EurobioPlex

SARS-CoV-2 Multiplex

REAL-TIME RT-PCR
For qualitative real-time RT-PCR

EBX-041
EBX-041-192

96/192 reactions

Version 4.00 of 2020/04/20

Validated on:

ƒ CFX96TM Real Time PCR detection system (Bio-Rad) with analysis on CFX
Manager version 3.1 (Bio-Rad)
ƒ LightCycler®480 Instrument II (Roche) with analysis on LightCycler® 480
software v1.5 (Roche)
ƒ Applied Biosystems® 7500 Real-Time PCR Systems (Applied Biosystems) with
analysis on 7500 Software v2
ƒ QuantStudio 5® (Applied Biosystems) with analysis on QuantStudio TM Design
& Analysis Software v1.4.0

Storage conditions:

Keep all reagents between -15°C and -22°C until use and after first use

Instructions for use

1
 TABLE OF CONTENTS

Introduction and intended Use …………………………………………………………………. ϯ

Principle of detection …………………………………………………………………….…………. ϰ

Description and content of the kit …………………………………………………………….. ϰ

Storage ………………………………………………………………………………………………..…... ϱ

Cautions and notes ……………………………………………………………………………….…. ϱ

Samples collection, transport and storage ……………………………………….…...…. ϲ

Procedure ………………………………………………………………………………..........………. ϳ

I-RNA extraction …………………………………………………………………..………….. ϳ
II-Real-time RT-PCR procedure……………………………….…………………..……. ϳ
II-1/ Diagram of the procedure ……………………………………………..…. ϴ
II-2/ Detailed procedure..………………………………………………..…………ϵ

Validation of the experiment …………………………………………………………………... ϭϬ

Data analysis and Interpretation ……………………………………………………………… ϭ1

Performance analysis………………………………………………………….…………………….. ϭ3

Bibliography …………………………………………………………………………….…………..….. ϭϲ

Waste disposal …………………………………………………………..…………….………………. ϭϲ

Symbols ……………………………………………………………..……………………………..….…. ϭϳ

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 INTRODUCTION AND INTENDED USE

SARS-CoV-2/Covid-19 virus appeared in China end of 2019 in the city of Wuhan. It

belongs to the Coronaviridae family and the sub-genera Sarbecovirus.
Its genome consists of 29903 ribonucleic acid bases (RNA). SARS-CoV-2 is the 7th
coronavirus infecting humans to be identified, since human coronavirus (HCoV) 229E,
NL63, OC43, HKU1, SARS-CoV (coronavirus responsible for severe acute respiartory
syndrome) and MERS-CoV (coronavirus inducing Middle-East respiratory syndrome).
Eighteen genomes have been isolated and reported, amongst which BetaCoV / Wuhan
/ IVDC-HB-01/2019, BetaCoV / Wuhan / IVDC-HB-04/2020, BetaCoV / Wuhan / IVDC-
HB-05/2019, BetaCoV / Wuhan / WIV04 / 2019 et BetaCoV / Wuhan / IPBCAMS-WH-
01/2019. These sequences of SARS-CoV-2 have similarities with the ones of
betacoronaviruses found in bats. SARS-CoV-2 is genetically distinct from other human
coronaviruses such as the ones related to SARS and to MERS.

Symptoms can come as a cold, fever, cough, difficulty breezing, pneumonia, to severe
respiratory syndrome, which can be fatal. The level of mortality is not precise at the
beginning of the epidemic, around 2 % end of January. This is much less compared to
10 % for SARS-CoV or 30% for MERS-CoV. SARS-CoV-2 is highly contagious with
more than 90 000 cases worldwide beginning of March 2020.
EurobioPlex SARS-CoV-2 is a test based on real-time reverse-transcription and
amplification designed for qualitative determination of absence or presence of SARS-
CoV-2 in a RNA extract. This test is indicated to diagnose the occurrence of this
infection in humans, or complement a proven or indeterminate diagnosis.
EurobioPlex EBX-041 was designed to detect all known SARS-CoV-2 published
sequences by alignment in silico with sequences of other coronaviruses.
A decision algorithm based on the detection of 3 targets (RdRp Gene target 1 and
RdRp target 2, identical to the ones recommended by the World Health Organization
(https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-
guidance/laboratory-guidance: published under Real-time RT-PCR assays for the
detection of SARS-CoV-2 Institut Pasteur, Paris (2 March 2020)), and N Gene) can be
used to determine SARS-CoV-2 patient status (see Data analysis and interpretation
and limitations). The diagnostic must always be made by a physician and using the
clinical context, history and symptoms of the patient.
The kit allows testing of 94 patients (96 tests) or 190 patients (192 tests) in addition to
the positive and negative controls.
Extracted RNA is the starting material for the EurobioPlex SARS-CoV-2 kit.
It is the user’s responsibility to choose extraction methods relevant to the type of
samples tested.

