METHOD: DETECTION OF SARS-COV-2 RNA BY ...2.0 REFERENCES • China CDC Primers and probes for the ORF1ab and N regions of SARS-CoV-2 (24 January 2020) • Diagnostic detection of Wuhan

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METHOD: DETECTION OF SARS-COV-2 RNA BY REVERSE TRANSCRIPTION QUANTITATIVE POLYMERASE CHAIN REACTION

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1.0 OBJECTIVE

The objective of this document is to lay down a method to detect SARS-CoV-2 RNA by Reverse

Transcription quantitative Polymerase Chain Reaction.

2.0 REFERENCES

• China CDC Primers and probes for the ORF1ab and N regions of SARS-CoV-2 (24 January 2020)

• Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR – Charité, Berlin Germany – regions RdRp, E and N of the (17 January 2020)

• US CDC panel primer and probes– U.S. CDC, USA – 3 targets in the N region (28 January 2020)

• Luna Universal Probe One-Step RT-qPCR kit (New England BioLabs, catalog no. E3006L)

3.0 ABBREVIATIONS

• SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2

• RNA: Ribonucleic acid

• RT-qPCR: Reverse transcription quantitative polymerase chain reaction

• CDC: Center for Disease Control

• WHO: World health organisation

• FAM: Fluorescein

• BHQ1: Black Hole Quencher 1

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• BBQ: Black Berry Quencher

• NTC: No Template Control

4.0 EQUIPMENT AND MATERIALS

• Luna Universal Probe One-Step RT-qPCR kit (New England BioLabs, catalog no. E3006L)

• Real-time PCR machine (Bio-Rad CFX96 Real-Time System C1000 Thermalcycler or

similar)

• Centrifuge with plate holders

• Micropipettes p20, p200 and p1000 (Gilson or similar)

• Disposable tips for micropipettes p20, p200 and p1000

• Microcentrifuge tube of 1.5 and 0.5 ml (Eppendorf, catalog no. 0030120086 and

0030121023 or similar)

• Nuclease-free water (Promega, catalog no. P119C or similar)

• Multiplate PCR plates 96-well clear (Biorad, catalog no. MLL9601)

5.0 SAMPLE DETAILS

SARS-CoV-2 MULTITARGET sequence with T7 promoter sequence (underlined) and

selected primers highlighted in different colors:

TAATACGACTCACTATAGGGAGA GGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTG ACAGGTACGTTAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTTACTGCGCTTCGATTGTGTGCGTACTGCTGCAATATTG

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GTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTG GGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTC GACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGA TTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGC AGATTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTC CCCTGTGGGTTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGT

Note: The SARS-CoV-2 MULTITARGET sequence (812bp) was synthesized and cloned into pEX-

A128 vector (2450bp, ampicillin resistance ORF). For the pEX-A128 vector map and full sequence

please go the following link of Eurofins:

https://www.eurofinsgenomics.eu/media/1587668/pex-a128_map_seq_v10.pdf full

For details on how to prepare this positive control RNA from the pEX-A128-

nCoV_MULTITARGET_2 linearized plasmid, please look at the Document No. 001 named:

PREPARATION OF SARS-CoV-2 MULTITARGET RNA.

6.0 PREPARATION OF REAGENTS

• Sequences of the primers and the probes along the SARS-CoV-2 MULTITARGET

sequence with their working concentrations used in the current protocol. Primers are

unmodified oligonucleotides and probes are modified oligonucleotides with fluorophores

(FAM) and quencher (BBQ or BHQ1). Primers pairs with corresponding probes are

highlighted in different colours in the table 1.

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• Primer pair for detection of cellular RNAse P (primers no 16, 17 and 18 in the table 1) has

been included as an RNA extraction control.

No Oligonucleotide name Sequence (5'---3')

Working concentration

(µM) 1 DrostenE1-Fwd ACAGGTACGTTAATAGTTAATAGCGT 0.4 2 DrostenE1-Probe FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ 0.2 3 DrostenE1-Rev ATATTGCAGCAGTACGCACACA 0.4 4 DrostenN-Fwd CACATTGGCACCCGCAATC 0.6 5 DrostenN-Probe FAM-ACTTCCTCAAGGAACAACATTGCCA-BBQ 0.2 6 DrostenN-Rev GAGGAACGAGAAGAGGCTTG 0.8 7 CDCUSN1-Fwd GACCCCAAAATCAGCGAAAT 1 8 CDCUSN1-Probe FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 0.5 9 CDCUSN1-Rev TCTGGTTACTGCCAGTTGAATCTG 1

