SARS-CoV-2 Quantification Standards
Quickly Quantify COVID-19 and Emerging Strains via RT-qPCRHighlights•

Ready-to-plate SARS-CoV-2 N-Gene RNA ladder for quantification ofsamples with unknown concentrations of SARS-CoV-2 RNA•

Guidelines for analyzing resulting RT-qPCR data, generating standard
curves, and calculating sample concentrations•Allows cross comparison of data between different PCR plates

Catalog Number:
R3016

Scan with your smart-phone camera toview the online protocol/video.

Table of Contents
Product Contents ........................................... 01
Materials Provided .................................................. 01
Specifications................................................. 02
Equipment and Reagents Needed (User Provided) ......... 03
Product Description ....................................... 04
Protocol: RT-PCR Set-Up .............................. 06
Before Starting ................................................ 06
Plate Setup ........................................................... 06
Real Time PCR Setup .............................................. 07
Protocol: Standard Curve Analysis .............. 08
Example Data ................................................. 08
Standard Curve Generation ....................................... 09
Calculating Sample Concentrations ............................. 10
Ordering Information ..................................... 11
Troubleshooting Guide .................................. 12
Guarantee ...................................................... 13

INSTRUCTION MANUAL Ver.1.0.6

Revised on: 8/31/2021

Product Contents
Materials Provided
SARS-CoV-2 Quantification Standards

Catalog No.

AmountStorage
Temp.1SARS-CoV-2 RNA Standard 1 – 5000 copies/µL

R3016-1-200

200 µL-80°C

SARS-CoV-2 RNA Standard 2 – 1000 copies/µL

R3016-2-200

200 µL-80°C

SARS-CoV-2 RNA Standard 3 – 200 copies/µL

R3016-3-200

200 µL-80°C

SARS-CoV-2 RNA Standard 4 – 40 copies/µL

R3016-4-200200 µL-80°C

SARS-CoV-2 RNA Standard 5 – 8 copies/µL

R3016-5-200

200 µL-80°C

No Template Control (NTC)

R3011-4-1

1000 µL-80°C

Reagent Storage and Handling. The SARS-CoV-2 Quantification Standards is to be shipped on dryice. If received in a condition other than the label indicates, or if damaged, contact Zymo Research Corp.
directly. Upon receipt, all components of the kit should be stored at ≤ -80°C. Reagents are stable for up
to three (3) freeze-thaw cycles.

Storage Temperature - Store all kit components (i.e., buffers, columns) at ≤ -80°C. Before use: Make sure iceor cooling block is prepared to store reagents.
11Specifications•SARS-CoV-2 RNA Quantification – Detection range from 80copies to 50,000 copies per reaction.

•Sample Source – Quantify SARS-CoV-2 RNA in purified RNA
samples such as wastewater, sputum and saliva.

•Compatibility – Purified, synthetic SARS-CoV-2 RNA ladder isvalidated for use with Bio-Rad’s CFX96 TouchTM Real—TimePCR Detection System with corresponding CFX Maestrosoftware and the Quick SARS-CoV-2 Multiplex Kit (R3013).
However, it can be used in any RT-qPCR system and RT-qPCR
master mix that targets the N-gene in SARS-CoV-2.

2Equipment and Reagents Needed (User Provided)
Item

Catalog No.

Manufacturer

Quick SARS-CoV-2 Multiplex Kit

R3013

Zymo Research

CFX96 Touch™ Real-Time PCR
Detection System (with optics capableof detecting HEX and Quasar 670) with
CFX Maestro software

1855195

Bio-RadMicrocentrifuge

Non-specificNon-specificMini Plate Spinner

Non-specificNon-specificHard Shell PCR Plate, 96-well, thinwall

HSP9601

Bio-RadMicroseal ‘B’ sealMSB1001

Bio-RadAerosol barrier pipette tips (NucleaseFree)

Non-specificNon-specificMicropipettes (2, 10, 200, 1000 μl)

Non-specificNon-specific

Disposable gloves, powder-free

Non-specificNon-specificVortex mixer

Non-specificNon-specificFreezer (≤ -80°C)

Non-specificNon-specific3Product Description
The SARS-CoV-2 Quantification Standards consists of a set of synthetic
SARS-CoV-2 nucleocapsid (N) gene RNA at varying concentrations. Thesestandards are ready to plate, allowing users to easily quantify levels ofSARS-CoV-2 present in their samples (Figs 1 and 2).
These standards are recommended to be used with the Quick SARS-CoV-2
Multiplex Kit (R3013, not provided), which can target both the N-gene in
SARS-CoV-2 and RNase P in human cells.