The EurobioPlex EBX-041 has been validated on the following specimen:

¾ Nasopharyngeal aspirate
¾ Bronchoalveolar fluid
¾ Sputum
¾ Nasal swab

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 PRINCIPLE OF DETECTION

The EBX-041 is a test using reverse-transcription and real-time amplification of viral RNA
of SARS-CoV-2 based on amplification of 3 genes (RdRp Gene Target 1, RdRp Gene
Target 2 and N Gene target 3). If at least one RdRp target is positive, a SARS-CoV-2
positive diagnostic can be called (see Data analysis and Interpretation).

The kit contains one oligomix to detect the 3 targets, as well as an encapsidated control
of RNA extraction and RT-PCR inhibition. The test is performed from extracted RNA
from a sample, using one RT-PCR reaction in a single distinct well/tube.

The oligomix allows amplification of 3 specific targets (RdRp Gene Target 1, RdRp
Gene Target 2 and N Gene target 3). This ensures sensitivity, and specificity with
regard to other coronaviruses known, such as HCoV, SARS-CoV, betacoronavirus
associated to bats (BtCoV) and MERS-CoV.
The RNA extraction and RT-PCR inhibition control allows to check for variations that may
occur during the RNA extraction step of biological samples and real-time PCR
amplification. Thus, it ensures that a negative result may be due to a bad RNA extraction,
and/or due to the presence of PCR inhibitors in too large quantities.

RNA of SARS-CoV-2 is detected with specific probes of each target, labeled

respectively with FAM (target 1/RdRp gene), HEX (tardet 2/RdRp gene) and Texas red
(target 3/N gene). CI-ARN is detected using a CY5 labeled probe. Probes emit a
specific fluorescence following their hydrolysis during the elongation of the
amplification product. The measurement of the intensity of real-time fluorescence
correlates with the accumulation of amplification products.

DESCRIPTION AND CONTENT OF THE KIT

The RT-PCR real-time EurobioPlex SARS-CoV-2 kit is ready to use and contains all
reagents and enzymes for the detection of this virus (Table 1).

Fluorescence is emitted and individually recorded through optical measurements

during the PCR. The detection of the amplified fragment is performed by a fluorimeter
using the channels shown in the Table 2.

Table 1:
Cap color Content of the kit 96 reactions 192 reactions Reconstitution
Red Enzyme Taq polymerase 1500 —l 2x1500 —l Ready to use
Brown Enzyme Reverse transcriptase 24 —l 48 —l Ready to use
Transparent Oligomix 600 —l 1200 —l Ready to use

Yellow Positive Control CP 160 —l 320 —l Ready to use

Blue Water = negative control (CN-H2O) 1mL 1mL Ready to use

White RNA Control (CI-ARN) 1200 —l 2x1200 —l Ready to use
Oligomix: contains the primers and probes for the 3 targets as well as for the internal control CI-ARN

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Table 2:
Target Fluorophor Excitation Emission
Target 1/ RdRp Gene FAM 495 nm 515 nm
Target 2/ RdRp Gene HEX 535 nm 555 nm
Target 3/ N Gene Texas red 585 nm 605 nm
Control RNA (CI-ARN) Cy5 647 nm 667 nm

Equivalent channels on different real time PCR cyclers:

