emergency use only in association with genefindertm covid

EMD-COVID19_Open_Compatibility_Simplified workflow 600-2020/01 EN Internal Use 1

This document is intended to provide information about the compatibility of the kit with not validated platforms and its use should be considered for research and emergency use only in association with GeneFinder

TM COVID-19 PLUS RealAmp KIT.

A. Intended use

The GeneFinderTM

COVID-19 PLUS RealAmp Kit is used for the Qualitative detection of COVID-19 (SARS-CoV-2) virus through

Reverse Transcription and Real-Time Polymerase Chain Reaction from RNA. A feasibility study was evaluated to define the assay

compatibility in combination with the most commercial instruments. Before use, the laboratory should validate the whole process.

B. Matrices

› Alveolar Lavage Fluid (BAL) › Nasopharyngeal swabs (NPS) › Sputum

C. Kit content

COVID-19 PLUS Reaction Mixture

(Purple)

COVID-19 PLUS Probe Mixture

(Brown)

COVID-19 PLUS Positive Control

(Red)

COVID-19 PLUS Negative Control

(Green)

Maximum Shelf-life: 12 Months

Reaction Mixture 1 tube of 1,050 µL 100 reactions per kit 10 freeze-thaw cycles

Probe Mixture 1 tube of 550 µL 100 reactions per kit 10 freeze-thaw cycles

Positive Control 1 tube of 50 µL 10 freeze-thaw cycles 10 tests per Kit

Negative Control 1 tube of 50 µL 10 freeze-thaw cycles 10 tests per 1 Kit

Storage Temperature:

-20°C

D. Master Mix Preparation

Reagent 1 Test Total reactions

COVID-19 PLUS Reaction Mixture 10 µL Sample N + 3 rxn*

COVID-19 PLUS Probe Mixture 5 µL Sample N + 3 rxn*

Total volume 15 µL *The automatic PCR plate setup could require more. Check below in the specific extraction system in use

E. Result Interpretation

RdRP E N Result

+ +

SARS-CoV-2 positive + +

+ + +

+

Repeat to confirm Positive + +

+

+ SARBeCoV

F. Performances

LoD (PCR) Reproducibility Repeatability

10 copies (RdRp, E, N gene) CV < 5% CV < 5%

Valid Positive sample call : Target Ct < 43 Valid Negative sample call : IC Ct < 35

G. Tube type collection

Copan Ref.

Description

608CS01R eNAT Swab*

359C UTM Swab *The eNAT medium contains guanidine thiocyanate

Note: Not use the Swab tube as primary tube but transfer 200 µL

of sample into sonicator tube.

H. Sample Pretreatment (optional)

Sample inactivation is not required as shown in WHO guideline.

The sample can be pretreated with guanidine thiocyanate or other

lysis buffers under a Biosafety cabinet of class II (BSC2)

1. Dispense 200 µL of sample into the sonicator tubes

2. Add 150 µL of buffer containing denaturants

3. Cover the sonicator tube with the cap

4. Load the sample into the instrument removing the caps

Note: This procedure needs to be validated as per WHO guidelines

I. References

- WHO Reference: “Laboratory biosafety guidance related to coronavirus disease 2019 (COVID-19), Interim guidance, 12 February 2020” - CDC Reference: “Interim Guidelines for Testing Clinical Specimens from Persons Under Investigation (PUIs) for COVID 2019”

- Copan Reference: “Specimen collection for novel Coronavirus (2019-ncov) testing in suspected human cases: up to date guidelines

underline the aptness of flocked swabs and viral transport medium”.

- Christian Drosten et al.,“Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR ”, Euro Surveill. 2020;25(3).

X 1 X 1 X 1 X 1

GeneFinderTM COVID-19 PLUS RealAmp kit Code: IFMR-45 used in association to Open Real Time Platforms


J. Semi-automated extractions compatibility

ELITe GALAXY is a closed instrument but an open protocol is ready for use with COVID-19 Plus kit. Please refer to your local ELITechGoup

support to ask the required protocol to be upload into the instrument.

Sample preparation

› ELITe GALAXY – Extraction kit (Ref. INT021EX) › Sample volume processed : 300 µL › Sample dead volume (2mL tubes): 100 µL › Sample dead volume (13x75 tubes) 300 µL › Sample dead volume (16x100 tubes) 600 µL › Total elution volume: 100 µL

PCR setup

› Master Mix volume: 15 µL › Master Mix Dead volume: 45 µL › Eluate volume: 5 µL › Controls dead volume: 30 µL

Qiagen Qiasymphony is a closed instrument but permits the usage of open protocols. Please refer to your local Qiagen support to ask an offer

to provide you the required protocol.