5000
copies/µL

1000
copies/µL

200
copies/µL

40
copies/µL

8copies/µL

Figure 1: Resulting amplification curves of SARS-CoV-2 Quantification Standards using
recommended Quick-SARS-CoV-2 Multiplex Kit master mix.

Figure 2: Example standard curve generated from the average Ct readings for a set of theSARS-CoV-2 Quantification Standards4

Each standard in this kit is also spiked with a small amount of human HeLa RNAfor stability. This spike-in can be used to gauge standard integrity. As eachstandard, regardless of the concentration of SARS-CoV-2 RNA, was spiked withequal amounts of HeLa RNA, the resulting Ct values should be near identical(Figure 3). Deviation from the expected average Ct of 27.5±2 would indicate loss
or degradation of SARS-CoV-2 RNA template.

Figure 3: Resulting amplification of HeLa RNA in the SARS-CoV-2
Quantification Standards.

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Protocol: RT-PCR Set-Up✓

Before starting, thaw frozen 2X CV Master Mix (not provided) on ice, mix 10 times byinversion, centrifuge briefly, and place back on ice, covered, until ready to use.

✓Thaw frozen SARS-CoV-2 Standards on ice until ready to use.

✓If this is your first time thawing these standards, aliquot each standard in 25µL
volumes and store at ≤ -80°C for future use to avoid excessive freeze-thawcycles.

✓Avoid exposing the 2X CV Mix and subsequent reactions to direct light and keep theRT-PCR plate on ice during preparation.

✓To prevent contamination, handle all reagents carefully and aliquot the necessary
master mixes in the RT-PCR plate before handling any test samples, standards, orcontrols.

Plate Set Up
1. Pipette 10µL of the 2X CV Master Mix into each well following thetemplate plate shown below.
2. Each standard curve control (STND1-STND5) will be plated induplicate; add 10µL of RNA sample or standard to each wellaccording to the template1.
1

2

3

4

5

6

A

STND1

STND2

STND3

STND4

STND5NTC

B

STND1

STND2

STND3STND4STND5NTC

7891011C
DEF
GH

3. Firmly seal the 96-well PCR plate with an optically transparent
sealing film.
4. Briefly vortex the 96-well PCR plate and centrifuge to eliminate
bubbles and bring any droplets to the bottom of the well.

1The exact layout is dependent on the number of samples being processed. It is strongly recommended to platestandards in technical duplicates; plating samples in technical replicates is up to user discretion.

612Real-Time PCR Set Up✓Ideally, have the PCR program and plate layout prepared prior to plating.
Otherwise, avoid exposing the prepared plate to light and keep on ice until ready.

1. Using the Real-Time PCR software (e.g., Bio-Rad CFX Maestro forthe CFX96), create a PCR program using the following parameters.
Step
1
2
3
4
5
6
7

Temperature
55°C
95°C
95°C
72°C
57°C
PLATE READ
END

Time (min:sec)
15:00
10:00
00:05
00:30
00:30

45 cycles

2. Assign each well the corresponding sample names, targets, andfluorophores.
Targets

Fluorophores

Excitation

Emission

N1HEX

535 nm

556 nm

RPQuasar® 670647 nm

666 nm

3. Enable all the light filters required to detect both fluorophores.
4. Load the RT-PCR plate into the Real-Time PCR machine and start
the program. When finished, the program will return Ct values forboth the HEX channel and the Quasar 670® channel.
5. Proceed to Standard Curve Analysis (page 8) to generate astandard curve from the HEX channel Ct values.

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Protocol: Standard Curve Analysis
Example Data
This section will cover generating a Standard Curve from the Ct values
obtained from your RT-PCR. It will also cover how to calculate theoriginal sample concentration.
The following sample data will be provided as reference for data analysis
and standard curve generation.
Table 1. Concentration (ng/µL) of SARS-CoV-2 Quantification Standards
HEX (N1)
Standard
Standard
Standard
Standard
Readings
1 (Red)2 (Orange)
3 (Yellow)
4 (Green)
Concentration

5000

1000

20040

Table 2. HEX channel Ct values for standards plated in duplicate.
HEX (N1)
Standard
Standard 2
Standard 3
Standard
Readings
1 (Red)(Orange)(Yellow)
4 (Green)Standard
5 (Clear)
8Standard
5 (Clear)

25.40

27.60

30.24

33.01

35.08

25.17

27.9330.19

32.88

35.68

25.29

27.77

30.52

32.95

35.38

Raw CtAverage Ct

Table 3. Quasar® 670 channel Ct values for standards plated in duplicate.
Standard
Standard 2
Standard 3
Standard
Standard
Quasar 670
Readings
1 (Red)(Orange)(Yellow)
4 (Green)
5 (Clear)
26.99

27.13

27.26

27.39

27.48

26.83

27.32

27.38

27.40

27.34

26.91

27.23

27.32

27.40

27.41

Raw CtAverage Ct

8Standard Curve Generation✓

Before starting, ensure the Quasar® 670 Ct values for each standard falls
within 27.5 ± 2 Ct. As can be seen in the sample data (Table 3), all values
fall within this range.