- Channel FAM (Systems ABI, SmartCycler II, Systems Mx, CFX96TM/Chromo4, T-COR8TM-
IVD), Channel 510 (LC 480), Channel Green (RotorGene)
- Channel HEX (Chromo 4/CFX96, Systèmes Mx, T-COR 8®-IVD), Canal VIC (Systèmes ABI),
Canal Alexa532 (SmartCycler II), Canal 580 (LC 480), Canal Yellow (RotorGene),
- Channel Texas Red (ABI 7500, Chromo 4/CFX96, Systèmes Mx, T-COR 8®-IVD), LC Red
610 (LC480), Channel Orange (RotorGene), Channel Rox (QuantStudio 5)
- Channel Cy5 (CFX96TM/Chromo4, Systems ABI, Systems Mx, T-COR8TM-IVD), Channel
Alexa647 (SmartCycler II), Channel 660 (LC 480), Channel Red (RotorGene)
Note: On LC480 instrument II, apply color compensation for the following channels: FAM-
HEX/VIC-TexasRed-Cy5 (465-510, 533-580, 533-610, 618-660).

Required material not provided:

¡ Biological Hood
¡ Real-time PCR instrument
¡ Micro centrifuge
¡ Vortex
¡ Plates / tubes for real-time PCR
¡ Micropipettes
¡ DNase-free RNase-free filter tips for micropipettes
¡ Sterile microtubes
¡ Gloves (powder free)

STORAGE

All reagents must be stored between -15 and -22°C.

All reagents can be used until the expiration date indicated on the label of the kit.
Many freezing/defrosting cycles (> 3x) must be avoided, and could lead to decrease in
sensitivity.

CAUTIONS AND NOTES

Read carefully instructions before starting.

¡ The experiment must be performed by competent staff, trained to technical and

safety techniques.
¡ The biosafety local regulations for SARS-CoV-2 testing must be followed
accurately under all circumstances, using appropriate equipment and
laboratories in this regard.

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 ¡ Instruments must have been properly installed, calibrated and maintained
according to the manufacturer's recommendations.
¡ It is the user’s responsibility if he/she uses other non-validated equipment, and
if so, the performances are not guaranteed.
¡ Clinical samples are potentially infectious and must be processed under a
laminar flow hood.
¡ The experiment must be performed according to good laboratory practices.
¡ Do not use this kit after expiration date, mentioned on the label.
¡ The kit is shipped with dry ice, and the components of the kits must arrive frozen.
If one or more components are defrosted, or of the tubes have been damaged,
contact Eurobio Scientific.
¡ Once defrosted, spin down briefly the tubes before use.
¡ It is recommended to define three working areas: 1) Isolation of RNA, 2)
Preparation of the reaction mix and 3) Amplification / Detection of amplified
products.
¡ It is recommended to open and manipulate the positive controls away from
biological samples to tests and from other components of the kit in order to avoid
cross-contamination.
¡ Use specific lab coat and gloves (powder free) in each working area.
¡ Pipettes, reagents and other materials must not cross each area.
¡ Specific caution is required to preserve the purity of the reagents and reaction
mixtures.
¡ Appropriate methods of preparation/extraction of RNA to produce high quality
RNA and to be followed by an RNA application should be used, particularly
avoiding all sources of RNase contamination.
¡ Always use RNase-free DNase-free filtered tips for micropipettes.
¡ Do not pipette with mouth and do not eat, drink or smoke in the area.
¡ Avoid sprays.

SAMPLES COLLECTION, TRANSPORT AND STORAGE

¡ Collect samples in sterile tubes.

¡ It is the responsibility of the user to master its own conditions of collection,
transport and storage of samples, and extraction of RNA by suitable systems to
produce RNA of good quality.
¡ The samples should be extracted immediately or stored following the
recommendations in the table below (Table 3).

Table 3:
Recommendations of maximum storage of samples before extraction
Room temperature 2h
+ 4°C 72 h
- 20°C (preferably - 80°C) Long term storage

¡ The user can refer to the World Health Organization or High Health Authority for
storing samples.
¡ Extracted RNAs have to be stored at -80°C.

