see Table 16.


Quick reference guide – manual set up

Step 1Place 96 well plate in a cool block

for the duration of reaction setup.

Step 2Carefully peel back the foil seal. It is

recommended to do this in sections

to minimise open wells during

pipetting. Check there is a reagent

bead in each well.

Step 5Carefully drop cool block 3 times from

1 cm height. Vortex the cool block/

plate for 3 x 15 seconds at full power

(total 45 seconds).

Step 8Centrifuge the plate for 2 minutes

at 2000 RPM.

Step 4Carefully peel optical film seal from its

backing and seal the plate. Use of a

microtitre sealing brayer or similar is

recommended.

Step 7Vortex the cool block/plate for

3 x 15 seconds at full power

(total 45 seconds).

Step 3Pipette 20 μL of extracted specimen

or control into the side of each well.

Add 20 μL of Nuclease Free Water

to any wells that do not contain a

specimen. Ensure all beads have

been rehydrated.

Step 6Carefully drop cool block 3 times from

1 cm height. Incubate on cool block

for 5 minutes.

Step 9Load the plate on the Real-Time PCR

instrument and start the program

immediately. Ensure all the run

parameters are correct before starting.


Notes


Notes


Genedrive® is a registered trademark of Genedrive Diagnostics Ltd.

Copyright © 2021 genedrive plc.

All rights reserved

Use By Single useConsult Instructions

for Use

Temperature limitation Contains sufficient for <n> tests

Product code

ManufacturerIn Vitro Diagnostic

Medical DeviceIVD Batch codeLOT

REF

Date issued: February 2021

IVD

Manufacturer Details

Genedrive Diagnostics Ltd. 48 Grafton Street,

Manchester M13 9XX United Kingdom

Tel +44 (0)161 989 0245

For general and technical enquires: [email protected]

For complaints: [email protected]

For distributor details please visit the website at:

www.genedrive.com

Prescription

Use only

Documents

Genedrive 96 SARS-CoV-2 Kit - Beckman