To generate a standard curve (Figure 4), plot standard
concentrations (Table 1) as an exponential function (Equation 1) of
the resulting average Ct for each standard in the HEX channel(Table 2). To better visualize the standard curve, the concentrations
can be plotted on a logarithmic (base 10) scale.
Equation 1. Standard curve relating Ct to template concentration (𝑪𝒕𝒆𝒎𝒑𝒍𝒂𝒕𝒆 ) withexponential coefficients 𝑎 and 𝑏.

𝑪𝒕𝒆𝒎𝒑𝒍𝒂𝒕𝒆 = 𝑎 × 𝑒 𝑏(𝑪𝒕)Figure 4: An example standard curve generated using the above formula.

In the case of the sample data (Table 2), the resulting regression curve is:
𝑪𝒕𝒆𝒎𝒑𝒍𝒂𝒕𝒆 = (4.48 × 1010 ) × 𝑒 −0.634(𝑪𝒕)This standard curve has a coefficient of determination of 𝑅2 = 1.000,
indicating a very strong correlation between template concentration(𝐶𝑡𝑒𝑚𝑝𝑙𝑎𝑡𝑒 ) and Ct. This allows accurate predictions of the concentrations of
unknown samples based on resulting Ct values.

Once the regression curve for this standard curve has been
generated, samples can then be quantified. The next section willprovide guidelines on how to account for any dilution factors toaccurately calculate the original sample concentration.

9Calculating Sample Concentrations✓Before starting, please have the following information available to quantify samples:oThe sample input volume 𝑉𝑠𝑎𝑚𝑝𝑙𝑒 (µL) that was initially processedoThe elution volume 𝑉𝑒𝑙𝑢𝑡𝑖𝑜𝑛 (µL) of the final extracted RNA
oThe standard curve calculated in the previous section.

✓The above information will be used to calculate:oThe original sample concentration 𝐶𝑠𝑎𝑚𝑝𝑙𝑒 (genomic copies per µL)
oThe template concentration 𝐶𝑡𝑒𝑚𝑝𝑙𝑎𝑡𝑒 (genomic copies per µL) which is
directly generated from our standard curve and is the concentration of theinput template directly added to the PCR reaction.

1.

The previously generated regression curve can be used to predict
the template concentration 𝑪𝒕𝒆𝒎𝒑𝒍𝒂𝒕𝒆 (copies/µL) by inputting thesample Ct into the resulting exponential equation (Equation 1).
Example: A sample of unknown SARS-CoV-2 concentration with a Ct valueof 30 would yield a template concentration as follows:
𝑪𝒕𝒆𝒎𝒑𝒍𝒂𝒕𝒆 = (4.48 × 1010 )𝑒 −0.634(𝑪𝒕)𝑪𝒕𝒆𝒎𝒑𝒍𝒂𝒕𝒆 = 𝟐𝟒𝟓 𝒄𝒐𝒑𝒊𝒆𝒔 𝒑𝒆𝒓 µ𝑳

However, this concentration represents the concentration of thetemplate (𝐶𝑡𝑒𝑚𝑝𝑙𝑎𝑡𝑒 ), not the sample concentration (𝐶𝑠𝑎𝑚𝑝𝑙𝑒 ).
2. The template concentration (𝐶𝑡𝑒𝑚𝑝𝑙𝑎𝑡𝑒 ) can be converted to thesample concentration (𝐶𝑠𝑎𝑚𝑝𝑙𝑒 ) using dilution factor D, which
relates input sample volume (𝑉𝑠𝑎𝑚𝑝𝑙𝑒 ) to elution volume (𝑉𝑒𝑙𝑢𝑡𝑖𝑜𝑛 ):𝐷=𝑉𝑒𝑙𝑢𝑡𝑖𝑜𝑛
𝑉𝑠𝑎𝑚𝑝𝑙𝑒Example: If a total volume of 500µL of our original sample was processed,
but eluted in 35µL, the dilution factor would be:

𝐷=35 𝜇𝐿= 0.07500 𝜇𝐿

3. Using this dilution factor, the original sample concentration(𝐶𝑠𝑎𝑚𝑝𝑙𝑒 ) can be related to the template concentration (𝐶𝑡𝑒𝑚𝑝𝑙𝑎𝑡𝑒 ):𝐶𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐶𝑡𝑒𝑚𝑝𝑙𝑎𝑡𝑒 × 𝐷
𝐶𝑠𝑎𝑚𝑝𝑙𝑒 = (245 𝑐𝑜𝑝𝑖𝑒𝑠 𝑝𝑒𝑟 µ𝐿) × (0.07)
𝐶𝑠𝑎𝑚𝑝𝑙𝑒 = 17.15 𝑐𝑜𝑝𝑖𝑒𝑠 𝑝𝑒𝑟 µ𝐿

10Ordering Information
Product Description

Catalog No.