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 ¡ Transport of clinical samples must obey local regulations. The biosafety local
regulations for SARS-CoV-2 must be followed.

PROCEDURE

I- RNA extraction

It is the user’s responsibility that the extraction system used is compatible with
downstream real time RT-PCR technology. For this kit, we recommend to use
extraction methods of viral RNA from respiratory samples, and refer to manufacturer’s
instructions of the extraction kit.

In the EBX-041 kit, CI-ARN on the CY5 channel can be added before extraction or in
the PCR reaction. It ensures that a negative result is not due to an extraction problem
or due to the presence of high amounts of RT-PCR inhibitors.
We recommend the addition of 10 ul CI-ARN and an elution volume of 50 ul after
extraction. If the CI-ARN is added to control the RT-PCR, CI-ARN is added to the
reaction mix (1 —l per PCR reaction). See real-time RT-PCR protocol for details.

CI-ARN is available from Eurobio Scientific (Ref EurobioPlex EBX-003).

II- Real-time RT-PCR Procedure

General comments:

- The positive control, and the RNA extraction and RT-PCR inhibition control (CI-ARN)
contain a very high concentration of matrix. Manipulations must be performed carefully
to avoid contamination.

- To control the functioning of the PCR, and steps of extraction and real-time RT-PCR
amplification, it is necessary to test the positive control, as well as the negative control
(water PCR provided = CN-H2O+ CI-ARN) (see II-2/6 for real-time RT-PCR protocol
for details).

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II-1/ Diagram of the procedure

1 - PREPARATION OF MASTERMIX
(a) For RNA samples extracted without (b) For RNA samples extracted with CI-
CI-ARN ARN

Number of reactions N+3 Number of reactions N+3

Enzyme Taq polymerase (N+3) x 12,5 —l Enzyme Taq polymerase (N+3) x 12,5 —l
Enzyme Reverse trancriptase (N+3) x 0,2 —l Enzyme Reverse (N+3) x 0,2 —l
Water (N+3) x 2,3 —l trancriptase
Oligomix (N+3) x 5 —l Water (N+3) x 2,3 —l
CI-ARN (N+3) x 1 —l Oligomix (N+3) x 5 —l
Total Volume Total Volume
(N+3) x 21 —l (N+3) x 20 —l
Mastermix Mastermix

2 - PREPARATION OF REACTIONS

Sample Sample
20 —l Mastermix 20 —l Mastermix
+ +
5—l RNA sample 5—l RNA sample

Positive Control Positive Control

20 —l Mastermix 20 —l Mastermix
+ +
5—l CP 5—l CP

Negative Control Negative Control

20 —l Mastermix 20 —l Mastermix
+ +
5—l Molecular biology water (CN-H2O) 4—l Molecular biology water (CN-H2O)
+
1 —l CI-ARN

3 – REAL TIME PCR INSTRUMENT

Program Temperature Duration Cycle(s)

Reverse
Transcription 45°C 10 min 1 -

Denaturation 95°C 3 min 1 -

95°C 15 sec -
Amplification 40
58°C 30 sec Acquisition de fluorescence

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II-2/ Detailed Procedure

1) Homogenize the tube of Enzymes, and vortex the Oligomix, CP, and CI-ARN
tubes before starting, and centrifuge briefly.
2) Prepare the Mastermix as below. N is the number of PCR reactions (positive
and negative controls included). Plan to prepare enough reagents for at least
N+3 reactions (refer to part 1-(a) or 1-(b) of the previous diagram according to
the case).

Case (a): For samples extracted without CI-ARN

Number of reactions N+3
Enzyme Taq polymerase (N+3) x 12,5 —l
Enzyme Reverse trancriptase (N+3) x 0,2 —l
Water (N+3) x 2,3 —l
Oligomix (N+3) x 5 —l
CI-ARN (N+3) x 1 —l
Total Volume
(N+3) x 21 —l**
Mastermix

Case (b): For samples extracted with CI-ARN

Number of reactions N+3
Enzyme Taq polymerase (N+3) x 12,5 —l
Enzyme Reverse trancriptase (N+3) x 0,2 —l
Water (N+3) x 2,3 —l
Oligomix (N+3) x 5 —l
Total Volume
(N+3) x 20 —l
Mastermix

** The volume difference between case (a) or (b) has no effect on performance.

3) Homogenize the Mastermix prepared in 2) and centrifuge briefly.

