GSD NovaPrime®
SARS-CoV-2 (COVID-19)
RT-PCRFor research use only

Product Number:

PCOV6033(96 Determinations)

CONTENTS
CONTENTS .................................................................................................................................................................. 2
ENGLISH ...................................................................................................................................................................... 3
1.
2.
3.
4.
5.

INTRODUCTIONINTENDED USE
PRINCIPLE OF THE ASSAY
MATERIALS PROVIDEDSTABILITY AND STORAGE
5.1.

6.
7.

8.

3
3
3
4
4

Materials and Equipment needed, but not provided ...................................................................................................... 4

SAMPLE COLLECTION AND PREPARATIONASSAY PROCEDURE

4
4

7.1.

Reaction Setup ............................................................................................................................................................. 4

7.2.

Programming the Real-Time PCR Instruments ............................................................................................................. 5

RESULTS

5

Run Validation Criteria ............................................................................................................................................................. 6
8.1.

9.

10.
11.
12.

Interpretation of Results ................................................................................................................................................ 6

SPECIFIC PERFORMANCE CHARACTERISTICS

6

9.1.

Precision ....................................................................................................................................................................... 7

9.2.

Limit of Detection (LoD) ................................................................................................................................................ 7

9.3.

Inclusivity ...................................................................................................................................................................... 7

9.4.

Cross-Reactivity ............................................................................................................................................................ 7

9.5.

Negative and Positive Percent Agreement (NPA, PPA) ................................................................................................ 7

QUALITY CONTROLTRADEMARKS AND DISCLAIMERS
PRECAUTIONS AND WARNINGS
12.1.

7
7
8

Disposal Considerations ............................................................................................................................................ 8

13. ORDERING INFORMATION
8
Bibliography ................................................................................................................................................................11
Symbols Key ...............................................................................................................................................................122

ENGLISH
1. INTRODUCTION
End of 2019, a novel respiratory disease emerged in the city of Wuhan, Hubei Province of the People’s Republic of China, andsoon spread rapidly within the country and worldwide. The causative agent was identified as severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2). SARS-CoV-2 (2019-nCoV), like the closely related SARS coronavirus (SARS-CoV), belongs to thegenus Betacoronavirus within the family of coronaviruses. The zoonotic reservoir of the virus appears to be bats.
Coronaviruses are enveloped, positive single‐stranded large RNA viruses that infect humans, but also a wide range of animals.
The common human coronaviruses NL63, 229E, OC43 and HKU1 are widespread especially throughout the winter months. They
are responsible for up to one third of all acute respiratory diseases, typically with mild symptoms (common cold). More than 80 %of the adult population have antibodies against human coronaviruses. The immunity from previous infections lasts only for a shortperiod of time. Therefore, reinfections with the same pathogen are possible just after one year.
SARS-CoV-2 is predominantly transmitted by droplet infection via coughing or sneezing and through close contact with infectedpatients. In theory, smear infection and infection through the conjunctiva of the eyes are also possible.
The incubation period is in the median 5–6 days (and up to 14 days maximum).
The clinical manifestations of SARS‐CoV‐2‐related COVID-19 disease include fever, cough, respiratory problems and fatigue. In
most patients the infection manifests with symptoms of a mild febrile illness with irregular lung infiltrates.
The initial clinical sign of COVID‐19 which allowed case detection was pneumonia. But it turned out that the course of the disease
is non-specific and varies widely, from asymptomatic courses to severe pneumonia with lung failure and death. However, basedon current knowledge, around 80 % of the illnesses are mild to moderate.
Although severe courses of the disease also occur in younger patients and people without previous illness, the following groupsof people have an increased risk of serious forms of the disease: elderly people (with a steadily increasing risk from around 5060 years of age), smokers and people with certain diseases of the cardiovascular system or the lungs, patients with chronic liverdiseases, diabetes mellitus, cancer, or patients with a weakened immune system (e.g. due to immune deficiencies or by taking
drugs that suppress the immune system).
Currently, there is no specific treatment or vaccine available against SARS-CoV-2 infection.
Species
SARS-CoV-2(severe acuterespiratory
syndrome
coronavirus 2)

DiseaseSymptoms e.g.