10 CDCUSN2-Fwd TTACAAACATTGGCCGCAAA 1 11 CDCUSN2-Probe FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1 0.5 12 CDCUSN2-Rev GCGCGACATTCCGAAGAA 1 13 CDCUSN3-Fwd GGGAGCCTTGAATACACCAAAA 1 14 CDCUSN3-Probe FAM-ATCACATTGGCACCCGCAATCCTG-BHQ1 0.5 15 CDCUSN3-Rev TGTAGCACGATTGCAGCATTG 1 16 RNAseP-Fwd AGATTTGGACCTGCGAGCG 1 17 RNAseP-Probe FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1 0.5 18 RNAseP-Rev GAGCGGCTGTCTCCACAAGT 1

19 CDCchinaORF1ab-Fwd CCCTGTGGGTTTTACACTTAA 1

20 CDCchinaORF1ab-Probe

FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1 0.5

21 CDCchinaORF1ab-Rev ACGATTGTGCATCAGCTGA 1

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Table 1: This table contains the sequences of primers and probes along the SARS-CoV-2

MULTITARGET sequence with their working concentrations used in the current protocol. Primers

pairs with corresponding probes are highlighted in different colors.

• Different pairs of primers have been tested and the linear range of amplification varies

between different primer sets in the experimental conditions used (see the figure below).

• On the bases of these results we report the Table with different ranges of RNA dilutions to

be used for standard curve preparation corresponding to each primers pair:

101 102 103 104 105 106 107 108 10910

20

30

40

SARS-CoV-2 RNA copies

Ct m

ean

Drosten NCDC US N1

CDC US N2

CDC US N3

Drosten E

CDC China ORF1ab

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7.0 PROCEDURE

7.1 General Considerations

The objective of this document is to lay down a method to detect SARS-CoV-2 RNA by Reverse

Transcription quantitative Polymerase Chain Reaction. RT-qPCR is the gold standard technique

for RNA quantification where RNA is first transcribed into complementary DNA (cDNA) by reverse

transcriptase and the cDNA is then used as the template for the qPCR reaction. RT-qPCR can be

performed in a one-step or a two-step assay. One-step assay combines reverse transcription and

qPCR in a single tube while in two-step assays, the reverse transcription and qPCR steps are

performed in separate tubes, with different reaction conditions. Luna Universal Probe used in this

procedure is one step RT-qPCR kit.

7.2 Procedure

7.2.1 Preparation of SARS-CoV-2 MULTITARGET RNA standard curve preparation

• Take an aliquot of SARS-CoV-2-MULTITARGET RNA from -80 °C and prepare serial

dilutions as per table 2 below using the nuclease-free water. These dilutions will be used to

prepare to a standard curve for RT-qPCR.

Note: Prepare serial dilutions in 100 µl aliquots andkeep them at -80 to avoid repeated freezing

and thawing.

Primerspair RNAdilutionsforstandardcurveDrostenE 1pg-10fgDrostenN 1pg-10fgCDCUSN1 100pg-1pgCDCUSN2 100pg-1pgCDCUSN3 100pg-1pgCDCChinaORF1ab 1pg-10fg

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• The corresponding RNA copy number was calculated on the basis of the concentration and

size of the SARS-CoV-2 MULTITARGET RNA (812 bps) using the NEBioCalculator

(nebiocalculator.neb.com) and Endmemo (endmemo.com).

• The NTC (No Template Control) is included as well in order to determine the limit of PCR

sensitivity and tests reactions for possible background signal that may occur during

amplification. The statistical significance of lowest detectable signal is determined by

employing the threshold cycle (Ct) of NTC. This control also assesses the generation of

primer-dimer PCR artefact or amplicon contamination of a kit reagent.

Concentration of SARS-CoV-2 MULTITARGET RNA RNA copy number per µl 1 µg/µl 2.E+12 100 ng/µl 2.E+11 10 ng/µl 2.E+10 1 ng/µl 2.E+09 100 pg/µl 2.E+08 10 pg/µl 2.E+07 1 pg/µl 2.E+06 100 fg/µl 2.E+05 10 fg/µl 2.E+04 1 fg/µl 2.E+03 100 ag/µl 2.E+02 10 ag/µl 2.E+01 1 ag/µl 2.E+00

Table 2: Concentration of the SARS-CoV-2 MULTITARGET RNA and corresponding copy number

used for a standard curve in RT-qPCR.

7.2.2 Preparation of RT-qPCR reaction mixture

• Dissolve lyophilized primers and probes in nuclease free water to the concentration of 100

µM. From that stock prepare a working solution of 10 µM in nuclease free water.

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• For standard curve use 1µl the SARS-CoV-2 MULTITARGET Template RNA of serial

dilutions previously prepared as per table 2.

• For sample use 5µl the Sample Template RNA previously prepared as per the Document

No. 001 named: PREPARATION OF SARS-CoV-2 MULTITARGET RNA.