4) Distribute 20 —L of Mastermix with a micropipette and filtered tips in each tube
or well of a microplate for real-time PCR.
5) Add 5 —L of extracted RNA sample.
6) In parallel test the following controls:

- Positive control:
ƒ 20 —l Mastermix + 5 —l CP.
- Negative Control:
o Case (a): For samples extracted without CI-ARN
ƒ 20 —l de Mastermix + 5 —l provided water (CN-H2O)
o Case (b): For samples extracted with CI-ARN
ƒ 20 —l de Mastermix + 4 —l provided water (CN-H2O) + 1 —l CI-ARN

7) Close immediately the tubes, or plate with an adhesive film to avoid all
contamination.
8) Centrifuge briefly to collect all the reaction mix at the bottom of the tubes or
plate.
9) Program the real-time PCR instrument as follows

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 Program Temperature Duration Cycle(s)
Reverse
Transcription 45°C 10 min 1 -

Denaturation 95°C 3 min 1 -

95°C 15 sec -
Amplification 40
58°C 30 sec Acquisition de fluorescence

Note 1: For the Applied Biosystems systems, select “NONE” in "PASSIVE REFERENCE".
Note 2: On Rotorgene ™, please calibrate the signal by clicking on “GAIN optimization”.
Note 3: On CFX96TM (Bio-Rad), start the run using the v1.6 or later version of CFX Manager software,
and analyze with v 3.1 (see § Validation of the Experiment)
Note 4: On LightCycler® 480 systems (Roche), two optical systems are available: only "System II" is
compliant with use of the kit.
Note 5: On LC480 instrument II, apply color compensation for the following channels: FAM-HEX/VIC-
TexasRed-Cy5 (465-510, 533-580, 533-610, 618-660).

VALIDATION OF THE EXPERIMENT

The analysis of data post-acquisition on a CFX96TM PCR instrument (Bio-Rad) must

be done with version 3.1 of CFX Manager software (Bio-Rad). In order to use this v3.1
version from a run started with an older version, follow the procedure below: at the end
of the run, the data file with “pcrd” suffix must be open and treated with version 3.1 of
CFX Manager (Bio-Rad).

If the run was done with CFX Manager v1.6 for instance, to open the data file with CFX
Manager v3.1, click on CFX Manager v3.1 icon. The screen below appears.

- Click on “File” and select “Open”, then “Data File”

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- Select the file you want to analyze and click on “Open”.
The “drift correction” option must be selected from the “Settings” Tab, as indicated on
the image below: click on “Settings”, then “Baseline Setting” then on “Apply
Fluorescence Drift Correction”.

Once this is done, the analysis can start.

The results for the controls must be the following (Table 4).

Table 4: Validation of the run

Positive Control
FAM Ct ” 32
HEX Ct ” 32
Texas Red Ct ” 30
Negative Control
FAM Undetermined Ct
HEX Undetermined Ct
Texas Red Undetermined Ct
Cy5 Ct ” 40

DATA ANALYSIS AND INTERPRETATION

RNA extraction and RT-PCR inhibition control in samples:

The proper functioning of RT-PCR reaction can be evaluated on the Cy5 channel
measuring the RNA extraction and RT-PCR inhibition control (CI-ARN) amplification.

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In some cases, it is recommended to repeat the extraction or to dilute the sample 5
times, because the result cannot be interpreted (NI) (See column « validity of the test »
on Table 5. All cases that can be encountered are described in Table 5.
For high viral loads detected on the FAM channel, the Ct value of CI-ARN can be
increased compared to the one in the negative control. This does not invalidate
positivity.