Transmission route

COVID-19

the course of the disease is unspecific,
diverse and varies greatly, fromasymptomatic courses to severe pneumonia
with lung failure and death

primary mode of transmission: droplet
infection;
smear infections and infections via theconjunctiva of the eyes aretheoretically possible

The presence of pathogen or infection may be identified by▪
Nucleic acid testing (NAT):
e.g. RT-PCR▪
Serology:
detection of antibodies by e.g. ELISA

2. INTENDED USE
The GSD NovaPrime® SARS-CoV-2 (COVID-19) RT-PCR is intended for the qualitative determination of SARS-CoV-2 (Severe
acute respiratory syndrome coronavirus 2) genomic RNA extracted from human respiratory (nasal wash/swab, nasopharyngeal
wash/swab, oropharyngeal swab and bronchoalveolar lavage) specimen types.

3. PRINCIPLE OF THE ASSAY
The qualitative determination of specific RNA is based on Real-Time reverse-transcription Polymerase Chain Reaction (RT-PCR)
technology. The kit contains specific primers and probes labelled with fluorescent reporter and quencher dyes for amplificationand simultaneous detection of specific RNA sequences. Furthermore, the assay contains a heterologous amplification target(Extraction Control, EC ) to identify possible RT-PCR inhibition by interfering substances contained in the sample or failure of
the preceding RNA extraction. Therefore, the EC is added to the specimen during RNA isolation.
The gene of interest specific probes are labelled with the fluorophore FAMTM. The Extraction Control specific probe is labelledwith the fluorophore Cy5TM thereby allowing parallel detection of both amplicons in the corresponding detector channels.

34. MATERIALS PROVIDED
Cap

SymbolComponent

Volume

green

E-MIX

(reaction buffer containing dATP, dCTP, dGTP, dTTP,
magnesium, reverse transcriptase, RNase inhibitor protein, hotstart DNA polymerase, stabilizers, and ROX Reference Dye)

1x

750 µL

blue

PP

Primer-Probe-Mix

1x1100 µL

yellowEC

1x

500 µL

red

PC

1x

100 µLtransparent

NFW

1x

500 µL

RT-PCR Enzyme Mix

Extraction Control(RNA-based internal lysis, extraction and amplification control)

Positive Control(plasmid DNA representing the N gene of SARS-CoV-2)

Nuclease Free Water

5. STABILITY AND STORAGE
The GSD NovaPrime® SARS-CoV-2 (COVID-19) RT-PCR kit is shipped on dry ice and all components should arrive frozen.
▪
All components have to be stored at -20 °C upon arrival.
▪
Repeated freeze thaw cycles of reagents (more than two) should be avoided, since this might affect the performance of
the kit. Reagents should be frozen in aliquots if they are used intermittently.
▪
Keep unfrozen storage (e.g. storage on ice) as short as possible.
▪
Keep the E-MIX and the PP in the freezer, until you are ready to use it.
▪

Protect E-MIX and the PP from light.

5.1.

Materials and Equipment needed, but not provided

▪
▪
▪
▪
▪
▪
▪
▪
▪

Real-Time PCR instrument (see 9.SPECIFIC PERFORMANCE CHARACTERISTICS)
Equipment and consumables for isolating virus RNA from respiratory specimens
Appropriate Real-Time PCR consumables (e.g. reaction plates, corresponding optical closing materials)
Benchtop microcentrifuge
Centrifuge with a rotor for microtiter plates
Vortex mixer
Adjustable pipettes in relation to reaction setup
Disposable DNase/RNase free pipette tips with filters
Disposable powder-free gloves

6. SAMPLE COLLECTION AND PREPARATION
▪
▪
▪

▪
▪

Extracted RNA or total nucleic acid is the starting material for the GSD NovaPrime® SARS-CoV-2 (COVID-19) RT-PCR
Kit. The quality of the extracted RNA has a crucial effect on the performance of the entire RT-PCR test system. Make
sure that the nucleic acid extraction method is compatible with Real-Time PCR technology.
For nucleic acid extraction a method suitable for extracting virus RNA from human respiratory specimen should be used.
The EC can be used for monitoring both the RNA extraction procedure and any potential PCR inhibition. Therefore,
the EC has to be added prior to the nucleic acid extraction procedure. Independent of the method / system used fornucleic acid extraction, the EC can be directly added to the specimen.
About 5 µL EC may be suitable, but should be carefully evaluated. Please refer to table under 8.1.
Since ethanol is a strong Real-Time PCR inhibitor, it is necessary to completely eliminate it prior to the elution of thenucleic acid during extraction. If using spin columns with washing buffers containing ethanol, it is highly recommended
to perform an additional centrifugation step of 10 min at approximately 17000 x g (~ 13000 rpm) before eluting the RNA.
For this additional centrifugation step, use a new collection tube.