• Prepare the PCR reaction mix as follows:

Components Volume (µl) Final concentration Luna Universal Probe One-Step Reaction Mix (2X) 12.5 1X

Luna WarmStart RT Enzyme Mix (20X) 1.25 1X

Forward primer (10 µM) variable variable Reverse primer (10 µM) variable variable Probe (10 µM) variable variable Template RNA 1 to 5

Nuclease-free water Up to 25

Table 3: RT-qPCR reaction mix preparation

7.2.3 Procedure and thermocycling protocol

• Prepare the standards and samples in microtubes.

• Prepare the PCR reaction mix.

• Put 25 µl of the reaction mixtures in the PCR plate, seal the plate and spin down in a centrifuge with

a plate adapter at 2000 rpm.

• Place the plate in the reaction chamber of the Real-time PCR machine and set the parameters as

per table 4.

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Step Temperature Time (minutes) Cycles

Reverse Transcription 55 °C 10:00 1 Initial Denaturation 95 °C 3:00 1 Denaturation 95 °C 0:15 45 Extension 58 °C 0:30

Table 4: Thermocycling protocol

8.0. RESULTS

8.1. Data analysis

• Analyze the data generated in an Excel Sheet by plotting a graph of Cycle number – Ct

value (Y axis) vs log of RNA copies (X axis) (see an Excel file attached).

• Calculate the % RSD between triplicates for standards and samples.

• Calculate the observed concentration values for standards using equation y = mx + b from

the graph for system suitability analysis.

• If the Ct value of the sample is inside the linear range of the standard curve calculate the

number of RNA copies in the sample using the linearity equation.

A typical example of standard curve and data analysis is shown below:

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8.2. Acceptance criteria

• The Ct value of NTC sample should be more than the Ct value of the lowest standard

• Percentage RSD between the Ct values of replicates of standards and NTC sample should

not be more than 5%

• The linear regression for the standard curve of Ct values (Y-axis) vs log of RNA copies (X

axis) should be equal to or more than 0.980

• Accuracy in terms of percent recovery of the Calculated values by the line graph for

standards should be between 70% and 130%

y=-2.9025x+44.269R²=0.98956

000

005

010

015

020

025

030

035

040

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Ct

LogRNAcopies

standardcurveNo. of RNA copies RNA pg ct1 ct2 ct3 ct mean std dev %RSD log RNA copies calc log RNA No. of RNA copies recovery

2,00E+06 1 26,53 26,34 26,35 26,35 0,01 0,03 6,30 6,18 1,50E+06 74,87

2,00E+05 0.1 28,50 28,41 28,39 28,40 0,01 0,05 5,30 5,47 2,93E+05 146,66

2,00E+04 0.01 31,56 31,69 31,62 31,66 0,05 0,16 4,30 4,35 2,22E+04 110,89

2,00E+03 0.001 35,04 34,94 34,93 34,94 0,01 0,02 3,30 3,22 1,64E+03 82,19

0,00E+00 NTC 37,17 36,53 36,53

sample 1 27,76 27,68 27,97 27,83 0,21 0,74 5,67 462871,89

sample 2 24,7 24,71 24,85 24,75 0,08 0,34 6,72 5293511,96

sample 3 26,3 26,4 26,56 26,42 0,13 0,50 6,15 1410997,59

sample 4 25,48 25,64 25,87 25,66 0,20 0,76 6,41 2571704,55

sample 5 36,17 35,91 36,32 36,13 0,21 2,80 635,31

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9.0. ADDITIONAL NOTES

• Basic expertise in working with RNA is highly recommended.

• Prepare samples under the clean laminar flow hood.

• Wear gloves.

• Use nuclease free water.

• Whenever possible, sterile disposable labware should be used in working with RNA in

order to avoid degradation.

• All reagents and equipment dedicated to the work with RNA should be RNase-free.

• Special care should be taken when handling the SARS-CoV-2 MULTITARGET RNA used

as a positive control in order to avoid contamination of samples and RT-qPCR reactions.

10.0. ATTACHED DOCUMENTS

• EXCEL FILE FOR DATA ANALYSIS (Covid_19_RTqPCR data analysis)

Disclaimer

All information, reagents and knowhow provided through ICGEB are intended for research use

only. The recipient agrees to use them in compliance with all applicable laws and regulations.

Every effort has been made to assure the accuracy of the sequences and quality of materials, but

ICGEB cannot provide any warranty regarding their accuracy or quality. Particularly, ICGEB makes

no express or implied warranties of any kind, including, but not limited to, the implied warranties of

merchantability or fitness for a particular purpose, or non-infringement. The recipient may

acknowledge the source of sequences in any oral presentations or written publications concerning

the research project by referring to the Laboratory of Molecular Virology, ICGEB, Trieste, Italy.

Documents

METHOD: DETECTION OF SARS-COV-2 RNA BY ...2.0 REFERENCES • China CDC Primers and probes for the ORF1ab and N regions of SARS-CoV-2 (24 January 2020) • Diagnostic detection of Wuhan