Sample preparation

› QIAsymphony - DSP Virus/Pathogen Midi kit › Sample volume processed: 400 µL › Total elution volume: 85 µL

PCR setup


Abbott m2000 is a closed instrument but permits the usage of open protocols. Please refer to your local Abbott support to ask an offer to

provide you the required protocol. The fluorescence channels as per the needs to be enables. See below:

Sample preparation

› Abbott m2000sp - mSample Preparation Systems DNA (CE) or mSample Preparation Systems RNA

› Sample volume processed: 400 µL – 600 µL › Total elution volume: 100 µL

Note: m2000 requires to extract the positive and negative controls. Substitute the controls with water and remove them at the end from the elution plate.

PCR setup

› Master Mix volume: 15 µL › Master Mix Dead volume: 100 µL › Eluate volume: 10 µL › Controls: 10 µL - Manual input

Note: Positive and negative controls are manually dispensed in the PCR plate after the automatic dispensation of the Master Mix and the patient samples eluates.

Siemens Versant kPCR permits the usage of open protocols. Please refer to your local Siemens support to ask an offer to provide you the

required protocol.

Sample preparation

› kVersant - Sample Preparation 1.0 Reagents Kit › Sample volume processed: 400 µL › Total elution volume: 100 µL

PCR setup


ELITechGroup ELITe GALAXY

Qiagen QiaSymphony

Abbott m2000sp

Siemens Versant kPCR


K. Real Time PCR thermal cycler compatibility

These settings could be theoretically compatible also with previous Rotorgene version (Corbett 6000 series). Verify before use.

Amplified sequence and Channels setting

Target Gene Channels Gain *

Target 1 RdRp gene Green 5.33

Target 2 E gene Orange 8

Target 3 N gene Yellow 5.33

Internal Control Rnase P gene Red 9.33

* Gain after 1st acquisition

Thermal Profile

Description Temperature Time

Hold Reverse Transcrition 50 °C 20:00

Hold 2 Denaturation 95 °C 05:00

Cycling Amplification and detection

45 cycles

95 °C 00:15

58 °C 01:00

Procedure

1. Thaw COVID-19 Plus-Reaction and Probe Mixed and COVID-19 Controls 2. Mix gently and spin-down 3. Reconstitute the MasterMix as per the paragraph D 4. Pipet 15 μL of Q-PCR-Mix in all microplate wells in use 5. Add 5 μL of extracted DNA in sample wells, 5 μL of molecular grade water in Negative Control well, and 5 μL of the Positive control in Positive control curve wells. Note: Each one has to be mixed by pipetting 3 times into the reaction mixture 6. Seal the microplate with the amplification sealing sheet

7. Transfer the microplate in the thermocycler and start

Interpretation settings

Setting Value

Threshold 0.15

Left Threshold 5 skip cycles

Start normalising from cycle 1

Noise Slope Correction No

No Template Control Threshold 0%

Reaction Efficiency Threshold Disabled

Normalisation Method Dynamic Tube Normalisation

These settings could be theoretically compatible also with other Roche similar instruments like Light Cycler 480 version II. Verify before use.


Target Gene Channels Threshold

Target 1 RdRp gene FAM (465 – 510) 1.5

Target 2 E gene T.Red (540 – 580) 2

Target 3 N gene VIC (540 - 610) 1.5

Internal Control Rnase P gene Cy5 (610 – 670) 0.2


Setting Value

Detection Format Multi hydrolysis probe

Background 2 to 6

Quant factor and Max integration time* 10 and 2 sec

Procedure



*Filter Combination List setting

Thermal profile

Description Analysis Cycles Temperature Acquisition Time Ramp Rate

Hold Reverse Transcrition none 1 50 °C none 20:00 4.40

Hold 2 Pre-Denaturation none 1 95 °C none 05:00 4.40

Amplification Denaturation

Quantification 45 95 °C none 00:15 4.40

Annealing 58 °C Single 01:00 2.20

Qiagen Rotor- gene Q

Roche Cobas z480


These settings could be theoretically compatible also with other similar instruments like the QuantStudio version 6/7. Verify before use.


Target Gene Channels Threshold Quencher

Target 1 RdRp FAM 10000 none

Target 2 E Texas Red 10000 none

Target 3 N VIC 10000 none

Internal Control Rnase P Cy5 10000 none

Thermal Profile

Description Temperature Time

Hold Reverse

Transcription 50 °C 20:00

Hold 2 Pre-Denaturation 95 °C 05:00

Cycling 45 cycles

Denaturation 95 °C 00:15

Annealing 58 °C 01 :00

Procedure




Setting Value

Passive Reference none

Baseline 6 – 15

Thermo Fisher QuantStudio 5Dx

emergency use only in association with genefindertm covid

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