7. ASSAY PROCEDURE
7.1.

Reaction Setup▪

Please read the instructions for use carefully before performing the assay. Reliability of results depends on following
strictly the instructions for use.
Before use make sure that all samples and reagents are thawed completely, mixed by up and down pipetting or vortexing
and centrifuged briefly.
It is highly recommended to pipet samples and controls in triplicates.
Pipette E-MIX slowly and carefully and use pipette tips suitable for pipetting viscous liquids.
The use of NFW as no template control (NTC) is highly recommended.
Define the positions of the wells on the plate for samples and controls ( PC or NFW ).

▪
▪
▪
▪
▪

4

Reaction Setup
E-MIX

7.5 μL

PP

10.5 µL

Sample or PC or NFW

12 μL

Total volume

30 μL

▪
▪

Close the optical 96-well reaction plate with an optical adhesive cover.
Centrifuge the optical reaction plate in a centrifuge with a rotor for microtiter plates for 60 seconds at approximately
1000 x g (~ 3000 rpm).

7.2.

Programming the Real-Time PCR Instruments

Regarding setup and programming of the Real-Time PCR instrument, please use the manual of the respective instrument.
RT-PCR Run Settings
RT-PCR Run Settings

Reaction Volume

30 μL

Ramp Rate

Standard

Passive Reference

ROXTM

Fluorescent Detectors/Dyes
Detector

Dye

Quencher

SARS-CoV-2 RNA

FAMTM

None

Extraction Control EC

Cy5TM

None

Temperature Profile and Data Collection
No. of Cycles

Temperature

Time

Data Collection

1

25 °C

2 min

-

1

50 °C

30 min

-

1

95 °C

2 min

-

95 °C

15 sec

60 °C

1 min

Fluorescence measurement
at the end of every cycle

40

Before starting the test run, please check the settings for cycles, temperature and time.

8. RESULTS
Data analysis should be performed with the software of the used real-time PCR device according to manufacturer’s instructions.

5

Run Validation Criteria
Test run is valid if PCR run complete
The GSD NovaPrime® SARS-CoV-2 (COVID-19) test protocol dictates that the controls be analyzed before patient sample results.
The PC , EC and NTC Ct values must meet the acceptance criteria in the table below for the assay to be valid.
If kit control(s) fail, the test is invalid and needs to be repeated.
Validation Criteria

Result/Acceptable Ct

Valid/Invalid

Measure

≥ 40

valid

-

< 40

invalid

Repeat test run

no Cy5TM signal

valid

-

no FAMTM signal

invalid

Repeat test run

valid

-

Sample result invalid

Repeat extraction and test run

Sample result invalid

Repeat extraction withless EC volume, repeat test run

NTC
FAMTM signal
PC

Cy5TM signal
28 < Ct ≤ 35
Cy5TM Ct > 35 andFAMTM signal ≥ 38
Cy5TM Ct ≤ 28 andFAMTM signal ≤ 38

EC

Patient sample data is analyzed and interpreted only after all the kit controls pass.

8.1.

Interpretation of Results

If test run is valid interpretation of sample results is as follows:
Detection Channel

Interpretation of Results

FAMTM

Cy5TM

Positive
Ct < 38

Positive
28 < Ct ≤ 35

The sample contains SARS-CoV-2 specific RNA.

Positive
Ct < 38

Negative
Ct > 35

The sample contains SARS-CoV-2 specific RNA.

Negative
Ct ≥ 38

Positive
28 < Ct ≤ 35

Negative
Ct ≥ 38

Negative
Ct > 35

The sample does not contain detectable amounts of SARS-CoV-2 specific RNA.
PCR inhibition or reagent failure.
A diagnostic statement must not be made.
The RT-PCR run should be repeated or a new sample must be analysed.

Diagnosis of an infectious disease should not be established only on the basis of a single test result. A precise diagnosis
should take into consideration clinical history, symptomatology as well as laboratory diagnostics.