Size

SARS-CoV-2 Quantification Standards

R3016

20 rxns

Quick SARS-CoV-2 Multiplex Kit

R3013
R3013-1K
R3013-10K

100 rxns
1,000 rxns
10,000 rxns

Complete Your Workflow
DNA/RNA ShieldTM
Cat. Nos. R1100-50, R1100-250,
R1200-25 R1200-125✓✓Abides by Center for Disease Control’s (CDC)
guidelines for pathogen inactivation
Stabilizes DNA/RNA

Zymo Environ Water RNA Kit
Cat. No. R2042✓✓✓

Inhibitor-free RNA extraction
Safely inactivates SARS-CoV-2
Enhanced viral enrichment and low elution
volume for low concentration samples

Quick SARS-CoV-2 Multiplex Kit
Cat. Nos. R3013, R3013-1K, R3013-10K✓✓

High sensitivity for low concentrations
Ready to use PCR Master MixOnline qPCR Concentration Calculator
Coming soon!
✓✓Calculates concentration of samples directly from Ct values
Generates standard curve graph and equation
11Troubleshooting Guide
Problem
BackgroundContamination

Possible Causes and Suggested Solutions
Workspace contamination:- Clean workspace, microcentrifuge, and pipettes with 10% bleachroutinely to avoid contamination.
- Use of kit in exposed environment without proper filtration can lead tobackground contamination. Check pipettes, pipette tips, microcentrifuge
tubes, workspace, etc. for contamination- Make sure all reagent tubes and bottles are properly sealed for storage.
Use of these outside a clean room or hood can result in contamination.

Loss of Volume
during PCR

Adhesive seal:- A loosened adhesive seal on the PCR plate can lead to sample
evaporation. Ensure that the plate seal is secure on every well during
targeted sequence amplification.
Lid pressure:- Inconsistent lid pressure. Ensure that the lid pressure on the real-timequantitative PCR instrument is consistent over the PCR plate according
to the manufacturer’s recommendation.

Low Coefficient ofDetermination

Degradation of standards:- Standard degradation can be checked by viewing Ct readings under theQuasar® 670 channel. Each standard should give a Quasar® 670 Ct
reading of approximately 27.5±2 Ct. A significantly higher Ct indicates lossof viable standard.
- Ensure that kit arrived on dry ice and that standards were stored at -80°C
upon arrival.
- Do not excessively freeze-thaw the standards. Standards are stable forup to 3 freeze-thaw cycles.
Plating contamination:- If Quasar® 670 readings appear normal, check plating techniques toprevent well to well contamination. Standards can be transferred fromoriginal tubes to strip tubes for use with multichannel pipettors.

High Standard
Deviation/No Signal
for Standard 5Limits of detection- Standard 5 (2 copies/µL) approaches the limits of detection when usedwith the recommended CFX96 TouchTM Real—Time PCR Detection
System with corresponding CFX Maestro software and the Quick SARSCoV-2 Multiplex Kit (R3013). It is not abnormal to have very low or nosignals with high standard deviation at this concentration.
Using a different RT-PCR detection system- These standards have been verified for use with the above system. Othersystems may have different limits of detection, leading to loss of signal at
the lower concentrations.

For technical assistance, please contact 1-888-882-9682 or email tech@zymoresearch.com

12100% satisfaction guarantee on all Zymo Research products,
or your money back.
Zymo Research is committed to simplifying your research with quality products
and services. If you are dissatisfied with this product for any reason, please call1(888) 882-9682.

Integrity of kit components is guaranteed for up to one year from date of purchase.
Reagents are routinely tested on a lot-to-lot basis to ensure they provide the highest performance and reliability.
This product is for research use only and should only be used by trained professionals. It is not for use indiagnostic procedures. Some reagents included with this kit are irritants. Wear protective gloves and eye
protection. Follow the safety guidelines and rules enacted by your research institution or facility.
™ Trademarks of Zymo Research Corporation, ® Registered Trademarks of Zymo Research Corporation
Other trademarks: Bio-Rad CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.), Quasar®
(Biosearch Technologies), SYBR® (ThermoFisher Scientific)

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