For clinical samples, and determination of presence or absence of SARS-CoV-2:

The following results are possible:

Cut-off Ct values for positivity:

Target 1 and Target 2 RdRp Gene and Target 3 N gene: Ct < 40

Table 5:
Target 1 Target 2 Target 3 CI-ARN Validity of Presence of SARS-CoV-2 or no
RdRp RdRp N the test possible interpretation (NI)

FAM HEX Texas Red CY5

+ +/- +/- +/- Yes YES
+/- + +/- +/- Yes YES
- - - + Yes NO

- - + + Yes Undetermined

- - +/- - Limited NI

NI : no possible interpretation because of RT-PCR inhibition or failed extraction: no

conclusion can be given. It is then recommended to proceed to a new sampling
and/or repeat the extraction and/or to dilute 5 times the sample.

Limitations of use and interpretation:

™ All samples must be treated as potentially infected by SARS-CoV-2, and

biosafety local regulations must be thoroughly followed.
™ Interpretation of results must take into account the possibility of false negatives
and false positives.
False negative can be due to:
- Inappropriate collection of samples, or bad storage
- Samples outside the viremic phase
- Incorrect extraction methods or use of non-validated PCR instruments
- Manipulations that do not rigorously follow all the indications of this
manual.
False positive can be due to:
- A contamination related to wrong manipulation of highly positive samples,
or from the positive control, or PCR products

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 - Procedures that do not rigorously follow precautions to avoid contamination
described in this manual
™ Results must be interpreted by medical professionals in the clinical context of
the patient, its history and symptoms.
™ This test does not exclude the presence of other pathogens than the SARS-
CoV-2.
™ A negative result for this test does not absolutely exclude a possible infection
with SARS-CoV-2.

PERFORMANCE ANALYSIS

¾ Limit of detection / Analytical sensitivity:

- Positive control: 30 copies/—l CP.

- A synthetic RNA comprising the 2 genes RdRp, and N of known
concentration (10e4 copies/ —l) spiked into a negative sample at dilution 1/10
(10 microl spiking, extraction using QIAamp viral RNA mini kit, and elution
into 60 microliters) was detected.
Sensitivity was < 16,6 copies RNA/ —l.
- A SARS-CoV-2 synthetic RNA (Twist Bioscience, USA) was detected with
a sensitivity of 12,7 copies ARN/—l

¾ Reproducibility

Inter-lot Ct variability between 3 lots of EBX-041 is the following:

Coefficient of variation % RdRp Gene RdRp Gene N Gene

Target 1 Target 2 Target 3
CFX96 0,77 1,04 1,59
ABI7500 1,16 1,63 3,31
Quant Sudio 5 3,69 1,86 0,94
LC480 2,24 2,54 2,38

¾ Diagnostics Specificity: (see also Limitations of use and interpretation)

This validation was carried out on:

- 56 negative samples for known coronaviruses, some of which are

positive for other respiratory viruses, in order to evaluate potential cross-
reactions.
- 13 samples positive other coronavirus than SARS-CoV-2: CoV NL63
(n=7), CoV OC43 (n=6) and 2 NATtrol™ RP Multimarker Respisratory
Controls (Zeptometrix) below.

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RNA extraction was performed on the following systems:
QIAamp viral RNA mini kit (Qiagen)
Magna Pure Compact (Roche Life Science)
EZ1 (Qiagen)

SARS-CoV-2
Number
Extraction Positive for Coronavirus status with
of cases
EBX-041
/ EZ1 RP1 NATtrol Negative Negative
/ EZ1 RP2 NATtrol Positive Negative
2 Magna Pure compact Adenovirus Negative Negative
2 Magna Pure compact RSV A ou B Negative Negative
4 EZ1 or QIAamp Influenzae A Negative Negative
3 QIAamp Influenzae B Negative Negative
16 QIAamp Parainfluenzae Negative Negative
2 Magna Pure compact Rhinovirus Negative Negative
3 Magna Pure or QIAamp Metapneumovirus Negative Negative
1 Magna Pure compact Bocavirus Negative Negative
2 Magna Pure compact Legionella pneumophilia Negative Negative
4 Magna Pure compact or Mycoplasma Negative Negative
QIAamp pneumoniae
4 QIAamp Bordetella pertussis Negative Negative
2 QIAamp Bordetella parapertussis Negative Negative
1 QIAamp Bordetella holmesii Negative Negative
1 QIAamp Parechovirus Type 1 Negative Negative
1 QIAamp Parechovirus Type 2 Negative Negative
1 QIAamp Parechovirus Type 3 Negative Negative
1 QIAamp Parechovirus Type 4 Negative Negative
1 QIAamp Parechovirus Type 5 Negative Negative
1 QIAamp Parechovirus Type 6 Negative Negative
4 QIAamp Enterovirus Negative Negative
7 QIAamp CoV NL63 Positive Negative
6 QIAamp CoV OC43 Positive Negative
An in silico analysis was used and demonstrates the specificity of the primers and
probes for SARS-CoV-2. For example, on MERS and SARS virus sequences, no
significant homology of the SARS-CoV-2 primers and probes, which could amplify
these viruses was found.