9. SPECIFIC PERFORMANCE CHARACTERISTICS
The determinations of the specific performance characteristics were done on ABI Prism ® 7500 SDS/Fast SDS (Applied
Biosystems) in standard mode (7500 Software v2.3).
To establish performance characteristics, RNA extraction was performed using the bioMérieux NUCLISENS® easyMAG® or
EMAG® instrument.
The results refer to the groups of samples investigated; these are not guaranteed specifications.
Use of other Real-Time PCR instruments or RNA extraction methods must be validated by the user. During validation asuitable EC volume which has to be added during RNA extraction has to be determined to obtain a valid Cy5TM signal as shownin table under 0.
For further information about the specific performance characteristics, please contact NovaTec Immundiagnostica GmbH.

6

9.1.

Precision

Precision data for specific RNA
SARS-CoV-2

nrun perday/replicates

Mean [Ct](day1/2/3)

Standard
Deviation

Coefficient of
Variation [%]Intra-Assay Variability
High positive sample

1/3

15.35/15.82/15.84

0.16/0.04/0.12

0.39/0.24/0.73

Medium positive sample

1/3

21.47/21.70/21.79

0.16/0.43/0.11

0.74/0.43/0.52

Low positive sample

1/3

27.30/27.27/27.55

0.21/0.01/0.01

0.75/0.04/0.05

High positive sample

3/9

15.67

0.10

0.67

Medium positive sample

3/9

21.65

0.12

0.56

Low positive sample

3/9

27.37

0.08

0.28

Inter-Assay Variability

9.2.

Limit of Detection (LoD)

The analytical sensitivity is expressed as Limit of Detection (LoD).
LoD[copies/reaction]
Standard cycling

9.3.

3.75

Inclusivity

The analytical sensitivity of the GSD NovaPrime® SARS-CoV-2 (COVID-19) RT-PCR is first and foremost ensured by the thoroughselection of the oligonucleotides. Inclusivity was evaluated by in silico analysis using all publicly available SARS-CoV-2 sequences(available on the 26th of April 2020) to determine the percent identity matches for targeted sequences of the GSD NovaPrime®
SARS-CoV-2 (COVID-19) RT-PCR assay. In total 73 SARS-CoV-2 genome sequences have been analyzed by alignment toprimers and probes.

9.4.

Cross-Reactivity

Cross-reactivity was evaluated by in silico analysis against normal flora or pathogens that cause similar symptoms or pathogens
related to SARS-CoV-2. Primer-probe-sets not exceeding 80 % identity to a potential cross-reacting sequence are not predicted
to cause a cross-reaction.
In addition to in silico analysis, GSD NovaPrime® SARS-CoV-2 (COVID-19) RT-PCR was performed on nucleic acid forcoronavirus 229E, coronavirus NL63, coronavirus OC43, and SARS. All pathogens tested by the GSD NovaPrime® SARS-CoV2 (COVID-19) RT-PCR assay did not generate detectable amplification signals.

9.5.

Negative and Positive Percent Agreement (NPA, PPA)

30 defined negative and 30 defined positive samples have been tested. 100 % agreement was achieved for all 60 samples tested.

10. QUALITY CONTROLIn accordance with NovaTec Immundiagnostica GmbH ISO-certified Quality Management System, each lot of GSD SARS-CoV2 RT-PCR has been tested against predetermined specifications to ensure consistent product quality.

11. TRADEMARKS AND DISCLAIMERSABI Prism® (Applied BiosystemsTM), (ThermoFisher Scientific)
Registered names, trademarks, etc. used in this document are to be considered protected by law even if not specifically marked
as such.

7

12. PRECAUTIONS AND WARNINGS
▪

▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪
▪

▪
▪
▪

The test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed.
The use of the test kit with other analysers than the ones mentioned under “9. SPECIFIC PERFORMANCE
CHARACTERISTICS” has to be validated. Any change in design, composition and test procedure as well as any use incombination with other products not approved by the manufacturer is not authorized; the user himself is responsible forsuch changes. The manufacturer is not liable for false results and incidents for these reasons.
Only for in-vitro diagnostic use.
Do not interchange reagents of different production lots.
No reagents of other manufacturers should be used along with reagents of this test kit.
Do not use reagents after expiry date stated on the label.
Wear disposable powder-free gloves, a laboratory coat and eye protection when handling specimens.
Always use DNase/RNase-free disposable reaction tubes and pipette tips with aerosol barriers.
Avoid microbial and nuclease (DNase/RNase) contamination of the specimen and the components of the kit.
In order to avoid contamination of working space with nucleic acids, reaction tubes/plates should not be opened after
amplification.
RT-PCR is highly sensitive to nucleic acid contamination. Therefore, positive/ potentially positive material needs to be
stored separate from all other components of the kit.
Dedicate supplies and equipment to the separate working areas and do not move them from one area to another.
This assay must not be used on the specimen directly.
Prior to using this assay the nucleic acid has to be extracted with suitable extraction methods from the original specimen.
Since ethanol is a strong Real-Time PCR inhibitor, it is necessary to completely eliminate it prior to the elution of thenucleic acid during extraction. If using spin columns with washing buffers containing ethanol, it is highly recommended
to perform an additional centrifugation step of 10 min at approximately 17000 x g (~ 13000 rpm) before eluting the RNA.
For this additional centrifugation step, use a new collection tube.
The result of this RT-PCR kit may be influenced by potential mutations in the genome of the pathogen if they are locatedin the primer / probe binding region. Underestimation and/or failure to detect the pathogen may occur.
PCR inhibitors may also elicit underestimation, false negative results or invalid runs. Therefore, only use nucleic acids
extraction kits, which remove PCR inhibitors and which are dedicated for downstream PCR processes.
The RT-PCR is only designed for qualified personnel who are familiar with good laboratory practice and trained in RealTime PCR.

12.1. Disposal Considerations
Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste isregulated through national and regional laws and regulations. Contact your local authorities or waste management companies
which will give advice on how to dispose hazardous waste.

13. ORDERING INFORMATION
Prod. No.:

PCOV6033

GSD NovaPrime® SARS-CoV-2 (COVID-19) RT-PCR (96 Determinations)

8

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BIBLIOGRAPHY
Gorbalenya, Alexander E.; Baker, Susan C.; Baric, Ralph S.; Groot, Raoul J. de; Drosten, Christian; Gulyaeva, Anastasia A. et
al. (2020): Severe acute respiratory syndrome-related coronavirus: The species and its viruses – a statement of the Coronavirus
Study Group (14). bioRxiv 2020.02.07.937862; doi: https://doi.org/10.1101/2020.02.07.937862
Gralinski, Lisa E.; Menachery, Vineet D. (2020): Return of the Coronavirus: 2019-nCoV. In Viruses 12 (2). DOI:
10.3390/v12020135.
RKI (Ed.): SARS-CoV-2 Steckbrief zur Coronavirus-Krankheit-2019 (COVID-19).
Available online at https://www.rki.de/DE/Content/InfAZ/N/Neuartiges_Coronavirus/Steckbrief.html. (accessed: 13.04.2020)
Wang, Guan; Jin, Xian (2020): The progress of 2019 novel coronavirus event in China. In Journal of medical virology 92 (5),pp. 468–472. DOI: 10.1002/jmv.25705.
WHO: Clinical management of severe acute respiratory infection (SARI) when COVID-19 disease is suspected. Interim
guidance. 13 March 2020.
Available online at https://www.who.int/publications-detail/clinical-management-of-severe-acute-respiratory-infection-whennovel-coronavirus-(ncov)-infection-is-suspected, accessed: 13.04.2020.
Zhou, Peng; Yang, Xing-Lou; Wang, Xian-Guang; Hu, Ben; Zhang, Lei; Zhang, Wei et al. (2020): A pneumonia outbreakassociated with a new coronavirus of probable bat origin. In Nature 579 (7798), pp. 270–273. DOI: 10.1038/s41586-020-2012-7.

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SYMBOLS KEY
Manufactured by

IVD

In Vitro Diagnostic Medical Device

LOT

Lot Number
Expiration Date
Storage Temperature

Protect from LightCE Mark

REF

Catalogue Number
Consult Instructions for Use

E-MIX

RT-PCR Enzyme Mix

PPPrimer-Probe-Mix

NFW

Nuclease free water

EC

Extraction Control

PC

Positive Control
Contains sufficient for “n” tests

NovaTec Immundiagnostica GmbH
Waldstraße 23 A6
63128 Dietzenbach, Germany
Tel.:
Email:
Internet:+49 (0) 6074-48760
info@NovaTec-ID.com
www.NovaTec-ID.com

Fax:+49 (0) 6074-487629
PCOV603xeng-20200504

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