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 ¾ Diagnostic sensitivity: (see Limitations of use and interpretation)

Evaluation was carried out on:

o 82 nasal swab samples from patients confirmed positive by a hospital using

either :

- For 19 : a « in-house » method based on the amplification of the 2

sequences on RdRp gene, recommended by WHO
(https://www.who.int/emergencies/diseases/novel-coronavirus-
2019/technical-guidance/laboratory-guidance: published under Real-
time RT-PCR assays for the detection of SARS-CoV-2 Institut Pasteur,
Paris (2 March 2020),

- For 65 : Allplex 2019 n-CoV kit (Seegene).

2 samples were evaluated with both methods.

Extraction was done using EasyMag (Biomérieux).

Resuls are the following :

EBX-041

POS NEG TOTAL

Pre-test “in house” Method POS 19 0 19

Pre-test Allplex 2019 n-CoV POS 65 0 65

o 34 negative samples of nasopharyngeal aspirate, broncho-alveolar fluid, sputum,

nasal swab, were supplemented with the RNA from a positive patient diluted 20-
folds, from which 10 —l added to 140—l sample. Extraction was then performed on
QIAamp viral RNA mini kit and eluted with 60 —l, from which 5 —l were tested with
EBX-041.

All 34 samples supplemented with RNA were positive for SARS-CoV-2 with Ct as
below.

RdRp Gene RdRp Gene N Gene

Target 1 Target 2 Target 3
Mean Ct +/- Standard Deviation 27.37+/-1.06 26.11+/-1.06 33.15+/-1.11
Coefficient of variation (n=34) % 3.9 4.0 3.4

Overall performances are:

ϭϱ
 EBX-041
SARS-CoV-2 SARS-CoV-2
POS NEG
SARS-CoV-2 POS 116 0
Pre-test
SARS-CoV-2 NEG 0 69

Sensitivity : > 99 % (116/116)

Specificity : > 99 % (69/69)
Concordance : > 99 % (185/185)

Tests were performed on CFX96TM (Bio-Rad).

BIBLIOGRAPHY

Chan JF, Kok KH, Zhu Z, Chu H et al. Genomic characterization of the 2019 novel
human-pathogenic coronavirus isolated from a patient with atypical pneumonia after
visiting Wuhan. Emerg microbes infect. 2020 Dec; 9(1):221-236.

Cohen J, Normile D. New SRAS-like virus in China triggers alarm, Science. 2020 Jan
17;367(6475):234-235.

Drosten C, Muth D, Corman VM, Hussain R et al. An observational, laboratory-based

study of outbreaks of middle east respiratory syndrome coronavirus in Jeddah and
Riyadh, kingdom of Saudi Arabia, 2014. Clin infect dis. 2015 Feb 1; 60(3):369-77.

Cryopréservation de tissus, cellules et liquides biologiques issus du soin,

Recommandations de bonnes pratiques, www.has-santé.fr.

WASTE DISPOSAL

Be in accordance with the law on the elimination of waste of clinical infectious material.

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 SYMBOLS

Reference

Batch number

Limits of storage temperature

Expiration Date

Content sufficient for « N » reactions

Keep protected from light

Manufacturer

CE labeled product

In vitro Diagnostic

Instructions for use

7, avenue de Scandinavie
ZA Courtaboeuf
91940 Les Ulis
FRANCE

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